Haematologica 2004;89: supplement no. 8 - Supplements ...

haematologicaThe origin and power of a nameAncient Greekαιµα [aima] = blood;αιµατος [aimatos] = of blood,λογος [logos]= reasoningScientific Latinhaematologicus (adjective) = related to bloodScientific Latinhaematologica (adjective, plural and neuter,used as a noun) = hematological subjectsModern EnglishJournal of Hematology2003 JCR ® Impact Factor = 3.453h

XVIII Congress of the Italian Society forHemostasis and Thrombosis ResearchRome, September 30-October 3, 2004

XVIII Congress of the Italian Society for Hemostasis and Thrombosis ResearchRome, September 30 - October 3, 2004"Centro Congressi Europa" Università Cattolica del Sacro CuoreORGANIZING COMMITTEE OF THE CONGRESSPresidentMaria Benedetta Donati, CampobassoCo-PresidentGiuseppe Leone, RomaHonorary PresidentBruno Bizzi, RomaMEMBERS OF THE SISET EXECUTIVECOMMITTEEVice-PresidentMarco Cattaneo, MilanoPast PresidentFrancesco Violi, RomaMembersAnna Falanga, BergamoCristina Legnani, BolognaMaurizio Margaglione, FoggiaPaolo Simioni, PadovaScientific SecretaryDomenico Prisco, FirenzeLOCAL SCIENTIFIC COMMITTEEChiara Cerletti, CampobassoGiovanni De Gaetano, CampobassoValerio De Stefano, RomaAugusto Di Castelnuovo, CampobassoLicia Iacoviello, CampobassoRaffaele Landolfi, RomaAntonio Mascioli, CampobassoSergio Storti, CampobassoBruno Zappacosta, CampobassoFrancesco Zito, Campobasso

September 2004Table of ContentsXVIII Congress of the Italian Society for Hemostasis and Thrombosis ResearchRome, September 30 - October 3, 2004Foreword....................................................................................................................................................................................................................aEducational SessionsUpdates in Cellular Biology............................................................................................................................................................................1Antithrombotic Therapy 2004........................................................................................................................................................................1Resistance to Antiplatelet Agents ...................................................................................................................................................................2Hemorrhagic Syndromes................................................................................................................................................................................4Infections and Thrombosis.............................................................................................................................................................................5Oral Anticoagulant Therapy...........................................................................................................................................................................7Atherothrombosis...........................................................................................................................................................................................8Blood Platelets.................................................................................................................................................................................................9Selected Oral CommunicationsInflammation and Vascular Risk...................................................................................................................................................................11Venous Thromboembolism..........................................................................................................................................................................13Blood Platelets and von Willebrand Factor ..................................................................................................................................................15Vascular Risk Factors and New Antithrombotic Agents..............................................................................................................................17Oral CommunicationsPlatelet Biochemistry and Physiology CO-001-CO-008 ..............................................19Cellular Biology of Hemostasis CO-009-CO-016 ..............................................23Hemophilia: Clinical Studies CO-017-CO-024 ..............................................27Thrombosis and Nutrition: Experimental and Epidemiological Studies CO-025-CO-031 ..............................................31Prevention and Therapy of Venous Thromboembolism CO-032-CO-039 ..............................................35Hereditary Thrombophilia: Epidemiological Aspects CO-040-CO-047 ..............................................39Hemorrhagic Coagulopathies: Molecular Defects CO-048-CO-054 ..............................................44Inflammation and Thrombosis CO-055-CO-062 ..............................................48Platelet Function Inhibitors CO-063-CO-070 ..............................................52Thrombophilia and Genetic Polymorphisms CO-071-CO-077 ..............................................56Anticoagulant Therapy: Clinical and Laboratory Problems CO-078-CO-085 ..............................................60Venous Thromboembolism: Risk of Relapse CO-086-CO-093 ..............................................64Vascular Risk Factors CO-094-CO-101 ..............................................69Homocysteine and Antiphospholipid Antibodies CO-102-CO-109 ..............................................73Hemorrhagic Syndromes: Diagnosis and Clinical Aspects CO-110-CO-117 ..............................................77Venous Thromboembolism: Epidemiology and Risk Factors CO-118-CO-125 ..............................................81Thrombosis and Cancer: Cell and Clinical Studies CO-126-CO-133 ..............................................85von Willebrand Factor and Disease CO-134-CO-141 ..............................................89PostersVascular BiologyPlatelets: Physiological and Clinical Aspectsvon Willebrand FactorHemorrhagic SyndromesGenetic Determinants of Cardiovascular RiskHomocysteineInflammation and Vascular RiskNutrition and Vascular RiskWomen’s Health Issues in Thrombosis and HemostasisThrombosis and CancerThrombosis and Myeloproliferative SyndromesAntiplatelet TherapyAcquired Coagulation DisordersAntiphospholipid AntibodiesCerebrovascular and Coronary DiseasesHereditary ThrombophiliaVenous Thromboembolism: EpidemiologyVenous Thromboembolism: Diagnosis and TherapyOral Anticoagulant TherapyControl of AnticoagulationTherapy of HemophiliaPO-001-PO-006................................................94PO-007-PO-015................................................97PO-016-PO-025..............................................101PO-026-PO-030..............................................107PO-031-PO-034..............................................109PO-035-PO-046..............................................111PO-047-PO-057..............................................117PO-058-PO-062..............................................123PO-063-PO-071..............................................126PO-072-PO-080..............................................131PO-081-PO-088..............................................135PO-089-PO-096..............................................140PO-097-PO-103..............................................144PO-104-PO-110..............................................147PO-111-PO-120..............................................150PO-121-PO-129..............................................155PO-130-PO-143..............................................160PO-144-PO-151..............................................167PO-152-PO-165..............................................172PO-166-PO-174..............................................179PO-175-PO-192..............................................184Haematologica 2004; vol. 89; supplement no. 8 - September 2004(indexed by Current Contents/Life Sciences and in Faxon Finder and Faxon XPRESS, also available on diskette with abstracts)http://www.haematologica.org/

XVIII Congress of the Italian Society for Hemostasis and Thrombosis ResearchRome, September 30 - October 3, 2004FOREWORDThis Supplement of Haematologica gathers the contributions of the members of the Italian Society forHaemostasis and Thrombosis Research (SISET) and of few, distinguished foreign guests on the occasion ofthe XVIII National Meeting of the Society.The Table of Contents of the abstracts (367 in total) reflects the structure of the Congress, with EducationalSessions (featuring State-of-the Art presentations or Forums on selected topics), Oral Communicationsand Posters.Four Sessions of Selected Oral Communications include presentations rated as of particularly high qualityby the Scientific Board of the Congress.The traditional support our Society gives to promote the research work of its younger members has beenexpressed this year by 12 SISET prizes awarded to Authors younger than 35, one prize made available by aprivate Foundation (Fondazione Polidoro - Città Sant'Angelo, Pescara) to promote cardiovascular preventionthrough life habit changes and two prizes awarded by the Federation of Centers Monitoring AnticoagulantTreatment (FCSA). These selected Abstracts are marked by a special label.The scientific content of this volume reflects the multidisciplinary approach of our Society, spanning fromfundamental research in cell and molecular biology to the clinical application of new diagnostic and therapeuticperspectives for hemorrhagic and thrombotic disorders. The results of some studies will be presentedfor the first time at our Congress.Together with the members of the Congress Scientific Committee, I wish you a stimulating reading andan enjoyable Congress!Maria Benedetta Donati, MD, PhD,President of SISET

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 20041XVIII Congress of the Italian Society for Hemostasis and Thrombosis ResearchRome, September 30 - October 3, 2004EDUCATIONAL SESSIONSUPDATES IN CELLULAR BIOLOGYTHE TISSUE FACTOR PATHWAY: NEW HYPOTHESESTremoli E*, Camera M*, Mussoni L, Banfi C*Dipartimento di Scienze Farmacologiche e *CentroCardiologico Monzino, Milan, ItalyTissue factor is a transmembrane protein expressedby blood cells and by cells of the vessel wall thatcontributes to thrombus formation and propagation.Endothelial cells, and circulating monocytes do notnomally express TF, but the synthesis of this proteincan be elicited by inflammatory stimuli, as cytokinesor bacterial endotoxin. Recently, it has been shownthat also blood platelets contain measurableamounts of TF. In addition, agonists of plateletaggregation, e.g. ADP calcium ionophore or TRAP,induce surface expression of TF in platelets, as shownby cytofluorimetry. Platelet TF has biological activity,as it binds factor VIIa and the TF/factor VIIa complexdirectly activates factor X. Drugs interferingwith platelet aggregation have discrete effects onplatelet TF expression. The stable PGI2 analog Iloprostcompletely prevents ADP-induced plateletaggregation, whereas short term aspirin incubationwith platelets does not exert any effect. Antagonistsof the GPIIbIIIa receptor have the paradoxical effectof increasing platelet TF surface expression. Interestingly,platelets contain TF mRNA, as measured byreal time PCR, which suggests that upon appropriateconditions platelets my undergo TF protein synthesis.Besides this, however, it remains difficult todefine the origin of intraplatelet TF. Indeed, the possibilityexists that intraplatelet TF may derive from TFthat circulates in blood, probably of monocyte orendothelial origin. In this context, it is worth mentioningthat after activation cells may secrete plasmamembrane fragments into the extracellularspace, which are called “microparticles”. These mayderive not only from monocyte or platelet vesciculationbut also from activated endothelial cells. Circulating“microparticles”, according to their contentmay govern the transcellular exchange of biologicalinformation. Indeed, monocyte-derived “microparticles”expressing tissue factor and PSGL-1 have beenshown to accumulate on activated platelets expressingP-selectin, thus concentrating tissue factor to alevel that triggers the initiation of blood coagulation.ANTITHROMBOTIC THERAPY 2004NEW ANTICOAGULANTS FOR TREATMENT AND PREVENTION OFVENOUS THROMBOEMBOLISMPrandoni PDipartimento di Scienze Mediche e Chirurgiche,Università di Padova, ItalyTreatment of venous thromboembolism (VTE) usuallystarts with concomitant administration ofheparin or low-molecular-weight heparin (LMWH)and an oral anticoagulant. Although the introductionof LMWH has simplified the initial treatment of VTE,problems remain that are awaiting proper solution.Indeed, LMWH must be given by daily subcutaneousinjection, and oral anticoagulants require routinecoagulation monitoring, which is inconvenient forpatients and physicians. Recently, three new anticoagulantshave been introduced in an attempt toovercome these limitations. They include ximelagatran,an orally active inhibitor of thrombin, fondaparinuxand idraparinux, which electively inhibit factorXa. These agents produce a predictable anticoagulantresponse, thus obviating the need for routinecoagulation monitoring. In addition, they do notcause heparin-induced thrombocytopenia. In availablerandomized clinical trials, all three compoundshave been shown to be as effective and safe asheparin or LMWH for the treatment of patients withestablished VTE. In addition, fondaparinux and ximelagatranhave recently been registered for preventionof VTE in patients candidate to major orthopedichaematologica vol. 89(suppl. n. 8):september 2004

2Educational Sessionssurgery. In available randomized clinical trials, bothof them have been shown to be significantly moreeffective and as safe as LMWH.GUIDELINES OF THE AMERICAN COLLEGE OF CHESTPHYSICIANSSchunemann HClinical Epidemiology & Biostatistics, McMasterUniversity, Hamilton, Ontario, CanadaThis paper describes the methodology for developmentof evidence-based guidelines using theexample of the 7 th American College of Chest Physicians(ACCP) Conference on Antithrombotic andThrombolytic Therapy. For the development of theseguidelines, authors began by specifying the population,intervention and alternative and outcomes foreach clinical question and defined criteria for eligiblearticles, including methodological criteria, foreach recommendation. Librarians, in collaborationwith guideline authors and methodologists, conductedsystematic searches for evidence. Guidelineauthors systematically evaluated the evidence, consideredthe full range of benefits, risks, inconvenienceand costs associated with alternative managementstrategies, considered patients' underlying values andpreferences, and made recommendations accordingly.To increase the likelihood that the recommendationsadequately represent patient values and preferencesthe development process included review ofrecommendations by research methodologists, practicinggeneralists and specialists. Authors paid carefulattention to the strength of underlying evidenceand the balance between risks and benefits, bothreflected in Grades of Recommendations. Thus,improvements to the process of making recommendationsfor the ACCP guidelines include explicit definitionof questions, transparent eligibility criteriafor including studies, and specification of values andpreferences underlying recommendations where particularlyrelevant. In combination with the previouspractice of grading recommendations according totheir strength, and the methodological quality of thesupporting studies, these innovations establish theseguidelines as, by and large, evidence-based.RESISTANCE TO ANTIPLATELET AGENTSRESISTANCE TO ANTIPLATELET AGENTS: REVIEW OF THELITERATURE AND SOME METHODOLOGICAL CONSIDERATIONSCattaneo MUnità di Ematologia e Trombosi, Ospedale San Paolo,Dipartimento di Medicina, Chirurgia e Odontoiatria -Università di Milano, ItalyIn the last few years, the problem of aspirin resistancehas been largely emphasized in the medicalliterature. More recently, clopidogrel resistance hasalso been investigated. The term aspirin resistancehas been given different definitions by differentresearchers. I propose that, lacking a reproducibleand highly sensitive and specific method to studyTxA2-dependent platelet function, the pharmacologicalresponse to aspirin treatment should beassessed by measuring the degree of inhibition ofTxA2 production. Therefore, the only acceptable definitionof aspirin resistance should rely on thedemonstration of an insufficient inhibition of TxA2production. Suboptimal reduction of urinary 11-dehydro TxB2 levels during aspirin treatment is associatedwith heightened risk for future myocardialinfarction and cardiovascular death; inadequateinhibition by aspirin of TxA2 biosynthesis can beobserved in patients on treatment with ibuprofen,due to competition of the two drugs at the COX-1level; observational studies and post-hoc analysissuggested that ibuprofen blunts the cardioprotectiveeffect of aspirin, although the question is stillcontroversial. Less well known than aspirin resistance,but certainly better characterized is clopidogrelresistance. The extent of the platelet aggregationresponse in vitro to ADP has usually been adoptedto define clopidogrel resistance. However, due tothe well-known general pitfalls of in vitro plateletaggregation testing and to the presence of two ADPreceptors on platelets, only one of which inhibited byclopidogrel (P2Y12), a better and more specific testwould be measurement of the extent of ADP-inducedinhibition of adenylyl cyclase, which is uniquelymediated by P2Y12. A recent study showed thatpatients with clopidogrel resistance are at increasedrisk for recurrent cardiovascular events. At present,aspirin and clopidogrel resistance should not belooked for in the clinical setting, because there is nodefinite demonstration of an association with clinicalevents conditioning cost-effective changes inpatient management.haematologica vol. 89(suppl. n. 8):september 2004

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 20043MOLECULAR MECHANISMS OF COX-1 AND ADP-INDEPENDENTPLATELET ACTIVATION IN THE PHENOMENON OF “RESISTANCETO ANTIPLATELET AGENTS”Gresele PDept. of Internal Medicine, Section of Internal andCardiovascular Medicine, University of Perugia, Perugia,ItalyPlatelet aggregation is initiated by soluble orsubendothelial agonists acting on specific surfacereceptors which in turn trigger a number ofintraplatelet signal transduction events leading toplatelet activation. Thromboxane A2 (TxA2) and ADP,released during the activation process, are consideredthe main amplification loops of the activationprocess and are the target of aspirin and clopidogrel,respectively. Interestingly, direct activation of signaltransduction mechanisms by some chemicals (e.g.PMA, a PKC activator) induces a platelet aggregationresponse insensitive to aspirin or thienopyridines.Over the last few years it has emerged that, besidesdirect platelet agonists, a number of substances existthat, although not able to induce aggregation ,facilitatethe response to subthreshold doses of full agonistsand act as an important system of modulationof the platelet response to stimuli. Several of thesesubstances are recognized as mediators primarilyinvolved in a series of pathophysiologic phenomenadifferent form haemostasis, such as inflammation orallergy (PGE2, cytokines), tissue catabolism or tumormetastasis (MMPs, beta-Hex, leptin), immunologicalresponse (sCD40L), growth arrest (Gas6), hemopoiesis(thrombopoietin). Platelet priming inducedby several of these substances involves molecularmechanisms insensitive to COX-1 and ADP-inhibition.Examples are: (1) Platelet CD40L expression isreduced by ADP inhibition but not by aspirin in vitroand especially in vivo; (2) The potentiation ofplatelet activation by MMP-2 or beta-Hex is not preventedby incubation with aspirin or by ADP-inhibition;in addition, aspirin does not suppress therelease of these mediators in vivo in humans, at alocalized site of platelet activation. The enhancedformation or release or a number of platelet primersin pathologic conditions may contribute to the resistanceto antiplatelet agents. Finally, activationinduced by high-shear stress is also largely insensitiveto inhibition, especially by aspirin.ASPIRIN RESISTANCE: ROLE OF THROMBOXANEPulcinelli FM, Violi FDipartimento di Medicina Sperimentale e Patologia,Università “La Sapienza”, Rome, ItalyThe antithrombotic property of aspirin has beenwell documented in patients with acute coronarysyndrome; however, laboratory and clinical evidenceshave demonstrated that not all the patients areequally sensitive to aspirin inhibitory action. For thisphenomenon the term “aspirin resistance” has beencoined, but to understand the exact role in cardiovasculardiseases it is necessary to clarify:a) the mechanisms involved; b) the methods tostudy this phenomenon ; c) the clinical relevance.a) The mechanisms of the platelet resistance toaspirin can be ascribed to two principal causes: 1. thecapability of platelets to still produce thromboxanedespite the patients are under aspirin treatment; thatit is possible to classify as true "aspirin resistance" ifafter an additional in vitro aspirin treatment thethromboxane is still produced or “non-responsiveness”to aspirin if the in vitro aspirin treatment abolishesagonists induced thromboxane production; 2.the concomitant participation of other metabolicpathways, independent of thromboxane, in inducingplatelet activation; that we can classify as pseudoresistance.Both mechanisms are probably involved butit seems that the prevalence is much higher for thefirst one. In fact, in patients undergoing coronaryartery bypass grafting the platelet aspirin resistanceis due to a disturbed inhibition of platelet COX-1 byaspirin (Zimmerman et al. Circulation 2003; 108 pg542) and in one of our studies, that will be presentedin this meeting, in 196 patients under aspirinchronic treatment, Collagen-induced thromboxaneproduction was 128.7±21.6 pg/10 8 cells, with valuesranging from 0.5 to 616.8 pg/10 8 cells and significantlycorrelated with platelet aggregation (r=0.44;p

4Educational Sessionsclinical relevance, different studies indicate that thisphenomenon may account for recurrent vascularthrombotic events,, during long-term follow-up, butad hoc clinical studies are necessary to understandthe exact role that aspirin resistance has in theatherotrombotic compliances. In conclusion at themoment several questions, regarding aspirin resistance,are open; in the future study this phenomenomusing appropriate methods will clarify most ofthem, expecially mechanisms and clinical relevance,and the Italian “Gruppo di Studio delle Piastrine” ishighly involved in the matter.HEMORRHAGIC SYNDROMESTHE HEMOSTATIC BALANCE: REVISITED THROUGH NATURALLYOCCURRING MODELSMannucci PMCentro Emofilia e Trombosi Angelo Bianchi Bonomi,Dipartimento di Medicina Interna e Dermatologia,IRCCS Ospedale Maggiore e Università di Milano, ItalyIt has been postulated more than 50 years ago thathemostasis is ensured by a balance between prohemostaticand anti-hemostatic factors, and thatthe derangement of this balance may lead to bleedingor thrombosis. Recent evidence obtained throughthe study of natural clinical models has providedimportant contributions to validate the existence ofa balance between hemostatic and antihemostaticmechanisms. The presence of thrombophilic mutationsor phenotypes attenuates hemorrhagic phenotypes.For instance, women carrying factor V Leidenbleed less at parturition than non-carriers, carriers ofboth sexes have less blood losses than non-carriersduring cardiac surgery and have less frequentlyintracranial bleeding; patients with hemophilia andother congenital coagulation defects have a lesssevere hemorrhagic phenotype when they also carrythrombophilic mutations. On the other plate ofthe balance, there are data indicating that patientswith hemophilia and obligatory hemophilia carrierswith reduced levels of factor VIII suffer less frequentlyfrom myocardial infarction than the normalpopulation. This is due to their decreased tendencyto form coronary thrombi, whereas they have no lessatherosclerosis than controls. The same observation(lack of protection from atherosclerosis, protectionfrom thrombosis) was made in carriers of such prototypicdefects of platelet adhesion and aggregationas severe von Willebrand disease and Glanzmannthrombasthenia. On the whole, it seems that the tendencyto bleeding or thrombosis may be attenuatedand/or modified by the coexistence of inheritedthrombophilic abnormalities.PATIENT STUDIES PROVIDING NEW INSIGHTS IN PLATELETPHYSIOLOGYVan Geet C, Freson K, Vermylen JCenter for Molecular and Vascular Biology, Universityof Leuven, BelgiumIn this era of “evidence-based”medicine, pathophysiologyoften takes second seat to epidemiology.Yet the history of thrombosis and haemostasis hascontinuously demonstrated that the careful study ofunusual patients with congenital disorders can providemajor insights into important physiologicalprocesses. Examples are the discovery of the clottingfactors, the elucidation of the cause of thrombasthemia,the familial thrombophilias, etc. Suchpathophysiological studies are now markedly aidedby the increased knowledge of the human genome,allowing identification of the genetic basis of thedisturbance and reproduction of the anomaly in animalmodels. A recent example* is the study of tworelated patients with mental retardation, a bleedingtendency, reduced platelet aggregation and a mildthrombocytopenia. Partial trisomy 18 p in thesepatients led to the search for possibly involved genesin this area. The patients indeed had three copies ofthe pituitary adenylate cyclase-activating polypeptide(PACAP) gene, increased PACAP mRNA levels infibroblasts, and elevated PACAP concentrations inplasma. The PACAP receptor in platelets is coupled toadenylyl cyclase activation; accordingly, increasedbasal cAMP levels were found in patients' platelets,providing a basis for the reduced aggregation.Megakaryocyte-specific transgenic overexpressionof PACAP in mice increased PACAP release fromplatelets, reduced platelet activation, and prolongedthe tail bleeding time. In contrast, the PACAP antagonistPACAP (6-38) or a monoclonal PACAP antibodyenhanced the collagen-induced aggregation of normalhuman platelets, and in PACAP knockout mice,an increased sensitivity toward collagen was found.Injection of antibody to PACAP in normal miceinduced marked thrombocytosis, indicating thatPACAP has a dampening role on thrombocytopoiesis.These findings suggest a potential role for PACAPinhibitors in managing bleeding problems.*J Cancer Inst, 2004,113;905-912.haematologica vol. 89(suppl. n. 8):september 2004

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 20045INFECTIONS AND THROMBOSISINFECTION AND THROMBOSIS: THE ROLE OF TISSUE FACTORSemeraro N, Colucci MDipartimento di Scienze Biomediche e OncologiaUmana, Sezione di Patologia Generale, Universitàdegli Studi, Bari, ItalyActivation of blood coagulation is a common featurein patients with severe infectious diseases andmay eventually result in thrombotic manifestations,ranging from localized events to sepsis-associateddisseminated intravascular coagulation. Moreover,some chronic infections have been related with atherosclerosisand arterial thrombosis. Recent studieshave provided new insights into the complex molecularevents linking infections and thrombosis. Thecausative micro-organism and/or a number of mediators,both micro-organism-derived and host-manufactured(mainly cytokines) can elicit several prothromboticchanges in endothelial cells, monocytes/macrophages(MM) and specific cells in targettissues, among which the synthesis and expression oftissue factor (TF) is one of critical importance. Awealth of available new knowledge, derived fromstudies in different animal models and in man, indicatesthat the procoagulant response to infectionleading to thrombin formation and micro-vascularthrombosis is solely mediated by the TF pathway andis own to the aberrant in vivo expression of TF by variouscells, especially MM. The shedding of procoagulant,TF-rich microparticles into the circulation andtheir interaction with other cells, e.g. platelets andneutrophils, may contribute to local propagationand/or dissemination of clotting activation. The centralrole of TF in infection is further supported byrecent observations indicating that the binding offactor VIIa to TF-bearing cells activates several intracellularsignal pathways eventually leading to proinflammatoryeffects and up-regulation of numerousgenes involved in regulation of growth, cell motility,synthesis of cytokines, proteolytic enzymes and evenTF itself. Although the precise pathophysiologicalimplications of these findings are presently unclear,there is some circumstantial evidence that, duringinfections, cell TF might directly influence inflammatoryevents. These insights into the pathogeneticmechanisms may have implications for the managementof infection-associated thrombosis. Indeed,potentially improved strategies directed at inhibitingTF have been developed and are being evaluated inclinical trials.INFLAMMATORY BOWEL DISEASE: ROLE OF ENDOTHELIALPROTEIN C RECEPTORFaioni EM, Ferrero S, Cattaneo MHematology and Thrombosis Unit and Pathology Division,DMCO University of Milano and Ospedale SanPaolo, Milano, Italy.Inflammation and coagulation are linked processes.Inflammatory mediators regulate the phenotypeof the endothelium, shifting the balance towardscoagulation, while procoagulant enzymes have proinflammatoryeffects. Conversely, inhibitors of coagulationbelonging to the protein C pathway haveanti-inflammatory and anti-apoptotic activity. EPCR,for example, was shown to be internalized in theendothelial cell and to modulate gene expression.Soluble EPCR binds to polymorphonuclear cells, preventingadhesion. Anti-apoptotic effect of activatedprotein C on the endothelium requires EPCR. Interestingly,EPCR is expressed also by intestinal dendriticcells (DCs). DCs are ubiquitous cells that linkinnate and acquired immunity, and are possiblyresponsible for self-tolerance. In the gut, pathogensand resident flora come in contact with the epithelialcells and with DCs, which in turn present antigento Tcells and elicit T cell responses. In vitro, EPCRis down-regulated on DCs upon contact with bacteria.In IBD (Crohn's disease and ulcerative colitis), anatural model of chronic inflammation, EPCR+ DCsare less abundant than in controls. The decrease inEPCR+ DC number seems correlated with the degreeof gut involvement. Structural homology of EPCRwith MHC class I proteins suggests EPCR may beinvolved in antigen presentation. Alternatively, sinceDCs have close contact with vessels of the mucosaand the intestinal lumen, one or two way EPCRmediatedsignaling between the intestinal lumen andblood components could occur. The unique EPCRpositivity of intestinal DCs, moreover, suggests a rolefor EPCR in the development of self-tolerance and inpreventing immune derangement. Since EPCR hasalso an important anti-coagulant role, it is not surprisingthat in diseases such as IBD local and systemicthrombotic events are a common feature sinceEPCR expression is extensively downregulated also inthe endothelium by chronic and acute inflammatorymediator production.haematologica vol. 89(suppl. n. 8):september 2004

6Educational SessionsTREATMENT OF SEVERE SEPSIS WITH NATURALANTICOAGULANTS: LESSONS FROM HUMAN STUDIESD'Angelo A, Della Valle P, Viganò D'Angelo SServizio di Coagulazione ed Unita' Ricerca Trombosi,IRCCS H S.Raffaele, Milan, ItalyCurrent strategies in the treatment of severe sepsisare aimed at the control of coagulation andinflammation. Based on animal models, modulationof major natural anticoagulant systems appears criticalfor survival. Three large double-blind studieshave investigated the potential benefit of a 96 hourinfusion of antithrombin, activated protein C andTFPI in patients with severe sepsis. The Kybersept trialenrolled 2314 patients, randomized to high-doseantithrombin concentrate (30,000 IU) or placebo. 28-day mortality was 38.7% in the placebo group and38.9% in the antithrombin group (ns). The Optimisttrial enrolled 1754 patients randomized to Tifacogin(recombinant human TFPI, 0.025 mg/Kg/h) or placebo.28-day mortality was 33.9% in the placebo groupand 34.2% in the TFPI group (ns). The Prowess trialenrolled 1690 patients randomized to Drotrecogin(human recombinant protein C, activated, 0.024mg/Kg/h) or placebo. 28-day mortality was 30.8% inthe placebo group and 24.7% in the APC group (p =0.005), with a relative risk reduction of 19.4%. Thebenefit of APC supplementation was evident inpatients older than 50 years, with APACHE II score>25, with 2 or more organ dysfunctions and withDIC. There were differences in baseline characteristicsof patients enrolled in the three studies, potentiallyaffecting the different overall mortality. In disagreementwith the final results, interim analyses ofthe Kybersept and Optimist trials were highlypromising, while APC showed a greater benefit inthe second half of the Prowess trial. In all studies,baseline presence of shock and, even more important,of major coagulopathy had a large influenceon 28-day mortality. About 2/3 of the patients ineach trial received concomitant heparin at prophylacticdoses. The mortality rate of these patients wasconsistently lower than that of patients not receivingheparin prophylaxis. Among the latter patients,there was a trend for a higher mortality in the TFPIand antithrombin groups than in the respectiveplacebo groups (OR = 1.21 and 1.13), while the benefitof APC treatment (OR = 0.85) did not reach statisticalsignificance. In contrast, among patients notreceiving heparin prophylaxis, the survival advantageof patients in the TFPI and antithrombin groupswas very close to statistical significance (OR = 0.71and 0.79), and the benefit of APC treatmentincreased dramatically (OR = 0.50). Irrespective ofconcomitant heparin prophylaxis, the incidence ofbleeding complications was higher in all studies withthe active treatment, in spite of the obvious exclusionof patients at high risk for bleeding.ORAL ANTICOAGULANT THERAPYNEW TRENDS IN STROKE PREVENTION IN PATIENTS WITHATRIAL FIBRILLATIONPengo VClinical Cardiology, University of Padova, ItalyAtrial Fibrillation (AF) is the most frequent sustainedarrhythmia and its rate increases with age.Patients with this rhythm disturbance may have mildsymptoms as thachycardia or fatigue but some timethey suffer from haemodynamic impairment leadingto dyspnoea and hypotension. In some case, patientswith AF are completely asymptomatic but anywayexposed to the risk of stroke. This is related to thefact that, in the absence of atrial contraction, fibrinrichthrombi could be formed in the left atrialappendage and then migrate to the cerebral circulation.AF, in fact, is the main cause of cardioembolicstroke. In the last 15 years, the occurrence ofstroke in patient with AF has been tremendouslyreduced with the use of oral anticoagulant drugs.More recently, new anticoagulant drugs (i.e. ximelagatran,a direct antithrombin drug not necessitatinglab control) have been shown to be as efficient as'old' anticoagulants in stroke prevention. Other drugswith different anticoagulant and/or antiplateletmechanism of action are currently being studied incomparison with old oral anticoagulants. Moreover,some nonpharmacological approach has being developedfor high risk patients. Among these, the use ofa device to close left atrial appendage or thetransthoracic closure of left appendage have beenused in our institution. Finally, a device shiftingembolic material from internal to external carotidartery is currently studied in experimental animals.PROPHYLAXIS AND TREATMENT OF THROMBOSIS IN PATIENTSWITH THE ANTIPHOSPHOLIPID SYNDROMEFinazzi GU.S. Emostasi e Trombosi, Div. di Ematologia OspedaliRiuniti, Bergamo, ItalyAntiphospholipid antibodies (aPL) are a heterogeneousgroup of immunoglobulins directed againstnegatively charged phospholipids, protein-phospho-haematologica vol. 89(suppl. n. 8):september 2004

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 20047lipid complexes or plasma proteins. The most clinicallyrelevant aPL are lupus anticoagulant (LA) andanticardiolipin antibodies (ACA), which are associatedwith a syndrome of arterial and venous thrombosisand recurrent fetal loss, named “antiphospholipidsyndrome”. Persistence of aPL, elevated levels of ACAand the presence of LA appear to increase the risk ofthrombosis. Another major predictor of vascular complicationsis a history of thrombosis: in a prospectivestudy, the annual incidence of both venous and arterialthrombosis was 0.9% per patient-year in asymptomaticindividuals and 5.4% in those who had a previousthrombotic event. A critical issue, therefore, isthe prevention of recurrent thrombosis in high-riskpatients. A beneficial role for high-intensity oral anticoagulationin achieving this goal has been shown inretrospective studies. Warfarin treatment of highintensity (to achieve an international normalized ratio[INR] of 3.0 or more) conferred better antithromboticprotection than standard intensity treatment (INR2.0 to 3.0) or aspirin. However, there is concern aboutthe implications of recommending such intensiveanticoagulation on the basis of retrospective andnon-randomised data. Fatal, cerebral and uncontrollablehemorrhaghes may occur and the cumulativerisk of major bleeding is expected to increase withduration and intensity of anticoagulant treatment.Recently Crowther et al. reported 114 patients withaPL and thrombosis treated with standard intensity(INR 2.0-3.0) or high intensity (INR 3.1-4.0) warfarintherapy and followed for an average of 2.7 years. Sixof 56 (10.7%) patients allocated to high intensitywarfarin and 2 of 58 (3.4%) allocated to standardintensity warfarin suffered an objectively confirmedrecurrent thromboembolism. Major bleeding wassimilar between the two groups. The Authors concludedthat standard intensity warfarin is appropriatefor patients with the antiphospholipid syndrome.In a similar way, we conducted a randomized trial inwhich patients with persistent LA and/or moderate tohigh ACA levels and a history of thrombosis were giveneither high intensity warfarin therapy (INR 3.0-4.0) or standard antithrombotic prophylaxis (warfarinPT INT 2.0-3.0 for venous and aspirin alone for arterialthrombosis). We sought to determine whetherintensive anticoagulation was superior to standardtreatment with regard to the combined outcome ofsymptomatic recurrent thromboembolism, majorbleeding and death. 109 patients (M/F 40/69; medianage 41 years) were eligible for randomization andmedian follow-up was 36 months (range 0-63). ActualPT INR of the 109 randomized patients wasassessed at 3, 6, 12, 24 and 36 months. Median PTINR values of patients randomized in the high-dosegroup (n=54) was 3.1, 3.2, 3.1, 3.3 and 3.3, whereasin the conventional group (n=55) was 2.3, 2.5, 2.6, 2.5and 2.5, respectively. All cause mortality (high-dose5.3% vs. conv. 3.6%) and thrombotic events (highdose8.9% vs. conv. 5.3%) were similar between thetwo groups, whereas bleeding complications weresignificantly more frequent in the patients assignedto “high-dose warfarin” (26.8% vs. 12.5%, p 4.5) were more frequent inunstable subjects than in controls (12.3% vs 0.4%;p

8Educational Sessionssioners, acenocoumarol vs warfarin, insufficientscore at AMT, unawareness of the reason for OAT,insufficient score at the questionnaire on comprehensionof OAT mechanisms. The genetic variants1/3 or 2/3 or 3/3 of cytochrome P450 CYP2C9 weresignificantly more frequent among cases than controls(29.9% and 15.0%, respectively; p = 0.042). Nodifferences were observed as regards schooling, familycomposition, diet and life habits, alcohol consumption,liver and renal function, other blood tests,mean daily dose of warfarin or acenocoumarol. Thequality of anticoagulation control significantlyimproved in unstable patients during the 4 monthsafter inclusion in the study. Conclusions. The reasonfor high intra-individual variability in OAT control ismultifactorial. Among these factors, a poor information/educationof patients on OAT appears to be ofprevalent importance since additional educationalcourses are effective in improving the quality of anticoagulationcontrol in highly unstable patients.ATHEROTHROMBOSISINFLAMMATION AND ATHEROTHROMBOSIS:PATHOLOGICAL PATTERNSpagnoli LG, Mauriello A, Palmieri G, Bonanno E,Fratoni S, Sangiorgi GCattedra Anatomia Patologica, Università di RomaTor Vergata, ItalyRecent research has furnished new insight into themolecular mechanisms that cause transition fromchronic to the acute phase of atherosclerosis andpoints to the inflammation as the playmaker in theevents leading to plaque destabilisation. Unlike thestable plaque that shows a chronic inflammatoryinfiltrate, the unstable plaque is characterised by achronic "active" inflammation. This "active" inflammationmainly involves T-lymphocytes and macrophageswhich are activated towards a pathway ofinflammatory response and secrete cytokines andlytic enzymes involved in the thinning of fibrous cap,predisposing the plaque to rupture and thrombosis.Despite the predominant hypothesis focusing on theresponsibility of a specific vulnerable atheroscleroticplaque rupture and thrombosis for acute coronarysyndromes, some recent studies performed in ourinstitute seem to suggest the possibility that theprincipal cause of coronary instability is not to befound in the vulnerability of a single atheroscleroticplaque, but in the presence of multiple vulnerableplaques in correlated with the presence of a diffuseinflammatory process involving the entire coronarytree. This new pathophysiological setting resultedfrom a systematic flow cytometric study in threegroups of autopsied patients: Acute MI, Old MI, Noischemic heart disease (controls). The coronaryplaques showed: A) higher proportion of inflammatorycells in Acute MI and Old MI than in Controls;B) higher percentage of T lymphocytes in Acute MIthan in Old MI and controls; C) diffuse cell activationin the whole coronary tree of Acute MI, but notof old Mi and controls. Moreover, these results havebeen confirmed by a morphological study provingthe presence of a high inflammatory infiltrate constitutedof macrophagic cells and T lymphocytesactivated in the whole coronary tree, also present inthe stable plaques of individuals died of acutemyocardial infarction. We conclude that T lymphocytesmay play a key role in coronary instability bydetermining activation of various cellular typesthroughout the coronary circulation. Activated T-lymphocytes and their products may well representa new target in both treatment and prevention ofacute coronary syndromes.CLINICAL RISK FACTORS OF ATHEROTHROMBOSISPalmieri V,* Tufano A,* Coppola A,* Celentano A,*°Di Minno G* #*Dipartimento di Medicina Clinica e Sperimentale,Università Federico II and °Centro TraumatologicoOspedaliero, ASL-Napoli 1, Napoli; # IRCCS Casa Sollievodella Sofferenza, S. Giovanni Rotondo (FG), ItalyAtherothrombosis is a progressive pathologicprocess of the vascular beds. The historical pathogenetichypothesis of the “lipid accumulation” hasevolved and new factors involved in the early phaseof the atherosclerosis and in its thrombotic complication,i.e. the atherothrombosis, have been studied.Among these, endothelial dysfunction and inflammationhave shown increasing scientific and clinicalinterest. The clinical consequences of atherothrombosisinclude acute coronary syndromes (unstableangina, acute myocardial infarction, and sudden cardiacdeath), ischemic stroke, and peripheral arterialdisease, the occurrence of which is unpredictableand potentially life-threatening. Atherothrombosisrather than arterial stenosis appears to account formost of the acute ischemic manifestations of theatherosclerotic process. However, fighting risk factorsof atherosclerosis, by dietary control, exercise, andsmoking cessation, body size, blood pressure andtotal cholesterol lowering and increase in HDL cholesterol,lowering homocysteine and blood glucose,and treating high risk patients with antithrombotichaematologica vol. 89(suppl. n. 8):september 2004

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 20049drugs, may help to prevent atherothrombosis. Therole of inflammation (C reactive protein, fibrinogenand tissue factor are new postulated markers of risk),and of platelet aggregation in the plaque instabilityand thrombus formation, address the search for newpotentially successful therapeutical strategies.where the contribution of genetic factors might beexpected to be more and that of environmental factorsless important than in older populations. Oneshould also consider the possibility that studies ofclusters of polymorphisms (haplotypes) or gene-environmentinteractions might appear more meaningfulthan those on single candidate genes.BLOOD PLATELETSPLATELET PHARMACOGENETICSde Gaetano G, Cerletti C, Iacoviello L, Donati MBLaboratori di Ricerca, Centro di Ricerca e Formazionead Alta Tecnologia nelle Scienze Biomediche, UniversitàCattolica, Campobasso, ItalyThe rapid development of techniques for geneticpolymorphisms has allowed, in the past decade, toinvestigate the possible relations between plateletgenotypes and phenotypes. In particular, candidategenes were identified and tested not only for theirprotective or deleterious influence on ischaemic cardiovasculardisease, but also as a way to identifyindividuals with different sensitivity to drugs. Thisapproach, referred to as “pharmacogenetics”, mightallow, e.g., to unreveal possible genetic regulationsof the so-called “aspirin resistance” as a functionalor clinical phenotype. A model example is that ofplatelet glycoprotein IIb/IIIa complex. PolymorphismP1A2 is a variant of the gene encoding for glycoproteinIIIa and is present in about 25% of the whitepopulation. This polymorphism has been associatedwith an increased risk of coronary heart disease,especially at young age and in patients undergoingcoronary bypass. The P1A2 allele has also been associatedwith an increased reactivity of platelets toaggregating stimuli and an altered integrin-mediatedplatelet adhesion and spreading. Moreover, thepresence of the P1A2 polymorphism influences theeffect of aspirin on plateletet aggregation and bleedingtime and may affect both safety and efficacy ofanti IIb/IIIa antagonists. Thus, introduction in clinicaltrials of genetic categories related to plateletfunction could provide a novel tool to predict drugefficacy and/or risk of bleeding and ultimately a betterchoice of the most appropriate anti-platelet drugfor subgroups of patients or even for each individual.Genetic regulation in multi-factorial diseases, suchas myocardial infarction, would affect a relativelysmall portion of the population; as a consequence,one should preferably study patients or families withfamilial history of the disease or younger subjects,PLATELETS BIOCHEMISTRY: NEW PERSPECTIVESDe Marco LCentro di Riferimento Oncologico, Aviano, (PN), ItalyCa ++ is one of the most versatile and importantintracellular messengers, as it is involved in the controlof many different cellular functions. Specificagonists can generate Ca ++ signals such as local andglobal cytosolic Ca ++ elevations which can be transient(spiking) or sustained. We and others haveinvestigated the mechanisms underlying the generationof local Ca ++ spikes and global Ca ++ transientsin platelets interacting with different substrates. Weperformed experiments by using FLUO3-AM loadedplatelets perfused onto a surface of either VWF oracid-soluble collagen type I. We analysed concurrentlythe instantaneous velocity and Ca ++ transientsin single platelet interacting with different substratesusing a real time video-imaging method characterizedby high speed image acquisition and a high performance,custom made, software. We describe thesequential cytoplasmic Ca ++ signals in a 2-stageplatelet activation process induced by the glycoprotein(GP)Ib alfa mechanoreceptor and focus on themechanisms that regulate signalling between GPIbalfa and alfa IIb-beta3 particularly on the role of thetwo ADP receptors: P2Y1 and P2Y12. We demonstratethat ADP may contribute to the initial stagesof platelet adhesion and activation mediated byimmobilized VWF through P2Y1, and to sustainedthrombus formation through P2Y12. In addition, therole of alfa2-beta1 and GPVI in generating calciumoscillations in platelets adhering to a collagen substrateunder flow and the signalling pathwaysinvolved will be discussed ad compared to the dataof the literature. We demonstrate that alfa2-beta1participates in the initial stages of platelet adhesionand activation which is subsequently reinforced byactivation though GPVI. This information may help inthe selection and evaluation of potential targets forpharmacological intervention aimed at preventingarterial thrombosis.haematologica vol. 89(suppl. n. 8):september 2004

10Educational Sessionshaematologica vol. 89(suppl. n. 8):september 2004

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 200411XVIII Congress of the Italian Society for Hemostasis and Thrombosis ResearchRome, September 30 - October 3, 2004SELECTED ORAL COMMUNICATIONSINFLAMMATION AND VASCULAR RISKCOS-01A POLYMORPHISM IN THE CYCLOOXYGENASE-2 GENE AS ANINHERITED PROTECTIVE FACTOR AGAINST MYOCARDIALINFARCTION AND STROKECipollone F,* Toniato E,* Martinotti S,* Fazia M,*Iezzi A,* Cuccurullo C,* Pini B,* Ursi S,* Vitullo G,*Averna M, # Arca M, § Montali A, § Campagna F, §Ucchino S,* Spigonardo F,* Taddei S,º Virdis A,ºCiabattoni G,* Notarbartolo A, # Cuccurullo F,*Mezzetti A**G. d’Annunzio University of Chieti; # University ofPalermo; § University of Rome "La Sapienza";ºUniversity of Pisa; ItalyMyocardial infarction (MI) and stroke are thoughtto be caused by matrix digestion by metalloproteinases(MMPs) leading to rupture of atheroscleroticplaques. Production of macrophage MMP-2 andMMP-9 is induced by cyclooxygenase 2 (COX-2) andprostaglandin (PG)E2 synthesis. Although COX-2expression may be genetically determined, the relationbetween COX-2 polymorphisms and the risk ofMI and stroke is unclear. To investigate the relationshipbetween the -765G→C polymorphism ofthe COX-2 gene and plaque rupture, we conducteda case-control study among 864 patients with firstMI or atherothrombotic ischemic stroke and 864hospitalized controls. The groups were matched forage, sex, BMI, smoking, hypertension, hypercholesterolemia,and diabetes. The G>C variant of the COX-2 gene was genotyped by restriction endonucleasedigestion of polymerase chain reaction products. Theprevalence of -765GC was 2.41 times higher amongcontrols than among cases (43,3% vs 17.9%,p

12Selected Oral Communicationslesterol and triglycerides adjusted vitamin E plasmalevels for people with 0, 1, 2, 3, 4, or 5 characteristicsof the metabolic syndrome were 4.26, 4.00, 3.96,3.44, 3.47 respectively [p (trend)

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 200413peripheral blood leukocyte number, and significantlyinhibited eosinophil recruitment (0.5±0.2 p

14Selected Oral Communications11-84); the median time between the first VTE andrecurrence or referral to our center was 3 years (A,range 1-21), 4 (B, range 1-40), and 5 (C, range 1-41).Patients with multiple defects had an increased riskfor recurrent VTE in comparison with patients withnormal genotype (hazard ratio 1.9, 95% CI 1.1-3.5);the risk for spontaneous recurrence after a first spontaneousVTE was 2.9-fold increased (95% CI 1.1-7.5).On the opposite, the risk for recurrence was notincreased among patients with deficiency of AT, PC,or PS in comparison with the reference group (hazardratio 1.2, 95% CI 0.7-2.2); after stratificationaccording to the circumstances of the first or therecurrent event the risk for spontaneous recurrenceafter a first spontaneous VTE was increased withoutreaching statistical significance (hazard ratio 1.8,95% CI 0.8-4.3). Therefore patients with PC or PSdeficiency do not seem firm candidates to long-termanticoagulation after a first VTE. The number ofpatients with AT deficiency is too low to draw anyconclusion: however the rate of spontaneous recurrenceafter a first spontaneous VTE was 67% (2 of 3)in them and 36% (4 of 11) in patients with PC or PSdeficiency. Further multicenter investigations arewarranted.COS-07HIGH RISK OF VENOUS THROMBOEMBOLIC RECURRENCE INRENAL TRANSPLANT RECIPIENTS: ROLE OFHYPERHOMOCYSTEINEMIA AND CLOTTING ACTIVATIONMARKERSPoli D, Antonucci E, Cecchi E, Marcucci R, Lapini I,Lari B, Lenti M, Zanazzi M,* Salvadori M,* Abbate R,Gensini GF, Prisco DCentro di Riferimento Regionale per la Trombosi,Dipartimento dell’ Area Critica Medico Chirurgica,*UO Nefrologia e Trapianti Azienda Ospedaliero-Universitaria Careggi, Florence, ItalyRenal transplantation is associated with anincreased risk of venous thromboembolism (VTE). Nodata are available about the optimal duration of oralanticoagulant therapy (OAT) in renal transplant (RT)recipients. Our study was performed to evaluate therisk of VTE recurrence in patients developing a firstepisode of VTE after RT. We prospectively studied 28RT recipients who had suffered from a first episodeof VTE after stopping OAT (Group 1). Group 1 wascompared with a group of 84 patients without historyof renal disease who had suffered from a firstepisode VTE matched for age, sex and type of thromboticevent (Group 2) and with a matched group of28 RT recipients without history of VTE (Group 3).After OAT withdrawal, thrombophilia (AT, protein C,protein S, prothrombin gene variant, factor V Leiden,homocysteinemia) and clotting activation markers(prothrombin fragment 1+2 (F1+2) and D-dimerplasma levels) were evaluated. During follow-up,14/28 patients of Group 1 and 8/84 patients ofGroup 2 experienced VTE recurrence (p=0.0001). Asthrombophilia is concerned, no difference was foundamong the 3 groups, except for homocysteine.Hyperhomocysteinemia was found in 24/28 Group 1patients, in 23/84 Group 2 patients (p

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 200415ia (239/475) (p=0.06). The hazard ratio for recurrencewas 1.48 (95% confidence intervals: 0.9-2.45)for patients with thrombophilia when compared withpatients without thrombophilia, while it was 1.76(95% CI: 1.05-2.75) among patients with RVT whencompared with patients without RVT, after adjustmentfor age, sex, duration of OAT, thrombophilia,and type of index event. When compared withpatients without thrombophilia and absent RVT, subjectswith thrombophilia and RVT had a hazard ratiofor recurrence of 2.1 (95% CI: 1.1 –5.7), after adjustmentfor sex, age, duration of OAT, D-dimer, type ofindex event and cancer. Conclusions: VTR is independentfrom thrombophilia as a risk factor forrecurrent VTE after OAT withdrawal.BLOOD PLATELETS AND VON WILLEBRAND FACTORCOS-09DIFFERENTIAL ROLE OF P2Y RECEPTORS IN AGONIST-INDUCEDTISSUE FACTOR EXPRESSION BY PLATELETSCamera M,^* Frigerio M,* Cattaneo M, #Abbracchio MP,^ Jacobson KA,° Biglioli P,*Tremoli E^*^Dept. Pharmacological Sciences, University ofMilan, *Centro Cardiologico Monzino IRCCS, Milan,#San Paolo Hospital, DMCO, University of Milan,°NIH, USARecent data indicate that human platelets containTissue Factor (TF),which can be exposed on the membraneby platelet agonists such as ADP, TRAP andtromboxane A2 mimetic U46619. ADP-inducedplatelet activation is initiated by the platelet P2Y1receptor and amplified in a synergistic manner bythe platelet P2Y12. These two receptors play animportant role also in platelet activation induced byagonists other than ADP. In the present study weinvestigated whether agonist-induced TF expressionon platelet surface is mediated by concomitant signalingthrough co-activation of both receptor subtypes.Platelet-associated TF was measured by flowcytometry using a specific monoclonal anti human TFantibody in whole blood (WB) and in platelet richplasma (PRP) obtained from healthy subjects. ADP(10 µmol/L) incubated for 15 minutes in WB or PRPinduced a consistent expression of TF (+170±40%versus unstimulated platelets, p

16Selected Oral Communicationssis, the others had normal platelet counts. Estimationof α-granule proteins was carried out by immunostainingfor TSP-1, PF4 and P-selectin on plateletsfrom seven subjects. Immunofluorescence showed amarked reduction of reactivity for TSP-1, PF-4 and P-selectin in the propositus, and, to a lesser degree, inhis father and three relatives of him (I-2, I-5, II-3),with respect to controls (Table).% plt with normal III-2 II-2 II-1 III-1 I-2 I-5 II-3immunofluorescence propositus father mother brotherreactivityTSP-1 6.8 63.2 96.2 93.8 68.9 71.5 76.8PF4 4.2 69.5 97.1 93.2 71.5 67.9 72.8P-selectin 36.5 71.7 101 90 82 79.2 76In the propositus a specific marked reduction ofplatelet aggregation and calcium mobilization bythrombin, PAR-1-AP and PAR-4-AP could be detected,whereas aggregation and calcium mobilization byADP, collagen, ristocetin, U46619, epinephrine andconvulxin were normal. His father had a less evidentreduction in platelet aggregation by thrombin, PAR1-AP and PAR4-AP. The three relatives showed only amarked reduction of platelet aggregation by PAR-4-AP (Figure). This novel family with GPS demonstratedthat: 1) GPS platelets are characterized by a specificreduced response to thrombin, PAR1-AP andPAR4-AP: 2) the reduced aggregation by PAR4-APcharacterizes the platelet defect in the relatives withmild phenotype and healthy carriers: 3) immunofluorescenceis a specific and sensitive method to estimatethe content of platelets α-granules; 4) there isa correlation between α-granules protein contentand defective response to PAR4-AP, useful to identifyindividual with mild phenotype 5) although thepenetrance may be variable, the inheritence of thisdefect seems to be autosomal dominant.COS-11REDUCED BINDING OF PURIFIED TYPE 2M VICENZAVON WILLEBRAND FACTOR TO PLATELET α2Bβ3Cozzi MR, Castaman G,* Mazzucato M, Steffan A,Rodeghiero F,* De Marco LCentro di Riferimento Oncologico, IRCCS, AvianoItaly; *Dipartimento di Ematologia, Ospedale SanBortolo, Vicenza, ItalyWe have studied the interaction of the congenitallyabnormal type 2M Vicenza von Willebrand Factor(VWF 2M V) molecule, characterized by the presenceof supranormal VWF multimers with the two bindingsites on platelets, the glycoprotein (GP)Ib-IX complexand the α2bβ3. Variant as well as normal (N)VWFwere purified from plasma. Experiments based onmeasuring the ability of the unlabeled VWF 2M V andNVWF species to inhibit the binding of I125-labeled-NVWF to platelets resulted in estimates for binding ofsubunit molecules per platelet at saturation (Bmax)and inhibition costant in moles\liter (Ki) and gave thefollowing results. In the presence of ristocetin NVWF:(Bmax) 6,023 and (Ki) 0.2×10 -8 ; VWF 2M V 6,143 and0.3×10 -8 respectively. In the presence of botrocetinNVWF 21,382 and 2.5×10 -8 ; VWF 2M V 23,670 and4.9×10 -8 . After thrombin stimulation, NVWF 16,593and 1.2×10 -8 ; VWF 2M V 14,756 and 7.0×10 -8 . Afterstimulation with ADP + epinephrine, NVWF 6,928 and1.1×10 -8 ; VWF 2M V 4,818 and 8.9×10 -8 . Our studiesshowed that VWF 2M V bound normally to the GP Ib-IX complex in the presence of ristocetin and with aslight decreased affinity in the presence of botrocetin.More remarkably VWF 2M V bound to plateletα2bβ3with a greater and significantly decreasedaffinity,in spite of the presence of supranormal multimers.Our results suggest the occurrence of anabnormality in VWF 2M V leading to a decreasedaffinity for platelet α2bβ3, which may in part impairthrombus formation and confirm the inclusion of thisVWD variant within type 2.COS-12IN VITRO EXPRESSION STUDIES OF TWO NATURAL OCCURRINGMUTATIONS ON ADAMTS13 GENE IN AN ITALIAN TTP PATIENTPeyvandi F,* Lavoretano S,* Canciani MT,*De Cristofaro R,ˆ Rossi E,° Dalla Giovanna S,*Mannucci PM**A. Bianchi Bonomi Hemophilia and ThrombosisCenter and Fondazione Luigi Villa, IRCCS MaggioreHospital and University of Milan; ^Internal Medicine,Catholic University, Rome, Italy: °Immunohematologyand Blood Transfusion Service, L. SaccoHospital, Milan, ItalyADAMTS-13, the specific von Willebrand factor(VWF)-cleaving protease, is a plasma metalloproteasewhich prevents the spontaneous formation ofplatelet thrombi in the microcirculation by cleavageof the adhesive ultralarge VWF multimers into smallerforms. Congenital thrombotic thrombocytopenicpurpura (TTP), a rare and severe disease, is associatedwith low ADAMTS-13 activity caused by mutationson the gene. Of the 49 reported mutations onlyeight have been expressed. We investigated the molecularbasis of ADAMTS-13 deficiency in an Italianwoman with a history of recurrent TTP. ADAMTS-13activity, measured by collagen binding assay, wasless than 6% without detectable levels of ADAMTS-13 inhibitors. Mutational analysis by direct sequenc-haematologica vol. 89(suppl. n. 8):september 2004

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 200417ing showed 2 missense mutations, one previouslyreported in the metalloprotease domain (Val88Met)and one is novel, located in the first CUB domain ofthe ADAMTS-13 protein (Gly1239Val), both in theheterozygous state. To determine the effects of theidentified mutations, wild type (WT) and mutantforms of recombinant ADAMTS-13 (rADAMTS13)were transiently transfected in HEK 293 cells, usingeach expression vectors (rADAMTS13-WT,rADAMTS13-Val88Met, rADAMTS13-Gly1239Val). Tomimic the effect of compound heterozygous stateof the patient both mutant forms of rADAMTS13were transiently expressed together. The enzymaticactivity of ADAMTS-13 in the medium of untransfectedcells showed lack of activity. However, themedium of cells transfected by rADAMTS13-Val88Met and rADAMTS13-Gly1239Val separatelyand together showed 10%, 7% and 10% respectively.These results are slightly higher than those ofpatient plasma and further analysis by more sensitiveassays is required for in vitro expression studies.Previous studies on mutations located on metalloproteaseand CUB domains suggest that the missensemutation Val88Met could cause structuraldefect in one allele, whereas Gly1239Val mutationon the second allele probably causes an alteration inthe secretion pathway. Both mutations togetherleads to a severe ADAMTS-13 deficiency (

18Selected Oral CommunicationsSISET PrizeCOS-14THE G20210A PROTHROMBIN GENE MUTATION: IS THEREROOM FOR SCREENING FAMILIES?Tognin G, Tormene D, Perlati M, Zerbinati P,Fadin M, Gavasso S, Rossetto V, Pagnan A,Prandoni P, Simioni PDept. of Medical and Surgical Sciences, 2 nd Chair ofInternal Medicine, University of Padua MedicalSchool, Padua, ItalyBackground. The G20210A prothrombin variant is acommon polymorphism associated with an increasedrisk of venous thromboembolism (VTE). A clear definitionof the thrombotic risk in heterozygous carriers ofthis defect is necessary to establish prophylacticguidelines. Methods. First-degree relatives of consecutivepatients referred to our Center with a history ofthrombotic disease and identified as heterozygouscarriers of prothrombin G20210A were enrolled in thestudy, provided that they were aged above 15 years.Before DNA testing, a medical history was taken withspecific attention to previous VTE (objectively documented)and concomitant risk factors. Results. A totalnumber of 294 individuals belonging to 88 familieswere studied. One-hundred and fifty-two were carriersand 142 had no mutation. A total of 4338patients–years were obtained in the carriers group ascompared to 4309 in the non-carriers. Out of the 152affected family members, 5 experienced objectivelydocumented VTE (one spontaneous). Three thromboticevents occurred in non-carriers (two spontaneous).The overall annual incidence of VTE was 0.11% in thecarriers of the prothrombin mutation, and 0.07% innon-carriers (RR=1.7; 95% CI, 0.4 to 7). The incidenceof risk period-related thrombotic events was 1.0% inthe carriers group and 0.3% in non-carriers (RR=3.6;95% CI, 0.4 to 32). When we considered the thromboticrisk before and after 45 years of age the relativerisk was 2.0 (95% CI 0.2 to 21.6) and 1.5 (95% CI0.3 to 9.3) respectively. Conclusions. The results of ourretrospective family-study suggest that screening relativesof symptomatic carriers of single G20210A prothrombinmutation has the potential to identifyasymptomatic carriers in whom a mild increased riskfor thrombosis possibly exist. Whether this informationmight offer a real advantage in the prevention ofVTE remains to be proven.SISET PrizeCOS-15MELAGATRAN IS A BETTER PROFIBRINOLYTIC AGENTTHAN HEPARINPiro D, Rossiello MR, Rotunno C, Semeraro N,Colucci MDipartimento di Scienze Biomediche, Sezione diPatologia Generale, Università di Bari, ItalyThrombin generation inhibits fibrinolysis throughdifferent mechanisms, including clot stabilization,alteration of mass-to-length ratio of fibrin fibers, andremoval of plasminogen binding sites from partiallydegraded fibrin (via TAFI). We compared the fibrinolyticactivity of melagatran (active form of the oral,direct thrombin inhibitor, ximelagatran, AstraZeneca)with that of unfractionated heparin in platelet-poor(PPP) and platelet-rich (PRP) plasma clot models. Clotswere formed in the presence of t-PA, tissue factor,phospholipids, Ca ++ and different concentrations ofthe anticoagulants, and fibrinolysis was monitored bythe reduction in turbidity. Fibrinolytic activity of drugswas expressed as clot lysis ratio (lysis time in theabsence of the drug divided by lysis time in its presence).Drug potency, assessed by aPTT prolongation,was expressed as aPTT ratio. In PPP clot model, bothheparin and melagatran caused a concentrationdependentshortening of lysis time. However, whenfibrinolytic activity was expressed in function of aPTTratio, heparin resulted markedly less efficient thanmelagatran. At concentrations giving an aPTT ratio of2, the clot lysis ratios for heparin (0.3 U/mL) and melagatran(0.37 uM) were 1.29±0.13 and 1.75±0.19,respectively (p=0.01). In PRP clot model, heparin wasvirtually ineffective even at concentration as high as0.6 U/mL (aPTT ratio: 3.1±0.43; clot lysis ratio:1.15±0.21), whereas melagatran displayed a fibrinolyticactivity comparable to that observed in PPPclots at all tested concentrations (0.05-0.75 uM). Theeffect of heparin was also abrogated when PPP wassupplemented with platelet membranes but not withplatelet cytosolic fraction or the releasate of activatedplatelets, thus ruling out the involvement ofplatelet factor 4. We conclude that melagatran issuperior to heparin in promoting fibrinolysis, particularlyin platelet-rich clots in which clotting factorsbound to platelet membranes are protected from inhibitionby antithrombin-dependent anticoagulants.haematologica vol. 89(suppl. n. 8):september 2004

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 200419COS-16LOW-MOLECULAR WEIGHT HEPARINS INHIBIT BREASTCANCER AND LEUKEMIA CELL-INDUCED ENDOTHELIALANGIOGENESISMarchetti M, Vignoli A, Pagnoncelli M, Falanga AHematology Dept, Ospedali Riuniti di Bergamo, ItalyClinical studies of thrombosis in cancer patientsshow that, besides their role as antithrombotics,heparins may have beneficial effects on survival ofcancer patients, with a major role by LMWHs comparedto UFH. In this study we investigated theeffects of two LMWHs, i.e. enoxaparin (ENX) and dalteparin(DLT), compared to UFH, on tumor cellinducedendothelial angiogenesis. Angiogenesis wasevaluated by the in vitro capillary-like tube formationassay in Matrigel. Human microvascular endothelialcells (HMEC-1) were incubated in 96-well plates precoatedwith Matrigel with: 1. tumor cell conditionedmedium (CM) from three human breast cancer celllines, i.e. MCF-7, ZR 75.1 and MDA.MB.231, and onehuman leukemia cell line, i.e. NB4; or 2. purifiedproangiogenic factors (VEGF, bFGF and TNFα). After24h incubation, tube formation was examined underphase-contrast microscopy and tube length determinedby image analysis software. Control CMinduced a modest tube formation (total tube length= 223 mm/cm 2 ), which was 33% decreased by ENX(p

20Oral CommunicationsCO-002LYSOSOMAL β-N-ACETYLHEXOSAMINIDASE IS A PRIMEROF PLATELET ACTIVATIONRossi R, 1 Giannini S, 1 Mencarelli S, 2 Falcinelli E, 1Orlacchio A, 2 Emiliani C, 2 Gresele P 11Department of Internal Medicine, Section ofInternal and Cardiovascular Medicine; 2 Departmentof Biochemical Sciences and MolecularBiotechnology, University of Perugia, Italy.Platelet contain, besides α- and δ-granules, lysosomeswhich store mainly acid hydrolases. Humanplatelets release their lysosomal content both in vitro,after stimulation with strong agonists, and in vivoat a localised site of vessel wall damage. β-N-acetylhexosaminidase(Hex) is the main lysosomal glycohydrolasereleased by human platelets. Hex canhydrolyse the glucidic moiety of glycoproteins, glycolipidsand glycosaminoglycans, but a role for extracellularHex has not been established yet. We studiedthe effect of β-Hex on platelet activation andthe platelet signal trasduction pathways. Hex fromhuman placenta was fractionated in its two isoformsby DEAE-cellulose, and further purified; in someselected experiments, Hex was purified from humanplatelets. Gel-filtered platelets were pre-incubatedwith Hex, at a concentration (2 mU/mL) similar tothat previously found to be released in vivo, or withits vehicle and tested for aggregation, P-Sel releaseand GPIIb/IIIa activation after stimulation with subthresholddoses of thrombin. For the p47 phosphorylationexperiments 32P-labeled platelets wereused. Hex did not induce platelet activation by itselfbut it did enhance platelet aggregation (+190.8±64.9%), P-sel release (+64.1±15.5%) and GPIIb/IIIaactivation (+138± 20.8%) induced by subthresholddoses of thrombin; preincubation with Hex endhancedp47 phosphorylation (+20% vs ctrl) too.Preincubation with the PKC inhibitor RO 31-8220,the PLA2 inhibitor aristolochic acid, and the Ca ++influx inhibitor NiCl2, completely abolished Hexinducedpotentiation; the PTK inhibitor tyrphostine,and the PI3-K inhibitor wortmannin partially reducedthe Hex priming, while the COX inhibitor aspirin, theLOX inhibitor NDGA, the p38-MAPK inhibitor SB203580, and the MAPK inhibitor PD 169316, did notaffect the Hex potentiating activity. Concerning theHex isoforms: both Hex A (α/β) and Hex B (β/β) werefound to prime platelet activation. In conclusion,lysosomal Hex acts as a proaggregatory agent withan activity involving PKC- and PLA2-dependentmechanisms and Ca ++ influx.CO-003REGULATION OF TISSUE FACTOR EXPRESSION IN HUMANPLATELET BY NITRIC OXIDEFrigerio M,* Tremoli E,* § Biglioli P,* Camera M* §Centro Cardiologico Monzino IRCCS, Milan;§Dipartimento di Scienze Farmacologiche,Università degli Studi, Milan, ItalyRecent data indicate that human platelets containTissue Factor (TF), the primary activator of the coagulationcascade. We have shown that intraplatelet TFcan be exposed on the membrane by platelet agonistssuch as ADP, Trap and tromboxane A2 analogueU46619. Nitric oxide (NO) was originally discoveredas a vasodilator product of the endothelium. Overthe last 15 years, this vascular mediator has beenshown to have important antiplatelet actions byactivating guanylyl cyclase and increasing intracellularcGMP. NO-donors are pharmacologically activesubstances that, in vivo or in vitro, release NO. In thepresent study we investigated whether different NOdonors modulate agonist-induced TF and P-selectinexpression on platelet surface. Both antigens weremeasured by flow cytometry using specific monoclonalantibodies in platelet rich plasma (PRP)obtained from healthy subjects. PRP incubated for 15minutes with ADP (10 µmol/L), TRAP (35 µmol/L) andU46619 (1 µmol/L) induced a consistent expressionof TF (+170±40%, +146±16% and +152±19% versusunstimulated platelets, respectively). ADPinducedTF and P-selectin expression was inhibitedin a comparable manner by S-nitrosothiols, such asSNAP (10 µmol/L, -40±16%) and GSNO (10 µmol/L,-53±8%). Similarly, TRAP-induced TF expression wasinhibited by SNAP and GSNO by 46±7% and72±20% respectively, while P-selectin expressionwas only modestly affected by both compounds.U46619-induced TF as well as P-selectin expressionwas almost abolished by SNAP and GSNO. Comparableresults were observed also with NCX-4016,which is a nitroderivative of acetylsalicylic acid (NOaspirin).Interestingly, this compound was able toexert an inhibitory effect on TF and P-selectinexpression also in whole blood. These findings suggestthat NO significantly reduces platelet-associatedTF expression in vitro; this might have relevancein vivo for the treatment of clinical conditions suchas acute coronary sindromes.haematologica vol. 89(suppl. n. 8):september 2004

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 200421CO-0042-SUBSTITUTED ADENINE NUCLEOTIDES: MODULATION OFPLATELET AGGREGATION THROUGH P2Y RECEPTORSPodda GM, 1 Vittori S, Lecchi A, 1 Costanzi S, 2Cristalli G, 2 Cattaneo M 31Centro Emofilia e Trombosi Angelo Bianchi BonomiIRCCS Ospedale Maggiore, Departimento diMedicina Interna, Università di Milano, 2 Dipartimentodi Scienze Chimiche, Università di Camerino;3Unità di Ematologia e Trombosi, Ospedale SanPaolo, Departimento di Medicina, Chirurgia eOdontoiatria, Università di Milano, ItalyAim of our study was to evaluate the response ofhuman platelets to nucleotide analogues, in thepresence or absence of ADP. We tested the six compoundsreported in Figure 1. The compounds (mono–,di– and tri-phosphate derivatives of 2-hexynyl and2-phenylethynyladenosine) were synthetizedthrough phosphorylation of the corresponding nucleosides.Shape change, aggregation and inhibition ofadenylyl cyclase induced by 10 microM ADP werestudied in washed human platelet suspensions.Nucleotides 2 and 3 caused platelet shape changeand aggregation, with an EC50 ranging from 10 to100 microM. The extent of platelet aggregationinduced by 100 microM of these compounds wasslightly lower than that elicited by 10 microM ADP,indicating that they activate both P2Y1 and P2Y12receptors. The 2-phenylethynyladenosine derivatives4-6 and the 2-hexynyl monophosphate derivative 1,at concentrations between 10 and 130 µM, did notinduce platelet shape change or aggregation. In contrast,they concentration-dependently inhibitedplatelet aggregation induced by 10 µM ADP, withthe following IC(sub)50: 14 µM (1), 130 µM (4), 60µM (5) and 85 µM (6). At a concentration of 800microM, nucleotides 1, 4 and 6 completely inhibitedboth platelet aggregation and shape change inducedby 10 µM ADP, while nucleotide 5 inhibited plateletaggregation by 90% without affecting shape change.At a concentration of 800 µM, all the nucleotidespartly (30-75%) antagonized the ADP-induced inhibitionof cAMP increase; lower concentrations hadno inhibitory effects. Therefore the tested inhibitorynucleotides displayed primarily an antagonisticeffect on the platelet P2Y1 receptor, while a partialinhibitory effect on the platelet P2Y12 receptor wasobserved only at the highest (800 µM) nucleotideconcentrations tested. The surprisingly differentbehaviour of 2, 3 (agonists) and 5, 6 (antagonists)can be explained in terms of different size and flexibilityof the substituent in 2 position.CO-005ROLE OF THE PLATELET P2Y1 RECEPTOR IN THROMBUSFORMATION UNDER FLOW CONDITIONSMarchese P,* Cattaneo M, # Gachet C,^ Ruggeri ZM**Division of Experimental Thrombosis andHemostasis, The Scripps Research Institute, La Jolla,California, USA; # Unit of Hematology and Thrombosis,Department of Surgery, Medicine and Dentistry,Ospedale San Paolo, University of Milan, Italy;^Institut National de la Sante et de la RechercheMedicale, Etablissement Francais du Sang-Alsace,Strasbourg, FranceP2Y1 is a receptor for adenosine diphosphate (ADP)on platelets required for normal shape change andaggregation following ADP stimulation. Moreover,P2Y1 deficient mice display defective platelet aggregationin response to ADP or low collagen concentrations,as well as reduced thrombus formation inarterial and venous models of vascular injury. Tocharacterize in more detail the role of P2Y1 inplatelet function, we tested P2Y1 deficient mouseblood in an ex vivo model of thrombus formation onbovine insoluble fibrillar collagen type I under flowconditions. In the absence of P2Y1, platelets formedsmaller than normal thrombi on surfaces with eitherlow or high collagen density when the wall shearrate was 1500 s -1 , but the defect was not detectableat the lower shear rates of 300 s -1 and 500 s(e) 1. Athigher shear rates, 6000-10000 s -1 , the defect waspresent at low but less apparent at higher collagenconcentrations. Real-time analysis of thrombus formationdemonstrated that the initial platelet adhesionand aggregation, within 30 s from the beginningof perfusion, were normal or only partially reduced,respectively, in the absence of P2Y1, while thereduced thrombus volume as compared to normalbecame progressively more apparent at later perfusiontimes. In fact, defective thrombus formation inthe absence of P2Y1, when present, was characterizedby instability of initially formed thrombi thattended to detach from the surface. Thus, the P2Y1ADP receptor plays a role in collagen-induced thrombusformation at medium to high shear rates by substantiallycontributing to thrombus stabilization.These results also indicate that P2Y1 function maybe more relevant for normal platelet function atmedium to high, rather than low, wall shear rates,and particularly on surfaces with lower collagen density.haematologica vol. 89(suppl. n. 8):september 2004

22Oral CommunicationsCO-006EXPRESSION OF PAR1 ON HUMAN CD133 + CORD BLOOD CELLSAND DURING MEGACARYOCYTE DIFFERENTIATIONDe Candia E,* Rutella S,° Bonanno G, #De Cristofaro R,* Leone G,° Landolfi R,* Scambia G #*Istituto di Medicina Interna e Geriatria,° Istituto diEmatologia, # Istituto di Ginecologia e Ostetricia,Università Cattolica del Sacro Cuore, RomeThe G protein-coupled receptor PAR1 is expressedon platelets, megakaryocytes (MK) and endothelialcells. Whether PAR1 is an early or late marker of theMK maturation is not known. We measured theexpression of PAR1 during in vitro MK maturationand compared it to that of the MK-restricted proteinsGpIIb and GpIb. Human cord blood CD133 +progenitor cells were purified by an immunomagneticseparation system (MACS: Myltenyi Biotec) andcultured under serum-free conditions in the presenceof 10-50-100 ng/mL thrombopoietin (TPO) orSCF 10 ng/mL + TPO 10 ng/mL. Expression of PAR1,GpIIb and GpIb on cultured cells was evaluated atday 0, 5, 7 by using, respectively, PE- PERCP- or FITCconjugatedanti-PAR1 SPAN12, anti-CD 41 and anti-CD 42b MoAbs and flow cytometry analysis. At day0, 70% of freshly purified CD133 + cells showed lowexpression of PAR1, whereas the other antigenscould not be detected. At day 5 of culture PAR1expression was markedly downregulated, beingdetectable in only 15% of total cells, 20% of totalcells expressed the GpIIb whereas the GpIb wasalmost undetectable (Figure 1). At day 7 the expressionof these markers was 35% of total cells forPAR1, 53% of total cells for GpIIb and 20% of totalcells for GpIb. The kinetics of protein expression wassimilar in all culture conditions; interstingly, cell culturedin the presence of TPO + SCF downregulatedthe expression of all the differentiation markers.These results demonstrate that PAR1 is present onthe surface of very early hematopietic progenitorsand that during MK differentiation it is first downregulatedto become later a specific marker of MKmaturation.CO-007POLYMORPHONUCLEAR LEUKOCYTE-PLATELET INTERACTIONIN PATIENTS WITH ESSENTIAL THROMBOCYTHEMIA ANDPOLYCYTHEMIA VERAFalanga A, Marchetti M, Vignoli A, Balducci D,Barbui TDept. Hematology, Ospedali Riuniti, Bergamo, ItalyThe pathogenesis of high thrombotic risk in ET andPV patients is not completely clarified. In vivo PMNactivation (i.e. increased PMN surface CD11b expressionand protease release) occurs in these patientsand is associated with a hypercoagulable state. SinceCD11b plays an important role in PMN/platelet interaction,as a ligand for GPIb-bound fibrinogen onplatelet surface, in this study we explored whetherthe CD11b increment on PMN may be associatedwith changes in PMN/platelet interaction and inplatelet cell adhesion molecules. Forty-three patientswith ET, 30 with PV and 50 control subjects werestudied. PMN and platelet activation was evaluatedby surface CD11b and CD62P expression, respectively.PMN-platelet aggregates were defined as the percentageof CD11b-positive PMN co-expressing aplatelet-specific marker (i.e. CD42b or CD62P). Theseparameters were studied by whole blood flowcytometryin both basal condition and after in vitroPMN stimulation by f-MLP. Significantly higherCD11b levels on PMN from ET and PV patients vscontrols were found. PMN/platelets aggregates werealso increased (p

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 200423CO-008EVIDENCE SUGGESTING THAT ACTIVATED PLATELETS CAUSEAPC-RESISTANCE IN VIVOLeone M,* Momi S,* Colucci M,° Gresele P**Department of Internal Medicine, Sect. of Internaland Cardiovascular Medicine, University of Perugia;°Department of Biomedical Sciences, Sect. ofGeneral Pathology, University of Bari, ItalyActivated platelets have been shown to cause APCresistancein vitro. In this study we investigated theeffect of platelets on the APC-anticoagulant pathwayin vivo in mice. Control in vitro experiments indicatedthat collagen-stimulated murine plateletsreduce the aPTT-based APC sensitivity ratio (APCratio)from 2.15±0.39 to 1.33±0.10 (p

24Oral Communicationsshowed that the proportion of PMN expressing TFwas increased by 185±10 and 241±50% of basal valuesafter P-selectin and fMLP stimulation, respectively.Conclusions: our data show that the procoagulantactivity observed in stimulated PMN wasassociated to the expression of functional TF.CO-010SRC-DEPENDENT SIGNALING IS NECESSARY FOR β2-INTEGRINFUNCTION AND PMN FIRM ADHESION IN FLOWTotani L, Piccoli A, Barone S, Pecce R, Federico L,Manarini S, Evangelista VLaboratory of Vascular Biology and Pharmacology,Mario Negri Sud, Santa Maria Imbaro, ItalyNeutrophil (PMN) tethering by P-or E-selectin promotesthe adhesive function of β2-integrins responsiblefor firm adhesion in flow. SRC kinases may regulateβ2-integrin function in PMN. We investigatedthe role of SRC kinases on PMN adhesion to surfaceadherentplatelet, HUVEC or E-selectin expressingcells (CHO-E), in flow. In our model after an initialrecruitment phase, the shear was stepped up to 5dynes/cm 2 and adherent cells perfused with freshmedium. Firmly adhered and rolling PMN were quantifiedin the high shear phase. Results. Up to 95% ofPMN recruited on adherent platelets at 2 dynes/cm 2remained firmly adhered when shear stress wasstepped up to 5 dynes/cm 2 . P-selectin was requiredfor PMN recruitment. Similarly to β2-integrin blockadeby monoclonal antibodies the SRC inhibitors PP1and PP2 but not their inactive analog PP3 barelyaffected the total number of interacting cells butdose-dependently reduced the number of firmlyadhered PMN and increased the rolling fraction.After initial recruitment at 0.25 dynes/cm 2 , P-selectin, β2-integrins and SRC kinase activity werealso necessary for PMN to firmly adhere to thrombin-activatedHUVEC. Like P-selectin, E-selectin mayalso trigger β2-integrin function. β2-integrin blockadedecreased to about 20±7% of the control firmadhesion of PMN to CHO-E. PP2 dose-dependentlyreduced both total PMN and firmly adhered fraction.PMN adhesion to CHO-E, was accompanied by β2integrin or SRC dependent tyrosine phosphorylationof the 110 kDa, focal adhesion kinase PYK-2. Finally,confocal microscopy study showed that two distinctactivation-dependent epitopes of β2-integrins, wereup regulated at the sites of CHO-E-PMN adhesion.These epitopes were practically absent in mixed cellconjugates formed by PMN treated with SRCinhibitors. SRC, allowing and/or stabilizing clusteringof activated β2-integrins, translate short-lived membraneto membrane contacts to firm PMN adhesionin flow. This process involves PYK2.CO-011GENE EXPRESSION ANALYSIS OF HUMAN LPS-STIMULATEDAND UN-STIMULATED HUMAN LEUKOCYTES BY MICROARRAYTECHNOLOGY: DEVELOPMENT OF A NEW BAYESIAN MODELGiusti B, Poggi F, Rossi L, Biggeri A,° Blangiardo M,°Magi A, # Sestini I, Cesari F, Ferrini S, Gensini F,Abbate R, Gensini GF, Pepe GDipartimento di Area Critica Medico Chirurgica,sezione di Clinica Medica Generale e ClinicheSpecialistiche, University of Florence, Florence;°Dipartimento di Statistica “G. Parenti”, Universityof Florence; # Unita’ Operativa di Genetica eCitogenetica, Azienda Ospedaliera Careggi,Florence, ItalyMicroarray technology (MT) allows monitoring ofexpression levels for thousands of genes in a singleassay. The basic strategy is to isolate RNA from twosources (control and test). mRNAs from the twostates are differentially labeled with two fluorescentmolecules (Cy3 and Cy5) and co-hybridized to themicroarray. The arrays are scanned and independentimages are generated for each pair of samples. Forimage quantitation, there are many commercial andfreely available softwares and developed methods.A challenging issue in MT is the cutoff value used todistinguish differential expression from natural variability.One of the most used method of analysis wasintroduced by Newton (1999). Aims of this studywere: to evaluate and validate the experimental andanalysis approach of MT by using an experimentalmodel of LPS-stimulated and un-stimulated humanleukocytes. Mononuclear cells were isolated fromperipheral blood of 20 controls. For experiments ofself-self hybridization, RNA was extracted from thepellets and pooled. For gene expression analysis ofLPS-stimulated and un-stimulated leukocytes, cellswere incubated at 37°C for 3 hours in presence orabsence of LPS (10 µg/mL) and then RNA wasextracted and pooled. Data analysis was performedby Newton method and two Bayesian models, onedeveloped by Tseng (2001) and one developed by us.We observed 14 differentially expressed genesaccording to Newton analysis, 40 genes according toTseng and 34 according to our model. Ten out of the14 Newton genes were identified by the three model;25/34 genes identified by our model were alsoidentified by Tseng model. For 12/25 genes identifiedby the two Bayesian models there were well establishedevidences in the literature confirming the differentialexpression after LPS stimulation (e.g. IL-1β,-RA; PAI-2; MIP-1A, -1B); for the other 13/25 therewere indirect evidences of involvement or new identifiedgenes up-or down-regulated after LPS stimulation.Bayesian models and in particular our modelseems to be more efficient in identifying differen-haematologica vol. 89(suppl. n. 8):september 2004

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 200425tially expressed genes. MT confirms its pivotal role asscreening approach to identify novel genes.This work was supported by a grant from Ministerodella Salute.CO-012DIVERSITY AND SIMILARITY IN SIGNALING EVENTS LEADINGTO RAPID COX-2 SYNTHESIS BY PMA AND TNFα IN HUMANENDOTHELIAL CELLSEligini S,* Barbieri SS,* Cavalca V,° Camera M,°De Franceschi M,° Tremoli E,*° Colli S**E. Grossi Paoletti Center, Department ofPharmacological Sciences and °Department ofCardiac Surgery, Centro Cardiologico FondazioneMonzino I.R.C.C.S, University of Milan, ItalyCyclooxygenase 2 (Cox-2) is a key enzyme in vascularhomeostasis, inflammation, tumorigenesis, andangiogenesis. Specifically, endothelial Cox-2 activityis crucial for the availability of prostacyclin and,consequently, for the modulation of platelet-vascularinteractions. Aim. This study examines whetherCox-2 synthesis and activity in endothelial cellsinvolves distinct signaling pathways when inducedby the representative proinflammatory cytokinetumor necrosis factor α (TNFv) or by the tumor promoterphorbol ester (PMA). Moreover, the hypothesisthat reactive oxygen species (ROS) and alterationof cell redox status are fundamental steps in Cox-2synthesis is addressed. Methods. Human endothelialcells isolated from umbilical vein (HUVEC) wereexposed to TNFα or PMA. Cox-2 protein synthesiswas evaluated by Western blot analysis and its activityby measurement of prostaglandin E2 (PGE2) and6-keto prostaglandin F1α levels. Intracellular ROSgeneration was detected by flow cytometry in cellsloaded with the oxidant sensitive 2'7'-dichlorofluoresceindiacetate and by reduction of nitroblue tetrazolium.Reduced and oxidized glutathione (GSH andGSSG) levels were measured by HPLC. Distinct signalingpathways are involved in early Cox-2 inductionby TNFα and PMA. PMA signals through theinvolvement of a small GTPase, protein kinase C (PKC)and mitogen activated protein kinase (MAPK) ERK1/2activation whereas p38 MAPK is critical when Cox-2 is induced by TNFα. Conversely, intracellular ROSgeneration and consequent reduction of GSH/GSSGratio represent a common step for Cox-2 inductionby both stimuli. In addition, evidence that phosphatidylinositol3(PI3)-kinase activation plays a regulatoryrole for Cox-2 synthesis induced by PMA andTNFα is provided. Cox-2 represents a critical regulatorof vascular homeostasis, inflammatory response,angiogenesis and tumor growth. The finding that twoindependent pathways, together with overlappingupstream events signal for Cox-2 induction in HUVECmay be of relevance to develop strategies aimed atselectively interfering with regulating pathways.CO-013SYSTEMIC SCLEROSIS: GENE EXPRESSION PROFILING BYMICROARRAY TECHNOLOGY OF ENDOTHELIAL CELLSGiusti B, Del Rosso M,* Fibbi G,*Matucci-Cerinic M,^ Rossi L, Poggi F, Attanasio M,Lucarini L, Saracini C, Lapini I, Pepe G, Abbate RDipartimento Area Critica Medico-Chirugica,Università degli Studi di Firenze; *Dipartimento diPatologia e Oncologia Sperimentale, UNiversitàdegli Studi di Firenze; ^ Dipartimento di MedicinaInterna, Università degli Studi di Firenze, ItalySystemic sclerosis (SSc) is a clinically heterogeneous,systemic disorder which affects the connectivetissue of the skin, internal organs and the wallsof blood vessels. Defective angiogenesis, resulting intissue ischemia, is prominent in the diffuse form ofSSc, but its pathogenetic basis is unknown. We isolatedmicrovascular endothelial cells (MVEC) fromthe dermis of healthy individuals (N-MVEC) andpatients with diffuse SSc (SSc-MVEC) in order toidentify differences in the gene expression profiling.For this purpose we used a 14,000 gene oligonucleotidearray (70-mers oligonucleotide, Operontechnologies). Total RNA from cultured MVEC wasisolated by RNeasy Kit (QIAGEN). Pooling of MVECtotal RNA from patients and controls was performed.One hundred and ninety nine genes showed alteredexpression levels between SSc-MVEC and N-MVEC:153 were up-regulated and 46 were down-regulated.More than 25 of the genes (e.g. MIF, LAMR1, IL-8, CTGF, CFL1, PEA15, PGAR, KLKs, PLAT, PLAU, etc)which were differentially expressed in SSc-MVEC aredirectly or indirectly involved with angiogenesis.Interestingly, in contrast with the clinical data indicatinga vascular desert-like pattern, many proangiogeneticgenes were found to be over-expressed inSSc. These results suggest that an epigenetic eventwhich occurs in SSc may be able to vanish the angiogenicattitude of MVEC. Preliminary data indicatethat the truncation of the urokinase-plasminogenactivator receptor (uPAR) in SSc-MVEC may account,at least in part, for defective angiogenesis.haematologica vol. 89(suppl. n. 8):september 2004

26Oral CommunicationsCO-014ENOS GENE AFFECTS BLOOD VISCOSITY AND RED CELLDEFORMABILITY: ROLE OF ENOS T-786C, G894T AND 4A/4BPOLYMORPHISMSMannini L,* Fatini C,* Sticchi E,* Cecchi E,* Ilari I,*Bruschettini A,* Costanzo M,* Leprini E,° Pagnini P,°Gensini GF,* Prisco D,* Abbate R**Dipartimento Area Critica Medico-Chirurgica,Università degli Studi di Firenze; °Dipartimento diScienze Chirurgiche Oto-Neuro-Oftalmologiche,Università degli Studi di Firenze, ItalyPlasma viscosity and erythrocyte aggregation playa key role in maintaining and regulating microcirculation.In vitro and in vivo studies suggested a role fornitric oxide (NO) in modulating flow-mediated vasodilatationand red blood cell deformability. Impaired NOavailability due to mutations in eNOS gene might contributeto altered hemorheological state. Aim of thisstudy was to investigate the role of eNOS T-786C,G894T and 4a/4b polymorphisms in influencinghemorheological state. We studied 80 Idiopathic SuddenSensorineural Hearing Loss (ISSHL) patients and80 healthy subjects. From the multivariate analysis asignificant association between eNOS 894T rare variantand ISSHL (OR 894TT+GT=2.08, p=0.03) wasfound. A higher percentage of altered erythrocyte filtrationtest (EFT) both in patients and in controls carryingthe eNOS rare variants was found in comparisonto subjects carrying the wild-type. Apart from thedisease, eNOS T-786C and G894T polymorphismsindependently affected the EFT (OR -786CC+TC=2.81,p=0.01 and OR 894TT+GT=2.5, p=0.02, respectively),in particular in subjects in whom the contemporarypresence of the two rare alleles was observed (OR -786CC+TC and 894TT+GT combined genotype =6.9,p

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 200427CO-016ADULT MESENCHYMAL STEM CELLS DERIVED FROM ADIPOSETISSUE HIGHLY EXPRESS TELOMERASE AND SPONTANEOUSLYDIFFERENTIATE INTO CONTRACTILE CARDIOMYOCYTES ANDMATURE ENDOTHELIAL CELLSMadonna R,* De Caterina R,° Willerson JT, § Geng YJ §*Internal Medicine, University of Texas MedicalSchool, Chair of Cardiology, University of Chieti,Houston, TX, United States, °Chair of Cardiology,University of Chieti, Italy, § Internal Medicine,University of Texas Medical School, Texas HeartInstitute, Houston, TX, USAChanges in adipose mass, involved in obesity andinsulin resistance syndrome, are achievied throughadipocyte differentiation. We hypothesized that theadipose tissue contains telomerase-positive multipotentstem cells, which can differentiate into contractingmyocytes and mature endothelial cells. Murine, pigand dog adipose tissue was processed by collagenaseto obtain adult mesenchymal stem cells (MSCs). After5 days culture, MSCs showed spontaneously higher levelsof early markers for endothelial differentiation(CD34 and VEGF-R2 ) and lower levels of CD31, vonWillebrand factor, endothelial nitric oxide synthase(eNOS) and nitrite production. Furthermore, MSCsshowed expression of biologically active telomerasecatalytic subunit (TERT) at a level close to that inembryonic stem cells (ESCs). Flow cytometry showedco-localization of TERT with the intracellular accumulationof DiI-acLDL in a subset of MSCs cells(2.9±0,2%). Unlike DiI-acLDL negative cells, in presenceof endothelial growth factors such as VEGF andbFGF, DiI-acLDL positive cells showed capability to differentiateinto mature, functional endothelial cells, byproducing eNOS and nitrite, expressing higher levels ofCD31, and efficiently repairing a linear scare performedon a confluent monolayer. Primary culture of MSCswere also evaluated for developmental plasticitytowards myogenic and cardiomyogenic lineages byexposing the cells to cardiomiogenic inductive mediafor 15-20 days. Upon inductive conditions, the adiposeMSCs expressed myocardin-A, a key transcriptioncofactor for myogenic cell development. Colonies withcontractile myogenic cells emerged from telomerasepositivebut not negative cells after 2-3 weeks in culture.Immunofluorescence and immunoblotting showedthe positivity of the cardiac myogenic markers, -sarcomericactinin, myosin, actin and the Rynodine receptorin the proteins extracted from beating colonies butnot from those without contractile cells.In conclusion,these observations suggest the possibility to modulatethe committment of adipocytes into cardiovascular celllineages, useful for cardiovascular therapies.Oral CommunicationsHEMOPHILIA: CLINICAL STUDIESCO-017INHERITED BLEEDING DISORDERS: THE EMILIA-ROMAGNAREGION REGISTERTagliaferri A,* Pattacini C,* Pozzoli D,* Biasoli C, #D'incà M,^^ Mancino A, $ Marietta M,^ Moratelli S,**Rossi A, ## Valdrè L,° Vincenzi D°°*Centro Emofilia Parma, # Cesena, ^^Reggio-Emilia,$Ravenna, ^Modena, **Ferrara, ## Piacenza,°Bologna, °°Faenza; ItalyTo assure high quality, homogeneous levels ofhemophilia care on the whole country, Emilia-Romagna Region (RER) defined in 2002 an organizationmodel (HUB and SPOKE), able to balancing toopposite requirements: a near-to-home distributionof services and concentration of specialistic activitiesin few highly qualified Centers. The inheritedbleeding disorders Register was estabilished to supportthis model of care. The primary aim of the registeris to better understand how to manage patientstracking the disease and its complications, healthrelated quality of life and social, epidemiologicaspects and health cost trend. Since January 2003the eight RER Hemophilia Centers (HC) used a computerizedclinical record EMOCARD (also used byAssociazione Italiana Centri Emofilia-AICE). HCextract every six months, by a dedicated algorithm,relevant data and send it to the Hub Center (HC-Parma) where are processed and published on theweb register public area (www.registroemofiliarer.it)in respect of Italian privacy laws. Responsible of HCand RER can access to a more detailed data privatearea with a personal password. The register identified495 patients: Haem.A=219, Haem.B=49,vWD=142, Rare bleeding disorders=46 Haem.carriers=33,Platelet disorders=6. 11 Haemophilia.Apatients present inhibitors (6 high responders, 5 lowresponders ), no Haemophilia B patients developedinhibitors. Immune-tolerance is ongoing in twohaemophilia A patient. In 2003 496 bleeds werereported: joint=267, muscular=102, Other=121. 65hospital admission, 165 DayHospital, 37 surgical procedureswere registered. Fifty-nine Haemophilia Aand 11 Haemophilia.B patients used recombinantfactor concentrate. Twenty-one Haemophilia.A, 8Haemophilia.B, 12 vWD, 10 rare bleeding disordersused plasma derived concentrate. Seven Haemophila.Awere in primary prophylaxis; 22 Haemophilia.Aand 6 Haemophilia.B in secondary prophylaxis. Ourhaematologica vol. 89(suppl. n. 8):september 2004

28Oral Communicationsstudy demonstrates the usefulness of a registry incollecting epidemiological data, health costs HRQoLand also to improve care delivered.CO-018PREVALENCE OF THROMBOPHILIC GENE MUTATIONS IN APOPULATION OF PATIENTS WITH INHERITED BLEEDINGDISORDERSCoppola A, Cimino E, De Simone C, Cirillo F,di Napoli F, Agnes R, Di Minno M, Tufano A,Di Minno GRegional Reference Centre for Coagulation Disease,Dep. of Clinical and Experimental Medicine;“Federico II” University, Naples, ItalyPhenotypic expression of inherited bleeding disordershas been thought to be affected by modifiergenes, able to mitigate their clinical severity. Amongthem, a role for thrombophilic gene mutations hasbeen proposed. We evaluated the prevalence of thewell defined thrombophilic mutations G1691A ofFactor V gene (FV Leiden) and G20210A of prothrombin(FII) gene in unrelated patients with severehemophilia (n=55) or von Willebrand disease (vWD,n=58, 41 women, 17 men) screened at our HemophiliaCentre. Prevalence of FV Leiden mutation was7.2% (4/55) in hemophiliacs and 1.7% (1/55) in vWDpatients, respectively. FII G20210A mutation wasdetected in 3.6% (2/55) of hemophiliacs and in 3.4%(2/58) of vWD patients (1 woman carried the homozygous20210A genotype). These gene frequencieswere comparable to those obtained in healthy subjectsfrom the same geographic and ethnic background(n=328, FV Leiden: 19/328, 5.8%; FIIG20210A: 17/328, 5.2%). On the whole, the 6 hemophiliacscarrying thrombophilic gene mutations didnot show clinical differences as far as severity or frequencyof bleeding episodes. Among the 3 vWDpatients with FV/FII mutations (all women), 2 had atype 1 disease with mild bleeding tendency; the thirdhad a more severe phenotype, with a long-lastinghistory of nose bleeding, but she had a type 3 disease.Three hemophiliac and 1 vWD patients had documentedatherosclerotic disease (1 coronary and 3peripheral artery disease): none carried thrombophilicgene abnormalities. A relatively low prevalenceof thrombophilic gene mutations is shown inthis population of patients with inherited bleedingdisorders. These data and the inherent limitations ofthe sample size make it difficult to define a possiblerole for thrombophilia in the phenotypic modulationof these diseases.CO-019A SKEWED X-INACTIVATION MECHANISM COULD EXPLAIN THESEVERE HEMOPHILIA B FEATURE OF A 3-YEAR OLD GIRL WITHA SEVERE HEMORRHAGIC DIATHESISMargaglione M, Schiavoni M,* Santacroce R,Di Perna P, Ettorre CP,* Ciavarella N*Genetica Medica Azienda Policlinico-Foggia, *U.O.S.Coagulazione-Med. Trasf. Azienda OspedalieraPoliclinico, Bari, ItalyHemophilia A and B are X-linked inherited hemorrhagicdiseases resulting from deficiencies of bloodcoagulation factor VIII or IX. Usually hemophilias aretransmitted by healthy heterozygous female carriersto affected males. Very rarely can a woman exhibithemophilia A or B and the most common explanationsfor how this can happen include compound heterozygosityand skewed X-inactivation. Few, isolatedcase-reports exist, so that we ignore the frequency ofhemophilia A and B in women and whether the clinicalphenotype is the same of that encountered inmales. Our experience regards a 3 year-old little girlsuffering from a severe hemorrhagic diathesis. Thefirst bleeding was an apparently spontaneoushemarthrosis of the left knee, afterward she presenteda large hematoma of left ilio-psoas muscle. Laboratorydata showed a prolonged PTT (R=1.97, n.v. 0.9-1.2) and the dosage of F.IX

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 200429During 1997-2000, subcutaneous ports were used atour Center by 23 children without feasible peripheralaccess. Ports removal was required, after a median of5 years, in 17 patients (74%) for infections and onedislocation. In 1999, we started to create AVF in 5 childrenwho had their ports removed. Since 2000, wehave introduced the use of AVF in patients needing avascular access either as first option or after portremoval. Prospective data have been collected to evaluatethe safety and feasibility of AVF as alternativevascular access. To date, 32 hemophiliacs (median age:3 years), 18 with inhibitors (56%), underwent the creationof 36 proximal AVF in the forearm. Mild forearmhaematomas were observed after 7 procedures (19%)in patients with inhibitors. Inadequate AVF maturationwas observed after 5/35 procedures (14%) in 4children. AVF were first accessed after a median of 45days (21-120) and regularly used at home by 30/31patients (97%) for a median of 39 months (2-56).Thrombosis of a venous branch occurred in one AVF(3%) after 9 months of uncomplicated use in a childwith inhibitor who spontaneously recovered fromsymptoms and still used AVF for 21 additional months.Mild symptoms of distal ischemia were transientlyreported by 3 children (9%) who needed no remedialintervention. Our data referred to a median follow-upperiod of 40 months (3-57) demonstrates that the useof AVF allowed long-term treatment at home in 97%of hemophiliacs.CO-021INHIBITOR DEVELOPMENT IN PREVIOUSLY TREATED PATIENTSWITH HEMOPHILIA A SWITCHED TO A B DOMAIN-DELETEDRECOMBINANT FACTOR VIIIGringeri A,* Tagliaferri A,° Tagariello G,^ Morfini M, §Santagostino E,* Mannucci PM* and theREFACTO-A.I.C.E. Study Group*Angelo Bianchi Bonomi Haemophilia andThrombosis Centre and Department of InternalMedicine and Dermatology, IRCCS MaggioreHospital and University of Milan, Milan, Italy;°Hemophilia and Thrombosis Centre, Ospedale diParma, Italy; ^Haemophilia Centre, Ospedale Civile,Castelfranco Veneto (TV), Italy; § HaematologyDepartment and Haemophilia Centre, UniversityHospital of Florence, ItalyBackground. There are recent reports of the unexpecteddevelopment of inhibitors in previously treatedpatients (PTPs) switched to a B-domain-deletedrecombinant factor VIII (BDD-rFVIII). Aim and methods.To evaluate inhibitor development we report theresults of a 6-month prospective study of 25 severePTPs and of a retrospective survey of 94 PTPs (53severe, 33 moderate and 8 mild hemophiliacs), allswitched from other products to BDD-rFVIII. Results.In the prospective study one patient, previously treatedwith plasma-derived concentrates only, developeda high titer inhibitor (30 BU) after 5 exposure days.Discontinuation of BDD-rFVIII led to inhibitor loss andallowed effective treatment with a plasma-derivedconcentrate, without reappearance of inhibitors. Theretrospective survey, carried out in the whole ItalianPTP population who had switched to BDD-rFVIII inthe recent past, involved high risk 19 PTPs becausethey were previously exposed to other FVIII concentratesfor up to 50 days and low risk 75 PTPs becausepreviously exposed for more than 50 days. One of 53severe haemophiliacs developed an inhibitor: he wasa high-risk PTP previously exposed to another recombinantFVIII for 3 days only. Among the entire low-riskpopulation of Italian PTPs with severe haemophiliaswitched to BDD-rFVIII (25 in the prospective study,49 in the retrospective cohort) only one patient developedan inhibitor (1.3%), an incidence similar to thatfound for other recombinant products. Conclusions.Overall these data indirectly support the views thatBD-rFVIII is equivalent to other recombinant productsin term of inhibitor development.CO-022IMMUNE TOLERANCE INDUCTION WITH RECOMBINANTFACTOR VIII IN HEMOPHILIA A PATIENTS WITH INHIBITORSRocino A,* Santagostino E,^ Mancuso ME^*Hemophilia and Thrombosis Center San GiovanniBosco Hospital, Napoli, Italy; ^Angelo BianchiBonomi Hemophilia & Thrombosis Center, IRCCSMaggiore Hospital, Milan, ItalyIntroduction: Inhibitors develop in up to 25-30% ofpatients with severe haemophilia A. Treatment optionsare effective in bleeding management but are not optimalin providing good long-term outcome. Inhibitoreradication by immune tolerance induction (ITI) representsthe best current treatment option. Few data existon ITI using recombinant FVIII (rFVIII). Objectives: toevaluate the success rate of ITI using rFVIII and theimpact of FVIII gene mutations on ITI response. Studydesign and patients: a retrospective/prospective studywas carried out in 2 Centers including 27 patients withsevere haemophilia A (age: 1-25 years, 19 with intron22 inversion), 25 high-responders and 2 low-responders,given rFVIII for ITI. Successful ITI was defined asthe achievement of an inhibitor titer

30Oral Communicationsmonths (2-39) and no relapse was observed. Amongpatients who achieved success, 13 (76%) had intron 22inversion. Conclusions: the observed rate of successfulITI using rFVIII was similar to the rates reported usingplasma-derived FVIII concentrates. Intron 22 inversiondid not reduce the chance of successful response to ITIin this cohort.CO-023IMPACT OF EARLY FVIII EXPOSURE, PROPHYLAXIS ANDPRENATAL/PERINATAL EVENTS ON INHIBITOR RISK INCHILDREN WITH HEMOPHILIA A: A CASE-CONTROL STUDYSantagostino E,* Rocino A,^ Mancuso ME,*Mazzucconi MG,° Mancuso G, # Messina M, §Tagliaferri A,°° Luciani M,^^ Boeri E,** Mannucci PM**Angelo Bianchi Bonomi Hemophilia & ThrombosisCenter, IRCCS Maggiore Hospital, Milan, Italy;^Hemophilia & Thrombosis Center, San GiovanniBosco Hospital, Naples, Italy; °Hemophilia Center,Policlinico Umberto I Hospital and La SapienzaUniversity, Rome, Italy; # Hemostasis & ThrombosisCenter, "G. Di Cristina" Hospital, Palermo, Italy;§Hemophilia Center, "Regina Margherita" Hospital,Turin, Italy; °°Hemophilia Center, Parma Hospital,Parma, Italy; ^^Hemostasis & Thrombosis Center,"Bambino Gesù" Hospital, Rome, Italy;**Hemophilia Center, "G. Gaslini" Hospital, GenoaIn a multicenter case-control study we investigatedthe impact of prenatal/perinatal events and earlyFVIII exposure on the inhibitor risk in hemophiliacs.Patients: 102 children (age:13-196 months) withhemophilia A (FVIII

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 200431Oral CommunicationsTHROMBOSIS & NUTRITION:EXPERIMENTAL & EPIDEMIOLOGICAL STUDIESCO-025COMPARISON OF GLOBAL RISK OF FATAL CARDIOVASCULARDISEASE IN THREE EUROPEAN POPULATIONS: RESULTS FROMTHE IMMIDIET PROJECTDi Castelnuovo A,* Zito F,* Dircks C, # Siani A,°Arnout J, # Cappuccio FP, § Donati MB,* Iacoviello L*on Behalf Of The IMMIDIET Study Group*Laboratory of Genetic and EnvironmentalEpidemiology, Center for High Technology Researchand Education in Biomedical Sciences, CatholicUniversity of Campobasso, Italy; # Center forMolecular and Vascular Biology, University ofLeuven, Campus Gasthuisberg, Leuven, Belgium;§Department of Community Health Sciences,St. George's Hospital Medical School, CranmerTerrace, London, UK; °Institute of Food Sciences,CNR, Avellino, Italy.Background and aim. An increase in global cardiovascular(CVD) risk from southern to northern Europeanpopulations is largely accepted. Recently theThird Joint Task Force of the European Societies onCoronary Prevention and investigators of the SCOREProject proposed a new risk chart for evaluation often-year risk of primary fatal CVD disease in Europe.The estimation models are based on gender, age,total cholesterol (TC), smoking and systolic bloodpressure (SBP). We aimed at investigating the differencesin the risk of fatal CVD using SCORE equationsin three primary care-based European populationsfrom the IMMIDIET project. Methods. TheIMMIDIET Project (Dietary Habit Profile in EuropeanCommunities with Different Risk of MyocardialInfarction: the Impact of Migration as a Model ofGene-Environment Interaction) includes a study on1602 subjects from 3 populations: Italy (N=542),Belgium (N=535) and England (N=525). The subjects,apparently healthy and aged 30-65 years, were freefrom diabetes, CVD or other major chronic diseases.Ten-year CVD risk was calculated applying SCOREequations to IMMIDIET subjects, separately for menand women. Differences among countries have beenadjusted for age and for multiple comparisons.Results. Some characteristics of the subjects areshown in the Table. Italians were the youngest andEnglish the oldest. Prevalence of smokers and SBPlevels were more elevated in Italy as compared withBelgium and England. Belgians showed the highestlevels of cholesterol. Although the prevalence ofindividual risk factors was lower in the English group,the age-adjusted CVD risk in females was higher inEngland (P

32Oral Communicationscompared to healthy subjects. Both serum folate andvitamin B6 levels were higher in healthy subjectsthan in patients, while there was no difference invitamin B12 concentrations. Concerning dietaryhabits we observed that total lipid intake was notadherent to Italian guidelines (

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 200433gen were determined by flow cytometry, and theirprocoagulant potential assayed by a fibrin generationtest. The total levels of MP were not different in thetwo groups. Endothelium-derived MP and total MP TFantigen did not change in both groups. The largestnumber of MP was derived from platelets and monocytesin both groups. Both platelet and monocyte MPwere significantly lower in the n-3 PUFA group216×10 6 /L (87-677) (median, 5-95 percentiles)p=0.016 and 265×10 6 /L (7-984) p=0.010 vs theirbaseline levels 420×10 6 /L (126-1796) and 478×10 6 /L(9-1681), respectively. MP supported the coagulationmainly via intrinsic pathway and to a lesser extentalso via TF-dependent extrinsic-pathway. However,MP TF-procoagulant activity was reduced in the n-3PUFA group respect to placebo group. A supplementationwith n-3 PUFA after myocardial infarctionexerts favorable effects by decreasing platelet- andmonocyte-derived MP, possibly limiting their proinflammatoryand pro-thrombotic potential.SISET PrizeCO-029BIOAVAILABILITY AND METABOLISM OF OLEUROPEIN IN RATSUNDER DIFFERENT DIET REGIMENGiordano L, De Curtis A, Murzilli S,* Rotilio D,Donati MB, Iacoviello LCenter for High Technology Research and Educationin Biomedical Sciences Catholic UniversityCampobasso; *Consorzio Mario Negri Sud,S. Maria Imbaro, ItalyExtra-virgin olive oil is a source of polyphenoliccompounds with a strong antioxidant activity. Thesecompounds can have a beneficial effect on processesinvolved in the pathogenesis of thrombosis. Weinvestigated the absorption and urinary excretion ofoleuropein (one of the most important polyphenoliccompounds in olive oil) and its major metabolite,hydroxytyrosol, in rats. The study also aimed at investigatingwhether the absorption of these phenoliccompounds could be affected by the soy oil used asa vehicle. The experimental design involved fivegroups of eight rats: two groups were treated per oswith 100 mg/Kg oleuropein using H2O or soy oil andH2O (1:1) as vehicle; three control groups were treatedwith standard diet (8 g), soy oil (2 mL) and soy oiladded to standard diet (8 g in 2 mL). Each rat wastreated four times with the same dose, alternatingwith a week of standard diet plus two days of washout.Urines were collected at 0 and 24 hrs after eachtreatment. We have also considered three groups ofrats treated for a month with: 1) standard diet witholive oil (32 mg/Kg), 2) standard diet with olive oilwithout polyphenols, 3) standard diet with olive oil(64 mg/Kg), respectively; urine were collected atbaseline, after 1 day, 10 days and 30 days. Oleuropein/hydroxytyrosollevels were measured by LC-MS/MS. We also evaluated the levels of the conjugatedform with glucuronic acid by enzymatichydrolysis with β-glucuronidase. Data obtained confirmedthat hydroxytyrosol was the major oleuropeinmetabolite detected in urine, even if a basal level ofthis compound was also found in control rat urine.Urinary hydroxytyrosol level increased of 80% in ratstreated with oleuropein in soy oil compared to thosetreated with only oleuropein, while oleuropein leveldecreased of 45%. The four single-doses of oleuropeinadministered in succession to the same ratdetermined an increase in urinary mean levels forhydroxytyrosol, starting from 209±114 µg for thefirst dose to 316±101 µg for the fourth dose. Urinarymean levels for rats treated with oleuropein in soy oilincreased from 340±139 µg for the first dose to728±280 µg for the fourth dose. An increase inhydroxytyrosol urinary mean levels was also foundfor rats treated for a month with olive oil from thefirst to the 30th day, with the greatest enhancementfor rats treated with regimen 3). These data indicatedthat oleuropein absorption and metabolism wasaffected by the vehicle of administration and repeatedsingle-dose ingestion determined an enhancementin oleuropein levels, as, revealed through itsmetabolite hydroxytyrosol.CO-30EFFECT OF POLYPHENOLIC COMPONENTS OF EXTRA VIRGINOLIVE OIL ON ARTERIAL THROMBOSIS IN RATSDe Curtis A, Murzilli S, 1 Giordano L, Rotilio D,Donati MB, Iacoviello LCenter for High Technology Research and Educationin Biomedical Sciences Catholic University Campobasso,1Consorzio Mario Negri Sud, S. Maria Imbaro,ItalyEpidemiological studies showed a low rate of cardiovasculardisease in Mediterranean populations ascompared to North European countries. This has beenassociated with the use of extra virgin olive oil as themain source of fat. While many studies have beenfocused on the protective effect of the monounsaturatedfatty acids (MUFA) content of olive oil, fewstudies have investigated the effects of olive oilpolyphenols, with an elevated anti-oxidant activity.The aim of our study was to investigate the effect ofthe supplementation with olive oil and its polyphenoliccomponents on thrombosis and primaryhaemostasis in rats. Animals were supplemented forsix weeks with 1) standard diet (32 mL/kg of soybeanhaematologica vol. 89(suppl. n. 8):september 2004

34Oral Communicationsoil) 2) a diet containing 32 mL/kg of extra virgin oliveoil; 3) or a diet with 32 mL/kg extra virgin olive oildeprived of the polyphenolic fraction and 4) a dietwith the polyphenolic fraction extracted from olives.Thrombosis was induced by insertion into theabdominal aorta of an artificial prosthesis, andocclusion time (OT) was recorded as a measure ofthrombosis tendency. Template bleeding time (BT)was measured as a function of primary haemostasis.Factor VII coagulant activity (FVII:C), fibrinogen levels,platelet adhesion to fibrillar collagen and lipidlevels were also measured. A significant reduction inthe occlusion time of the aortic loop (63 h vs 79 h,p

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 200437od was 106 days (range 30-323 days). In 20 patientsALN filters were not removed for different clinicalreasons. ALN filter offers a broad range of clinicalapplications; it can be used as a definitive filter or itcan be safely removed after a medium term periodof placement, whereas retrieval after 3 months canbe unsuccessful. Indications for retrieval and maximumimplantation time require further studies.CO-036LMWH OR XIMELAGATRAN VERSUS VKA IN THE PREVENTIONOF VTE AFTER MAJOR KNEE SURGERY: A META-ANALYSIS OFRANDOMIZED CONTROLLED TRIALSIorio A, Ferrante F, Conti S, Nenci GGSezione di Medicina Interna e Cardiovascolare,Università di Perugia, ItalyIntroduction: Low molecular weight heparins(LMWH) have shown a better efficacy and safety profilethan UFH and are the most commonly used prophylacticagent in Europe after orthopedic surgery.On the contrary, vitamin K antagonists (VKA) still havethe preference of surgeons in North America for kneereplacement. This is likely to be due to the higher rateof bleeding at the surgical site for LMWH than forVKA, particularly if heparin is started soon aftersurgery or even before, and this in spite of the higherrate of VTE occurring with the use of VKA as prophylacticagent. Moreover, the new thrombin inhibitorXimelagratran has also been recently compared toVKA in knee surgery. Aims: To perform a meta-analysisof the randomized controlled trials evaluatingsafety and efficacy of VKA in comparison to LMWH orximelagatran in the prevention of venous thromboembolismafter major knee surgery. Materials andmethods: Searches of MEDLINE and EMBASE wereperformed. The analysis was performed on venographicallyevaluated patients. Standard metanalytictechnique was applied. Results: Nine studies (6 withLMWH and 3 with ximelagatran) were included in theanalysis for a total of 10291 patients (5033 and 5258,respectively). Statistical heterogeneity was found onlyfor symptomatic PE. For total DVT, VKA resulted lesseffective than LMWH (RR of 1.32 [1.18 – 1.47], P

38Oral CommunicationsCO-038PRACTICE VARIATION IN USA AND EUROPE FOR THEPREVENTION OF VENOUS THROMBOEMBOLISM INHOSPITALISED MEDICAL PATIENTS: FINDINGS FROM IMPROVEPini M, 1 Froehlich J, 2 Bergmann JF, 3 Monreal M, 4Piovella F, 5 Huang W, 6 Anderson F Jr, 6 for theIMPROVE Investigators1Ospedale di Fidenza Medicina Interna, Fidenza,Italy, 2 University of Massachusetts Medical Center,Division of Cardiovascular Medicine, Worcester,USA, 3 Hôpital Lariboisiere Clinique Thérapeutique,Paris, France, 4 Hospital Germans Trias I Pujol,Badelona, Spain, 5 IRCCS Policlinico San MatteoServizio Malattie Tromboemboliche, Pavia, Italy,6University of Massachusetts Medical School,Worcester, USAIntroduction. The clinical practices for venousthromboembolism (VTE) prophylaxis in acutely illhospitalised medical patients, a population at riskfrom VTE, are unknown. To investigate VTE prophylaxispractice variation between Europe and USA inIMPROVE, an international prospective cohort studyof hospitalised medical patients. Patients >18 yearsold, and hospitalised for >3 days with an acute medicalillness have been enrolled consecutively sinceJuly 2002. Data are recorded at discharge and 3months after discharge. A central adjudication committeereviews key clinical endpoints, includingsymptomatic VTE, bleeding and death. Data from1036 patients enrolled up to 30 September 2003 at12 hospitals in 5 European countries (France, Germany,Italy, Spain and UK) and 835 patients in 7 UShospitals are presented in this analysis. Europeanpatients were less likely than US patients to befemale (46% vs 54% respectively), were older (medianage 71 years vs 58 years), had a longer durationof both immobility (11 days vs 6 days) and hospitalstay (11 days vs 6 days) and were more likely to beimmobile for ?3 days in total (54% vs 46%). Only49% of European and 51% of US patients receivedprophylaxis. In Europe, low-molecular-weightheparin was the favoured prophylactic regimen,whereas in the USA, unfractionated heparin use wasmore common. US patients also received intermittentpneumatic compression or aspirin for VTE prophylaxissignificantly more often, and elastic stockingssignificantly less often than European patients.Despite being a population at risk of VTE, only onehalf of IMPROVE patients received VTE prophylaxis.Similar proportions of patients receiving prophylaxiswere observed, but practices vary considerablybetween USA and Europe with UFH, IPC and aspirinbeing received more by US patients and LMWH andelastic stockings being given more to Europeanpatients. These remarkable geographical variations inpractice patterns highlight the need for increasedawareness of guideline recommendations for VTEprophylaxis in medical patients.Funding: IMPROVE is supported by an unrestrictededucational grant from Aventis Pharma.CO-039MANAGED HEMOSTASIS “POINT-OF-CARE” TESTING IN ALARGE HOSPITAL: A THREE-YEAR EXPERIENCE AND THEASSESSMENT OF CLOTTING TESTSPaniccia R,* Costanzo M,* Blagojevich J,*Beconcini S,* Ciuti G,* Carboni E,* Filoni C,*Tronchin M,° Messeri G,° Abbate R,* Gensini GF,*Prisco D**Dipartimento Area Critica Medico-Chirurgica,Università degli Studi di Firenze; °Laboratorio diAnalisi Biochimico-Cliniche, AziendaOspedaliero-Universitaria Careggi, Florence, ItalyPoint-of-care (POC) automated systems for coagulationevaluation are key tools in surgical and/orintensive health care units. Activated Clotting Time(ACT) is the most common hemostasis POC test toquickly monitor heparin anticoagulation during cardiopulmonarybypass, interventional cardiology andhemodialysis. Recently, these POC systems have beenupdated to rapidly perform aPTT and PT on wholeblood, to obtain a shortening of time of results. Toensure safe and appropriate patient care, the clinicalsafety of POC tests in hemostasis is based onachievement of accurate and reliable results. Thisreport was aimed to show the routine quality control(QC) testing and tracking, as a part of a comprehensivequality assurance program, for hemostasisPOC devices, performed in Careggi Hospital(Firenze, Italy). Twenty-one Hemochron devices(International Technidyne Corp, USA) were located in8 buildings. More than 18,500 analysis/year wereperformed in the different Units. A management stafffor hemostasis POC was designed to control the useof these devices. To practice instrument, specific andrepeated training courses were held to hospital staff.QCs for each instrument and for each new lot of testcartridges were performed by the POC supervisor.Within-day CVs from normal and abnormal controlswere respectively: for ACT

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 200439relationships were found between values obtainedfrom POC tests and those from centralized laboratory:for aPTT (n=60) r=0.98, p

40Oral Communicationsjects; (ii) subjects with a diagnosis of PC deficiencyconfirmed at a molecular level present a clearlyincreased risk of both DVT and SVT when comparedto subjects with phenotypic PC deficiency alone. Weconclude that molecular analysis is particularly usefulfor the characterization of subjects with a PCdeficiency phenotype.CO-041VENOUS THROMBOEMBOLISM IN PATIENTS WITH FACTOR VLEIDEN, PROTHROMBIN MUTATION AND NEGATIVETHROMBOPHILIA SCREENING. SARDINIANTHROMBOEMBOLISM STUDYBarcellona D, Fenu L, Contu P,* Vannini ML,Mameli G, Marongiu FDipartimento di Scienze Mediche, PoliclinicoUniversitario, Università di Cagliari; *Dipartimentodi Igiene Pubblica, Università di Cagliari, ItalyThe aim of this study was to obtain a clinical profileof patients, affected by venous thromboembolism(VTE), carrying factor V Leiden, the G20210A allele ornegative thrombophilia screening. Considered thepeculiarity of the Sardinian genotype, in general, thisstudy also evaluated the percentage of Sardinianpatients with these thrombophilic defects or negativethrombophilia screening with the aim to better knowour clinical reality. We enrolled 436 consecutive,unrelated patients (157 M, 279 F, mean age 48 ± 17years) affected by VTE, referring to our ThrombosisCentre for investigation of possible causes of thrombophilia.Patients without an objective documentationof VTE were excluded from the study. Occasionalrisk factors such as oral contraceptive use, pregnancyor puerperium, immobilisation, surgery, andtrauma were recorded. If clinical and instrumentaldocumentation was available, a previous episode ofVTE and the age of appearance, were also recorded.As a control group, we studied 466 unrelated healthysubjects (205 M, 261 F, mean age 48 ± 14 years).Comparison between VTE cases and healthy controlswas carried out calculating Odds Ratios (O.R.) and95% Confidence Intervals (95% C.I.), according toConrfield. Fisher’s exact test was used when appropriate.Our findings showed that 72 patients (16%)carried the G20210A prothrombin mutation and 2patients were homozygous for this mutated genotype.Factor V Leiden was found in 42 patients (9.5%)and 2 of them were homozygous for the G1691Amutation. In healthy subjects the prevalence of themutated prothrombin and the factor V Leiden was5% and 2%, respectively. No homozygous werefound. The risk to develop a thrombotic episode inpatients carrying the prothrombin mutation was 3.64,while it was 4.86 in patients with factor V Leiden. Therisk for VTE in Sardinian patients for at least two generations(314 patients, mean age 49 ±17 years, 442sardinian controls, mean age 48 ±14 years) was similarto that obtained in all studied patients showingthat our findings represent the prevalence of the twomutated genotype in our region. Thrombophiliascreening was found negative in 258 patients (59%).The mean age of onset of VTE was similar in the threesubgroups of patients considered (45 years in patientswith the G20210A allele and in patients having anegative thrombophilia screening, 42 years inpatients with factor V Leiden). Spontaneous VTE werefound in 52% of patients carrying the factor V Leiden,29% of patients carrying the mutated prothrombinand 44% of patients with negative thrombophiliascreening. Patients with factor V Leiden andpatients with negative thrombophilia had a similarrisk for spontaneous VTE (p=0.3), while this risk was2.7 (C.I. 95% 1.2-5.9, p=0.01) and 1.9 (C.I. 95% 1.1-3.4, p=0.03) when patients carrying the factor V Leidenand those with negative thrombophilia wererespectively considered in comparison with patientscarrying the mutated prothrombin. The same resultswere obtained when we considered episodes of VTEdue to an occasional risk factor. The recurrence ofVTE was more frequent in patients carrying the factorV Leiden (43%) than in patients with G20210Aallele (24%) or in patients with negative thrombophilia(27%). The risk to have a recurrent VTE was2.4 (C.I. 95% 1.1-5.4, p=0.036) and 2 (C.I. 95% 1.0-4.0, p=0.038) in patients with factor V Leiden whencompared with patients carrying the mutated prothrombinand patients with negative thrombophiliascreening, respectively. No differences were foundbetween patients with negative thrombophilia andpatients with mutated prothrombin. The risk for VTEwas similar among the three subgroups of patientswhen we considered oral contraceptives, pregnancyor puerperium, immobilisation and trauma. A significantdifference was found among patient subgroupswhen surgery was considered. The prevalence ofpatients with an episode of VTE after surgery was2.2% in patients with factor V Leiden compared with19% in patients carrying the G20210A mutation and17% in patients with negative thrombophilia. The riskto develop a thrombotic episode after surgery was10 (C.I. 95% 1.2-7.8, p=0.009) and 8 (C.I. 95% 1.1-63, p=0.01) in patients with prothrombin mutationand negative thrombophilia screening when comparedwith those carrying the factor V Leiden. Finallywe found that 26% and 20% of patients with prothrombinmutation and negative thrombophilia,respectively, had an episode of pulmonary embolismwhile this percentage was only 9.5% in patients withfactor V Leiden. The risk to have a pulmonaryembolism was 3.4 (C:I: 95% 1.1-11, p=0.03) inpatients carrying the mutated prothrombin whenhaematologica vol. 89(suppl. n. 8):september 2004

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 200441compared with patients carrying the factor V Leidenwhile there was no statistical significant differencewhen patients with the G20210A allele were comparedwith patients having a negative thrombophiliascreening. A trend toward statistical significantdifference was found when patients with factor VLeiden were compared with patients having a negativethrombophilia screening (p=0.09). Our findingsshow that patients with the G20210A allele have ahigher risk for secondary VTE, particularly aftersurgery, and for pulmonary embolism when comparedwith patients with the G1691A mutation. Patientswith factor V Leiden have a higher risk for spontaneousVTE and the rate of recurrences is higher thanin patients with mutated prothrombin. Patients withnegative thrombophilia have some features similarto those with factor V Leiden and mutated prothrombinleading to the hyphotesis that perhaps otherunknown defects are present. Finally, the prevalenceof the prothrombin mutation is higher in Sardinianpatients compared with the prevalence reportedin the literature, while factor V Leiden seems to bea rare inherited defect in our region.CO-042THE INCIDENCE OF VENOUS THROMBOEMBOLISM IN RELATIONTO ANTITHROMBIN, PROTEIN C, PROTEIN S DEFICIENCY ANDFACTOR V HR2 HAPLOTYPE: THE RESULTS OF A FAMILYCOHORT STUDYTormene D, Fortuna S, Tognin G, Zerbinati P,Fadin M, Gavasso S, Petronella K, Rigo F, Pagnan A,Prandoni P, Simioni PDept. of Medical and Surgical Sciences, 2 nd Chairof Internal Medicine, University of Padua MedicalSchool, Padua, ItalyBackground. Antithrombin (AT), protein C (PC) andS (PS) defects are well recognized risk factors forvenous thromboembolism (VTE). HR2 haplotype is acommon genetic defect that seems to increase therisk of thrombosis in carriers of factor V Leiden butthe available data are controversial. Valid estimate ofthe thrombotic risk in carriers of AT, PC and PS associatedwith HR2 haplotype is still lacking. Methods.First and second degree relatives of consecutivepatients referred to our Center with a history ofthrombotic disease and identified as carriers of AT,PC and PS deficiency were enrolled in the study, providedthat they were aged above 15 years. A medicalhistory was taken with specific attention to previousVTE and concomitant risk factors. Then DNA test forHR2 haplotype was performed. Those subjects whoreported no prior venous thromboembolism in theirmedical history were eligible for inclusion in theprospective evaluation. Results. Three hundred andfifty-three individuals belonging to 66 families wereevaluated in the retrospective study. A total of 768years were observed in the carriers of AT, PC or PSand HR2 haplotype, 3433 in the carriers of a singleAT, PC or PS deficiency and 3783 in the non-carriers.The relative risk for spontaneous VTE was 9.85 (95%CI 0.9 to 109) for double carriers and 9.92 (95% CI1.26 to 78.2) for single AT, PC and PS defects. Threehundred and twenty-six patients were enrolled inthe prospective follow-up. A total of 1030 observationyears were obtained in the AT, PC and PS deficientgroup, 277 in the double–carriers group and1303 in the non-carriers group. The relative risk forspontaneous VTE was 10.1 (95% CI 1.27 to 80.8) and9.41 (95% CI 0.86 to 103) for AT, PC and PS deficientgroupand for the double-carriers group respectively.Conclusions. In this selected population the risk ofVTE in carriers of AT, PC and PS deficiency is higherthan in non-carriers and does not appear to be modifiedby the presence of HR2 haplotype.CO-043EPIDEMIOLOGICAL STUDY ON CONGENITAL AND ACQUIREDRISK FACTORS RELATED TO THROMBOEMBOLIC DISEASES INLIGURIA REGION (ITALY)Armani U,* Bertolini S,* Lo Pinto G, • Molinari A,Mandich P,° Perrone L, @ Leoncini G, § Intra E,Mori M, + Colla G, = Devoto GL,^ Ravera GB, #Badolati G #*Dipartimento di Medicina Interna, Centro di Ricercasulla Trombosi e sulla Arteriosclerosi, °Dipartimentodi Neuroscienze, Oftalmologia e Genetica,Sezione di Genetica Medica, § Dipartimento di MedicinaSperimentale, Cattedra di Biochimica, # Dipartimentodi Scienza della Salute, Cattedra di Statisticae Cattedra di Igiene e Medicina Preventiva, Universitàdi Genova; • Divisione di Medicina GeneraleCentro Trombosi, @ Laboratorio di Genetica Umana,+Servizio di Medicina di Laboratorio - E.O. OspedaliGalliera di Genova; Laboratorio di Emofilia, IstitutoGiannina Gaslini, Genova; Servizio di Medicina diLaboratorio, Ospedale Evangelico Internazionale,Genova; =Servizio di Medicina di Laboratorio,Azienda Ospedale S. Martino, Genova; ^Servizio diMedicina di Laboratorio, Ospedale di Lavagna, ItalyThromboembolic diseases represent one of themost relevant public health Italian issues. Accordingto recent epidemiological studies, cardiovascularmortality is still the leading cause of death, accountingfor 44% of all deaths, with a major contributionof ischemic heart disease (28%) and stroke (13%)(Ital Heart J, 2003). Hypertension, hypercholesterolemia,diabetes, obesity, smoking habit andsedentary physical activity have been defined ashaematologica vol. 89(suppl. n. 8):september 2004

42Oral Communicationsindependent risk factors of thromboembolic diseasesin many epidemiological studies. Genetic factorshave been also associated to a higher incidence ofthromboembolic diseases. In Italy, studies on theprevalence of coronary and cerebrovascular events,have been performed in population of differentregions (WHO Monica Project, National Institute ofHealth, ISS). The Liguria Region, sponsored by ItalianHealth Ministry, promotes an epidemiological studyon hereditary and acquired risk factors related tothromboembolic diseases. Twenty public hospitalcenters, homogeneously spread in the region, willcollect data with the aim of evaluating the prevalenceof thromboembolic diseases (myocardialinfarction, stroke, peripheral artery disease, venousthromboembolism) and the role of risk factors, particularlyof the genetic risk factors. The followingpurposes will be pursued: 1.Creation of an informaticdatabase for collection of clinical, laboratoristicand geneticinformation of patients with thromboembolicdiseases; 2.Definition of a regional registryfor thromboembolic diseases; 3. identification ofgenetic risk factors predisposing to thromboembolicdiseases; 4.Evaluation of the role of environmentaland behavioural factors interacting with geneticbackground in the pathogenesis of thromboembolicdiseases; 5.Defintion of regional guidelines for thediagnosis and therapy of thromboembolic diseases;6.Identification of intervention for primary and secondaryprevention and educational programs for thepopulation; 7.Educational events and informationsupport for general practitioners and personnelinvolved in the clinical managing of patient; 8.Creationof multidisciplinary centers for prevention,diagnosis and therapy of thromboembolic diseases.CO-044HEREDITARY AND ACQUIRED THROMBOPHILIA IN CHRONICTHROMBOEMBOLIC PULMONARY HYPERTENSIONPiovella F, Barone M, Librè L, Piovella C,D'Armini AM, Emmi V, Ghio S, Campana C,Cerveri I, Dore R, Braschi A, Aprile C, Viganò MServizio Malattie Tromboemboliche, Cattedra diCardiochirurgia, Servizio di Rianimazione I, Serviziodi Radiodiagnostica, Istituto di Malattie dell’ApparatoRespiratorio, Istituto di Cardiologia, Istituto diRadiologia - IRCCS Policlinico San Matteo, MedicinaNucleare, Fondazione Maugeri - Università diPavia, ItalyChronic thromboembolic pulmonary hypertension(CTPH) is a rare disease which results from obstructionof the major pulmonary arteries by incompletelyresolved or organized pulmonary emboli whichhave become incorporated into the pulmonary arterywall, eventually causing an increase in pulmonaryvascular resistances. Pulmonary ThromboEndarterectomy(PTE) is an effective surgical procedurefor CTPH. Although CTEPH likely has a thromboembolicorigin, the role of thrombophilic factors is lessclear. The presence of hereditary thrombotic risk factorshas been shown to be increased in some studies,but no greater than control subjects in otherstudies. Anti-phospholipid antibodies (APLA), however,are common, and have been reported in up to20% to 50% of patients. APLA are strongly associatedwith thrombosis. Recently, we started in Paviaa program in which members of a multidisciplinaryteam work in close interaction on the challengingproblems these patients present in the evaluative,surgical, and post-operative phases of their care.From May 1996 to May 2004, 105 patients, all inNew York Heart Association (NYHA) class III or IVwere treated with PTE. Overall operative and perioperativemortality is 9.5% (10/105). At the 3 monthexamination, most surviving patients were in NYHAclass I or II. At 3 years, postoperative hemodynamicvalues were substantially maintained. All patientsare maintained on chronic oral anticoagulant treatment.We assessed the presence of thrombophilia on87/105 patients of our series, the rare deficiencies ofnatural coagulation inhibitors (AT, PC and PS) weredetectable in less than 5% of patients. Factor V Leidenwas present in 12%, and the prothrombin20210A allele in 5% of our patients. Mild hyperhomocysteinemiaassociated with the homozygousC677T polymorphism in the MTHFR gene was presentin 11% of patients. The prevalence of anti-phospholipidantibodies (APLA), either isolated or in associationwith other coagulation defects, was strikinglyelevated (38%.CO-045THROMBOPHILIAS AS RISK FACTORS FOR RECURRENTSPONTANEOUS ABORTIONSantoro R, Iannaccaro P, Sottilotta G, Leo FCentro Emofilia – Centro di Riferimento Regionaleper la Patologia Emorragica e Trombotica Ereditaria,Azienda Ospedaliera “Pugliese-Ciaccio”, Catanzaro,ItalyIntroduction. In the recent years inherited thrombophiliashave been suggested as a possible conditionof increased susceptibility to adverse pregnancyoutcomes such as fetal loss, recurrent pregnancyloss, abruptio placentae, intrauterine growth restriction(IUGR) and early-onset, preeclampsia. The aimof this study was to investigate the prevalence ofFactor V Leiden (FVL), G20210A prothrombin mutation(FII G20210A), comparated with the prevalencehaematologica vol. 89(suppl. n. 8):september 2004

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 200443of the same mutations in our control group. Methods.We retrospectively examined 173 women withrecurrent abortions (2 or more) occurred in the firsttrimester of pregnancy, after the VII week of gestation,mean 3, range 2-9. The control group included115 healthy women (age 15-45) without history ofpregnancy loss. The prevalence of FVL was 2.6% (3pts); the prevalence of FII G20210A was the same(2.6%, 3 pts). Results. In our group prevalence of FVLwas 6,3% (11/173). The prevalence of FII G20210Awas 6.9% (12/173). In the group of the women withearly pregnancy loss the difference in prevalence ofFVL and of FII G20210A was not statistically significantin comparison with controls (for both FVL andFII G20210A, p 0.14 and 0.10 respectively ). Conclusions.This date are not compatible with the hypotesisthat prothrombotic mutations may play a role insome cases of recurrent abortions. However, furtherand larger studies are necessary in order to verifythe real impact of FVL and of FII G20210A mutationsin women with early pregnancy loss.CO-046ROLE OF THROMBOPHILIA IN THE OCCURRENCE OF SUDDENAND PROGRESSIVE HEARING LOSSGrandone E, Cerullo M, Colaizzo D, Cappucci F,Vergura P, Cocomazzi N, Vecchione G, Magaldi L,D’Ecclesia A, Vigliaroli L, Margaglione MIRCCS Casa Sollievo della Sofferenza,S. Giovanni Rotondo, ItalySudden hearing loss (SHL) is a neurosensorial hearingloss of variable entity with an onset of less thanthree days.In most cases (about 90%) it is ofunknown etiology (idiopathic sudden hearing loss,ISHL). Little is known about the progressive form ofhearing loss. One of the hypothesis about both theseforms is vascular. In this respect, it could be possiblethat a thrombogenic damage could be responsiblefor this condition. We carried out a study aimedat evaluating whether narkers of inherited ( ProteinC, Protein S, antithrombin, Factor V Leiden, FIIA20210 mutation) and acquired (antiphospholipidantibodies) thrombophilias were more frequent inpatients who suffered from ISHL. Moreover, we evaluateda possible relationship between homocysteineplasma levels and the occurrence of these conditions.Overall, 106 patients (64 females, 42 males)were studied; 24 of them suffered from SHL and 82from progressive form. All patients underwent anaudiological evaluation, impedenzometric examination,a speech audiometry, a pure tone audiometry,an ABR audiometry and MNR, in addition to a largepanel of autoantibodies. For comparison, a group of106 apparently healthy controls was also tested forthe same variables. Overall, 3 patients (2.8%) showedFV Leiden (all SHL), 9 (8.5%) FII A20210 (all with progressiveform), and 9 (8.5%) had homocysteine abovethe 97.5 centile of our reference population ( 2 werein the SHL group). Two showed the contemporarypresence of FII A20210 mutation and levels of homocysteineabove 97.5 centile. None showed naturalanticoagulants deficiency. Six (5.7%) patients hadborder-line titre of aCL IgG (all were with progressiveform), 1 LA ( in SHL). In the control group, 2 FVLeiden and 3 FII A20210 carriers were recorded,where 6 of them showed homocysteine above the97.5 centile. Three controls showed border-line titreof aCL IgG, none the LA. Overall, OR for the presenceof inherited causes of thrombophilia was 2.6 ( 95%C.I. 0.9-7.6). Although further invetigation is needed,common inherited thrombophilia seems to beassociated with the occurrence of hearing loss.CO-047DOES THROMBOPHILIA PLAY A ROLE IN IDIOPATHIC SUDDENHEARING LOSS?Marcucci R, Leprini E,* Berloco P,* Bruschettini A,Socci M,* Attanasio M, Paniccia R,Alessandrello Liotta A, # Mannini L, # Rogolino A, #Abbate R, Prisco DDipartimento Area Critica Medico Chirurgica;°Dipartimento Scienze Oto-Neuro Oftalmologiche;#Dipartimento Cardiologico e dei vasi; AziendaOspedaliero-Universitaria Careggi, Florence, ItalyOver the last years there has been a significantincrease of sudden hearing loss (SHL) in Westerncountries with an incidence of 20 out of 100,000persons affected every year. However, no clear pathogeneticcauses for this disease have been found thusfar in patients in whom an infectious episode oracustic neurinoma have been excluded. Aim of thisstudy was to investigate a number of acquired andinherited thrombophilic risk factors (lipoprotein(a);fasting homocysteine; plasminogen activatorinhibitor-1; antiphospholipid antibodies; antithrombin,protein C and S; factor V Leiden, factor II polymorphism)in patients (pts) with idiopathic SHL. Weinvestigated 168 consecutive pts ( M/ F; age: 55 (19-79) yrs) with a diagnosis of idiopathic SHL within 30days from the onset of symptoms, and 168 controls(ctrl) ( M/ F; age 54 (19-78) yrs). In no pt a deficiencyof physiological clotting inhibitors antithrombin,protein C and protein S was found. No significantdifferences between pts and ctrl were observed forLp(a) plasma levels [116 (1-817) mg/L vs 102 (9-695)mg/l] and for the presence of FV Leiden (4.2% vs 4%)and factor II polymorphisms (3% vs 2.4%). In ptsfasting homocysteine levels were significantly (p athaematologica vol. 89(suppl. n. 8):september 2004

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XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 200445CO-049IDENTIFICATION OF 18 NOVEL MUTATIONS IN HEMOPHILIA ACastaman G, Giacomelli SH, Ghiotto R, Simioni M,Rodeghiero FDipartimento di Ematologia, Ospedale S. Bortolo,Vicenza, ItalyA wide range of different mutations has been identifiedin patients with Hemophilia A, providing thegenetic basis for the extensive variability of the phenotype.Due to its considerable size (7.2 kb of thecoding sequence, with 26 exons), mutation detectionin gene of FVIII represents a challenge that is onlypartially met by conventional screening methods (forexample DGGE and SSCP). Denaturing high performanceliquid chromatography (DHPLC) is a newmethod for a fast and sensitive analysis of PCRamplifiedDNA fragments. We have analysed all the26 exons (33 fragments) of the FVIII gene in 69hemophilia patients by DHPLC. Only those fragmentswith a different pattern from the wild type weresequenced. 18 novel mutations were detected, inparticular 11 missense, 1 nonsense, 2 small deletions,2 small insertions and 2 splicing mutations. Of the 6patients with mild phenotype 5 show missensemutations (953CTC→CCC Leu299Pro; 2102ATG→ACG Met682Thr; 5419AGC→GGC Ser1788Gly; 6685CTT>TTT Leu2210Phe), and only one showsa splicing mutation (IVS4 +5 G>A). Of the 7 patientswith moderate phenotype 6 have a missense mutation(1313ATT→ACT Ile419Tyr; 2132 TGC→TACCys692Tyr; 5260TTC→CTC Phe1735Leu; 6563TGT>TAT Cys2169Tyr) and one has a small insertionin exon 8 (1189 Ins C). Finally, of the 11 severe hemophiliacs5 show missense mutation (1332 AAA→AACLys425Asn; 1966TGG→GGG Trp637Gly; 5321CAT→CTT His1755Leu), 2 a splicing mutation (IVS9 +1G→C), 2 have small deletion, one in exon 4 (427-428Del GA) and the other in exon 14 (2216 Del A), onehave a small insertion in exon 17 (5676 Ins T) andone show a non sense mutation (368-369TCC→TGASer104Stop). Only six patients showed no evidencefor a causative mutations. In four of them a possibleVWD type 2N was suspected on the basis of laboratoryphenotype. In conclusion, DHPLC is a rapid andefficacious method to identify causative mutationsin Hemophilia A.CO-050PREVALENCE OF THE INVERSION OF INTRON 1 AT THE FVIIILOCUS IN ITALYSantacroce R,* Tagliabue L,° Santagostino E,°Peyvandi F,° Margaglione M*^Unità di Aterosclerosi e Trombosi, IRCCS CasaSollievo della Sofferenza, San Giovanni Rotondo(FG);° A. Bianchi Bonomi Hemophilia and ThrombosisCentre, Milan; ^Genetica Medica, Università diFoggia, ItalyRecently, an intrachromosomal recombination thataffects the intron 1 and disrupts the FVIII gene, leadingto severe Hemophilia A has been reported in4.8% and in 5% of British and Spanish patients,respectively. To calculate the frequency of the mutation,we have investigated a large setting of Italianpatients (n=219) with severe Hemophilia A (residualactivity

46Oral CommunicationsCO-051DESMOPRESSIN SUBCUTANEOUS ADMINISTRATION IMPROVESPLATELET RESPONSE IN VIVO TO PLATELET-ACTIVATINGFACTOR, SOLUBLE P-SELECTIN RELEASE AND CAUSES A RISEIN INTERLEUKIN-6 PLASMA LEVELS IN HAEMOPHILIA A ANDVON WILLEBRAND DISEASE TYPE IMusso R, Cultrera D,* Chiarenza A,* Cipolla N,Burgio N, D'Arpa S, Giustolisi R*Haemophilia and Thrombosis Regional ReferenceCenter, *Dept. of Haematology, University ofCatania, ItalyIt has been reported that elevated plasma plateletactivatingfactor (PAF) concentrations are significantlyhigher in children with haemophilia A (HA)and Von Willebrand disease (VWD) than in healthychildren (Kavakli K. Thromb Haemost 1999; 81). It alsobeen also postulated that increased cellular PAFactivity may be an adaptative response by the organismin order to prevent hemorrhages and assist primaryhaemostasis (Kavakli K. Haemophilia 2001; 7).In conjunction with Von Willebrand Factor (VWF),the adhesion molecule P-selectin, stored in endothelialWeibel-Palade bodies and platelet α-granulemembrane, is suggested to regulate platelet biologyin VWD. In addition, it has been shown desmopressinto cause a rise in plasma interleukin-6 (IL-6) levels,which correlates with an increase of VWF and FactorVIII (FVIII:C)(Newby. Thromb Haemost 2000; 84).We here report that in vitro PAF at different concentrationinduced a marked increase of platelet aggregation(PA) in mild HA patients (n=14) and in VWDtype 1 subjects known to be responders to thedesmopressin (n=14, 6 males and 8 females). PA wasperformed in platelet-rich plasma (PRP) at250,000/mL cells count by using the Aggrecoder PA3210 in presence of subcritical (0.2 mM) and optimal(1 mM) concentrations of PAF acether (C18:0). PAwas performed before and 1 h after subcutaneousdesmopressin (Emosint. Sclavo) injection (0.4 mg/Kg).aPTT, FVIII:C, VW:Ag, RiCof, soluble P-selectin andIL-6 (ELISA method) were also measured. As expected,all FVIII complex plasma activities were significantly(p

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 200447charged polar side chain. Therefore, we speculatedthat the substitution might lead to an abnormalthree-dimensional conformation of the FVII molecule,which would have a significant impact on theprotein structure. In transfected cells, expression ofthe Arg110Cys mutation showed a reduction of theamount of intracellular and secreted FVII protein(48% and 18%, respectively). Likewise, cells transfectedwith the Asp123Tyr mutation gave rise to lowintracellular (40%) and extracellular (4%) FVII antigenlevels. In conditioned media, FVII procoagulantactivity was accordingly reduced (10% and

48Oral Communicationssuring the pKa values of the ionizable groups affectingthe catalytic cycle of the enzyme. Although protonsglobally affected the amidase activity of bothWT and DK9 thrombin in a similar way, the calculatedvalues of the pKa values were found significantlydifferent. His57 showed in fact in the free enzymea pKa value of 7.53 in the mutant, and 6.86 in WTthrombin. Likewise, the pKa of the N-terminus groupin the mutant undergoes a moderate pKa increasefrom 8.45 to 8.87 in the free enzyme and from 9.04to 9.36 in the substrate-bound form, for WT andmutant thrombin, respectively. The present findingssuggested that Lys9 deletion allosterically affectedthe protonation equilibrium of the active site His57,enhancing its affinity for protons and affecting thecatalytic cycle of the enzyme and its functional propertiesin vivo.Oral CommunicationsINFLAMMATION & THROMBOSISCO-055PLATELET MIGRATE IN LUNG TISSUE IN RESPONSE TOALLERGEN, IN A MODEL OF ALLERGIC INFLAMMATION IN MICEPitchford SC, Momi S,* Page C, Gresele PDepartment of Internal Medicine. Section ofInternal and Cardiovascular Medicine, University ofPerugia. Italy; *Sackler Institute, King’s College,London, UKPlatelets are necessary for airway wall remodelingin a murine model of allergic inflammation, and arerecovered in the lavage fluid of allergen-sensitizedand -challenged animals, implying that platelets canmigrate into tissue. The chemotactic response ofplatelets from allergen-sensitized mice was studiedin vitro and their presence in lung tissue in vivo. MaleC57BL/6 mice were immunised with ovalbumin(OVA) and lungs removed for histology at varioustime points. From other groups of immunized mice,blood was collected by cardiac puncture and theobtained platelet suspensions were placed in the topwells of modified Boyden chambers, separated fromthe bottom wells containing OVA or BSA (1 µg/mL)by a 3 µm micropore filter, to study platelet migrationin response to the sensitizing or an aspecificallergen. Some platelet preparations were incubatedin the presence of an antimouse IgE antibody(R35-92) or control antibody. After incubation, thefilters were washed with saline and plateletsimmunostained for the CD41 antigen (SantaCruzBiotech). The number of platelets per field wasrecorded at a depth of 0-249;m below the filter surface.6 fields were counted for each filter using ax25 objective. Data are expressed as means SEM(platelets/m 2 ). Significant migration occurredthrough the filter with OVA sensitised PRP/ OVA(bottom well) (384.4;40.5) compared to OVA sensitisedPRP/ BSA (31.7;9.3 p

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 200449inflammatory stimulus in vivo and in vitro, a phenomenonprobably related to the interaction of IgEwith platelets.CO-0564G/5G POLYMORPHISM OF PAI-1 GENE AS A RISK FACTOR FORTHE DEVELOPMENT OF IgE-MEDIATED ASTHMAPampuch A,*° Di Castelnuovo A,* Kowal K,°Bodzenta-Lukaszyk A,° Donati MB,* Iacoviello L**Center for High Technology Research andEducation in Biomedical Sciences,CatholicUniversity, Campobasso, Italy and °Department ofAllergology and Internal Medicine, Bialystok, PolandBackground. Bronchial asthma is a complex diseaseinvolving genetic an environmental risk. Theinflammatory process observed in the course ofbronchial asthma may affect the fibrynolysis system.Plasminogen activator inhibitor-1 (PAI-1) plays anessential role in tissue remodeling and regulation ofthe fibrynolytic pathway. PAI-1 gene is an importantfactor in the regulation of plasma levels of PAI-1 inresponse to different stimuli and can be consideredas a candidate in the genetic predisposition to developasthma. Aims. In the present study we investigatedwhether the risk of asthma could be geneticallydetermined by PAI-1 polymorhism and PAI-1genotypes relationships with PAI-1 antigen and IgElevels. Methods. This study was performed in 127house dust mite sensitive allergic asthmatics, 73allergic rhinitis patients. Control group consisted of89 healthy nonatopic subjects. PAI-1 genotype wasdetermined by an allele specific PCR. Plasma PAI-1antigen, total IgE and specific IgE concentration wereevaluated using ELISA and UniCap system respectivly.Bronchial challenge tests with histamine and Dermatphagoidespteronyssinus(Dp) allergen were performedaccording to the methods of Ryan and Cockroftrespectively. Results. The frequency of 4G allelewas significantly higher in allergic asthma patients(0.688 95%CI 0.62-0.75) than in healthy controls(0.55 95%CI 0.477-0.623; p = 0.003) and in allergicrhinitis patients (0.554 95% CI 0.474-0.635; p =0.007). In univariate analysis, PAI-1 polymorphismwas associated with an increased risk of asthma;odds ratio (OR) for 4G/5G genotype compared withthe 5G/5G was 1.8 (95%CI 0.77-4.19), while comparisonbetween 5G/5G and 4G/4G genotypesshowed an OR of 2.55 (95%CI 1.10-5.93). Afteradjustment for sex and age, the association was evenstronger [(OR for 4G/5G (2.59; 1.03-6.50) and for4G/4G (2.80; 1.13-6.98)]. IgE levels were associatedwith PAI-1 genotype in cases. Indeed, cases carryingthe genotype 4G/4G showed significantly higher levelsof IgE (547.1±341 kU/L) than 5G/5G homozygotes(318.9±310.5 kU/L; p = 0.0128) Preliminarydata on PAI-1 antigen show that in both controlsand cases, 4G/4G genotype was associated withhigher levels of PAI-1. Both nonspecific bronchialreactivity in response to histamine challenge andspecific bronchial reactivity in response to Dp challengewere higher in homozygotes for 4G/4G allelethan in homozygos for 5G/5G allele. Morning plasmaPAI-1 concentration correlated significantly withPC20(r=-0,3913; p=0,0001) and with total serumIgE(r=0,3037; p=0,0018). In patients challenged withDp allergen significant increase in plasma PAI-1 antigenconcentration was seen at EAR, which continuedto be increased even 24 hours after the challenge inthose who developed a LAR. Conclusion. The resultsobtained in this study support the hypothesis thatthe 4G/4G PAI-1 genotype is associated with anincreased risk of allergic asthma and bronchialhyperreactivity.CO-057PPAR α AND γ ACTIVATORS DIFFERENTLY AFFECT THEINFLAMMATORY RESPONSE BY HUMAN VASCULARENDOTHELIUM. POSSIBLE ANTI-ANGIOGENIC EFFECTS OFROSIGLITAZONEMassaro M, # *Scoditti E,* Zampolli A,*Carluccio MA,* Storelli C, # Distante A,*De Caterina R°*#University of Lecce, Lecce, Italy, *C.N.R. Institute ofClinical Physiology, Pisa and Lecce, Italy,°d’ Annunzio University, Chieti, ItalyPeroxisome proliferator-activated receptors (PPA-Rα and γ) are highly expressed in the atheroscleroticintima, and likely involved in inflammation, cellularproliferation and differentiation. Both PPARs mayregulate the expression of cyclooxygenase(COX)-2,which was found increased in atherosclerotic lesions,colocalizing with matrix metalloproteinase(MMP)-9in the endothelium of vasa vasorum, thus probablyfavoring plaque neo-vascularization, progression andinstability. Because of the involvement of a prostaglandin(PG)E/cAMP-dependent pathway in MMP-9 activation, we investigated the effects of thePPARgamma agonist rosiglitazone (0.1-50 mmol/Land of the PPARα agonist clofibrate (1-500 mmol/L),on the stimulated expression of COX-2 and MMP-9protein and activity in human umbilical veinendothelial cells (HUVEC) stimulated by phorbolmyristate acetate (PMA). Results. In the absence ofany toxicity, rosiglitazone, at ⁄10 mmol/L, but notclofibrate, significantly (*p

50Oral Communicationsthe Table, as% of stimulated controls (PMA). Theinhibitory effect by rosiglitazone was completelyreverted by the PPARγ antagonist bisphenol A diglycydilether (BADGE; 50 mmol/L) which, in the sameexperimental conditions, did not affect the PMAmediatedinduction of COX-2. In parallel, only rosiglitazonereduced the release of MMP-9 without affectingthe activity of other MMPs investigated (MMP-1and 2 and 3) at gelatin zimography and enzymeimmunoassay. Rosiglitazone reduces the inducedexpression of COX-2 as well as the stimulated releaseof MMP-9 in HUVEC. This effect occurs through aPPARγ-dependent mechanism since it is completelyreverted in the presence of BADGE. These antiinflammatoryand anti-angiogenic effects may contributeto vasculoprotection by PPARγ agonists invivo.CO-058PREVALENCE OF INFLAMMATORY GENE POLYMORPHISMS INNONVALVULAR ATRIAL FIBRILLATION PATIENTSGori AM, Gensini F, # Cecchi E, Innocenti D, Poli D,°Sticchi E, # Michelucci A, Padeletti L, Porciani MC,Prisco D, Abbate R, Gensini GFDepartment of Medical and Surgical Critical Area,University of Florence; °Dipartimento Cardiologicoe dei Vasi, Azienda Ospedaliera Universitaria, Florence;# Department of Clinical Pathophysiology,University of Florence, ItalyNonvalvular atrial fibrillation (NVAF) is the mostcommon arrhythmia in clinical practice and is apotential cause of thromboembolic events. Recently,the involvement of inflammatory processes inNVAF has been suggested, but no data are availableon genetic polymorphisms of the inflammatorymarkers in NVAF. We investigated the prevalence ofC-Reactive Protein (CRP) 1059G/C polymorphism,Interleukin 6 (IL-6) -174G/C promoter polymorphism,and Interleukin 1β (IL-1β) -511 C/T promoter polymorphismin 195 patients with AF (mean age 73±8;107 males and 88 females) and in 390 apparentlyhealthy subjects. One hundred and six NVAF patientshad history of ischemic event(s): ischemic stroke(n=67), and transient ischemic attack (TIA) (n=39). In89 NVAF patients no history of arterial ischemia wasregistered. CRP 1059G/C, IL-6 -174G/C and IL-1β -511 C/T polymorphisms were analyzed by PCR and bymicroarray technology using electronic chip (Nanogentechnology). Both in patients with NVAF and incontrol subjects the analyzed genotype distributionswere in Hardy-Weinberg equilibrium. The analysis ofCRP 1059G/C and IL-6 -174G/C polymorphismsshowed no significant differences in genotype distributionbetween NVAF patients and control subjects.The prevalence of IL-1Beta -511 TT and CTgenotypes in NVAF patients was lower (62.0%) butnot significantly (p=0.051) different than that foundin control subjects (69.2%). No significance differencesin genotype distribution of CRP 1059G/C, IL-6-174 G/C and IL-1β -511 C/T polymorphismsbetween NVAF patients with history of ischemicevents and NVAF patients without history of arterialischemia were observed. Similarly, the genotypedistribution of the inflammatory gene polymorphismsdid not differ between patients with chronic(n=142) or paroxysmal (n=53) NVAF. The percentageof AF patients with more than two rare variantsof inflammatory genes was significantly (p

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 200451and CD, EPCR staining was less intense on theendothelium and the dendritic cells expressing EPCRwere less numerous. TM was present on the endotheliumof the vessels only, and the intensity of colouringwas lower in IBD than in controls. Immunofluorescencedetected 23.8±10.4 CD21 + /EPCR + dendriticcells per high power field in controls, 12.0±3.6(p=0.03) in UC and 19.0±5.6 in CD. IBD had lower levelsof EPCR mRNA (RT-PCR) and higher levels of solublereceptors than controls. In conclusion, since EPCRexpression is present on dendritic CD21-positive cellsin normal intestinal mucosa which are decreased inIBD, we hypothesize that EPCR might have a role inmodulating innate immune responses in the intestine.Downregulation at the protein and mRNA levelof EPCR and TM in the vessels of IBD mucosa maypredispose to thrombosis, contributing to mucosalischemia.CO-060RISK FACTORS FOR THROMBOSIS IN PATIENTS WITH THENEPHROTIC SYNDROMEPodda GM, 2 Lussana F, 1 Moroni G, 3 Zighetti Ml, 1-2Faioni EM, 1 Ponticelli C, 3 Cattaneo M 11Unità di Ematologia e Trombosi, Ospedale San Paolo,DMCO, Università di Milano; 2 CentroEmofilia e Trombosi Angelo Bianchi Bonomi IRCCSOspedale Maggiore, Departimento di MedicinaInterna, Università di Milano; 3 Divisione diNefrologia Ospedale Maggiore IRCCS, Milan, ItalyArterial and venous thrombosis are frequent complicationsin patients with the nephrotic syndrome(NS). Although some abnormalities of the hemostaticsystem have been described, the causes for thehigh thrombotic risk in these patients are stillunclear. We investigated the prevalence of somewell-established thrombosis risk factors in patientswith NS. We enrolled 84 patients with NS and 84age- and gender-matched healthy subjects. The followingvariables were investigated: plasma levels oftotal homocysteine (tHcy), protein C, protein S,antithrombin, fibrinogen, factor VIII, vitamin B6 andserum levels of creatinine, albumin, folate, vitaminB12, cholesterol; anticoagulant response to activatedprotein C (APC); glomerular filtration rate (GFR).tHcy, creatinine, factor VIII, fibrinogen, protein C,protein S, cholesterol were significantly higher, whilethe anticoagulant response to APC, GFR, serumfolates, albumin and plasma vitamin B6 were significantlylower in NS patients than in controls. In NSpatients, the anticoagulant response to APC was correlatedwith factor VIII levels, while tHcy correlatednegatively with GFR, albumin and folate. In order torule out the influence of renal insufficiency on thestudied parameters, we restricted our analysis tosubjects with normal GFR (≥ 75ml/min): inthis subgroup of subjects, vitamin B6 and folate levelswere significantly lower and factor VIII, fibrinogen,protein C, cholesterol, triglycerides levels higherin NS patients (n=23) than in controls (n=62).Therefore, in agreement with previous reports, wefound that several thrombosis risk factors are commonlyfound in patients with NS. Some of them areindependent of renal insufficiency, which is a commoncomplication of NS. Among them, low plasmalevels of vitamin B6 have recently been shown to beassociated with a heightened risk for both arterialand venous thrombosis.CO-061MARKERS OF INFLAMMATION, D-DIMER AND RECURRENCESAFTER A FIRST EPISODE OF VENOUS THROMBOEMBOLISMCosmi B, Legnani C, Guazzaloca G, Pili C, Conti E,Palareti GDept. of Angiology & Blood Coagulation “MarinoGolinelli”, University Hospital S. Orsola-Malpighi,Bologna, ItalyIntroduction: D-Dimer (D-d) has been shown tohave a high negative predictive value for recurrenceafter oral anticoagulant therapy (OAT) withdrawalfor a first episode of venous thromboembolism (VTE).Elevated D-d may express a chronic hypercoagulablestate induced by mediators of inflammation such ascytokines. Aim of the study: to determine whethermarkers of inflammation are increased in patientswith recurrent VTE and whether they correlate withD-d after OAT withdrawal for a first episode of VTE.Materials and methods: Symptomatic outpatientswith a first episode of venous thromboembolism(deep vein thrombosis of the lower limbs DVT and/ orpulmonary embolism-PE) were prospectively followed-upafter withdrawal of OAT. Plasma concentrationsof interleukin 6 (IL-6), interleukin 8 (IL-8),monocyte chemotactic protein 1 (MCP-1), C-reactiveprotein (C-RP) (Turbiquant, Dade Behring, MilanItaly) and D-d (Vidas D-Dimer, BioMerieux, Rome,Italy) were measured at the time of OAT withdrawal(T0) and 3 months afterwards (T3). IL-6, IL-8 andMCP-1 were measured with commercial ELISA methods.Recurrent events were diagnosed in case ofsymptoms of DVT or PE by objective methods duringa 2-year follow-up. Results: 207 patients wereincluded in the study (M/F 109/98; mean age:66; agerange:23-93). Total follow-up was 240 patient/years.Recurrent events were recorded in 14 patients (7%).IL-8 and MCP-1 did not differ significantly either atT0 or T3 between patients with a recurrence andpatients without recurrence. D-d, IL-6 and C-RP lev-haematologica vol. 89(suppl. n. 8):september 2004

52Oral Communicationsels increased significantly at T3 when compared toT0 in patients with recurrences when compared topatients without recurrences. C-RP and IL-6 correlatedsignificantly with D-D at T3 in patients whoexperienced a recurrent event (r = 0.50). Conclusions:Our data indicate that C-RP and IL-6 correlatewith increased D-Dimer levels and may be a risk factorsfor recurrence after a first episode of VTE.SISET PrizeCO-062INCREASED SOLUBLE CD40 LIGAND LEVELS IN CYSTICFIBROSISFalco A,* Romano M,* Iapichino L, Collura M,Davì G**Center of Excellence on Aging, University of Chieti"G. d' Annunzio", Chieti; Cystic Fibrosis Center, DiCristina Hospital, Palermo, ItalyChronic inflammation represents a key pathogeneticevent in the progression of lung disease incystic fibrosis (CF). To identify novel mechanisms ofthe inflammatory reaction in CF and analyze its relationwith coagulative activation, we carried out across-sectional study to evaluate circulating levels ofthe inflammatory mediators soluble (s) CD40L, C-reactive protein (CRP), interleukin (IL)-1β, the coagulationmarkers activated factor VII (FVIIa) and prothrombinfragment (F) 1+2, as well as urinary 11-dehydro-thromboxane (TX)B2, an index of in vivoplatelet activation, in 34 CF patients and 34 matchedhealthy subjects. We observed that CF patients displayedsignificantly increased circulating levels ofsCD40L compared to controls [2.8 (0.4-15.6) vs 1.1(0.2-2.7) ng/mL, p = 0.0003]. sCD40L levels inverselycorrelated with forced expiratory volume at 1 second(FEV1) (ρ= -0.788, p = 0.0001), whereas it directlycorrelated with CRP and IL-1β levels (ρ =0.621, p=0.0004; and ρ = 0.745, p = 0.0001, respectively),which were also elevated in CF patients. CF patientshad also enhanced levels of FVIIa and F1+2 comparedto controls [39.2 (22.6-69.8) vs 22.3 (16.2-32.4)mU/mL, p = 0.0001; 0.60 (0.30-1.80) vs 0.17 (0.10-0.40) nmol/L, p = 0.0001, respectively]. A direct correlationwas observed between sCD40L and both plasmaFVIIa (rho = 0.691, p = 0.0001) and F1+2 (rho =0.545, P = 0.0017) as well as between sCD40L and urinary11-dehydro-TXB2 (rho = 0.433, p = 0.0129). Ourfindings suggest that in CF patients, sCD40L couldrepresent a biochemical link between the inflammatorystate, and endothelial damage and coagulativeactivation, leading to progressive impairment of pulmonaryfunction.Oral CommunicationsPLATELET FUNCTION INHIBITORSCO-063DETECTION OF A CRITICAL DEGREE OF PLATELETTXA2- PRODUCTION CAPACITY BY THE PFA100 INASPIRIN-TREATED SUBJECTS: POSSIBLE RELEVANCE FORTHE MONITORING OF ASPIRIN RESISTANCELeone M, Mezzasoma AM, Gresele PDepartment of Internal Medicine, Section ofInternal and Cardiovascular Medicine Universityof Perugia, ItalyAspirin resistance is attracting considerable attentionfor its potential role in recurrent ischemic eventsunder aspirin treatment. While the definition ofaspirin resistance is controversial, a widely held viewis that persistent production of platelet TxA2 is ahallmark of aspirin resistance. Early pharmacologicstudies have shown that ex vivo platelet TxA2 productioncapacity must be reduced by aspirin by~95% in order to exert in vivo antithrombotic effects(Reilly IA et al. Blood. 1987). Reliable methods toassess platelet thromboxane production (such asradioimmunoassay of serum TxB2 or the measurementof urinary Tx A2 metabolites) are cumbersomeand time-consuming. The Platelet Function Analyser100 (PFA100) can detect aspirin effects, but no systematicdata exist on the degree of inhibition ofplatelet TxA2 capacity that it can detect. Aim of ourstudy was to assess the threshold level of plateletTxA2 inhibition in vitro and ex vivo which can reliablybe detected by the PFA100. In vitro, citratedhuman whole blood samples were incubated withincreasing doses of the COX-1 inhibitors indomethacin,indobufen and aspirin and tested in thePFA100 (CEPI cartridges) and for thrombin-inducedmaximal TxB2 formation (by RIA). In vivo, two groupsof healthy volunteers (four each) were treated withaspirin at either 25 mg or 300 mg once a day for 5days and blood samples were taken at baseline andthen daily, 1 hour after morning aspirin intake, up today 5; serum TxB2 and PFA100 were assessed. In vitro,all the tested COX-inhibitors reduced dose dependentlyTxB2 formation, and the PFA100 CEPI wasprolonged to ⁄ 300 sec only when, and each timethat, platelet TxB2 production capacity was suppressedby ⁄97%. Ex vivo, only when a ⁄97% inhibitionof serum TxB2 was reached, prolongation to⁄300 sec of PFA100 was detected in all the volunteerstested. In conclusion, the PFA100 may theoreticallyhaematologica vol. 89(suppl. n. 8):september 2004

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 200453be used for the rapid detection of adequate in vivoinhibition of platelet TxA2 by aspirin. Only prospectiverandomised trials correlating PFA100 resultswith long-term incidence of events, on one hand,and the evaluation of whether PFA100-based adjustmentof aspirin dosage affects the rate of success oftreatment, on the other, can validate the use ofPFA100 for the detection of aspirin resistance.CO-064COLLAGEN-INDUCED PLATELET THROMBOXANE A2PRODUCTION IN PATIENTS CHRONICALLY TREATED WITHASPIRINPulcinelli FM, Riondino S, Celestini A, Pignatelli P,Trifirò E, Di Renzo L, Violi FDipartimento di Medicina Sperimentale e Patologia,Università degli Studi di Roma "La Sapienza", ItalyA certain number of patients chronically treatedwith aspirin shows a reduced sensitivity to itsantiplatelet effect. Aim of the present study was toverify whether such a reduced sensitivity dependson a persistent thromboxane A2 (TxA2) formationand whether this production is caused by an incompleteinhibition of cyclooxygenase (COX)-1, or by thepresence of COX-2. We enrolled 196 patients takingaspirin (100-330 mg/day) in whom collagen-inducedplatelet aggregation and thromboxane A2 productionwere measured. In a subgroup of 96 patientsplatelet aggregation and TxA2 production were alsoinvestigated after in vitro treatment with a TxA2receptor antagonist (13-Azaprostanoic acid), aspirinor a COX-2 inhibitor (NS-398). Results: Patients weredivided into quartiles defined by the distribution ofthe TxA2 values. Platelet still produced TxA2 inresponse to collagen (128.7±21.6 pg/10 8 cells); TxA2production is significantly correlated with plateletaggregation (Spearman correlation coefficient=0.44; p

54Oral CommunicationsCO-066NITROPRAVASTATIN (NCX 6550), A NITRIC OXIDE-RELEASINGSTATIN WITH POTENT ANTIPLATELET/ANTITHROMBOTICACTIVITYMomi S, 1 Rossiello MR, 2 Guglielmini G, 1Caracchini R, 1 Monopoli A, 3 Ongini E, 3 Semeraro N, 2Colucci M, 2 Gresele P 11Department of Internal Medicine, Sect. of Internaland Cardiovascular Medicine, University of Perugia,Perugia; 2 Department of Biomedical Sciences, Sect.of General Pathology, University of Bari, Bari; 3 NicOxResearch Institute, Milan, ItalyStatins decrease ischemic cardiovascular eventsboth in hypercholesterolemic and normocholesterolemicsubjects but with a slow onset of action.Recently a new class of drugs combining a statinwith a NO-donating moiety has been described. Aimof our study was to evaluate the in vitro and in vivoeffects of the novel NO-releasing pravastatin (NCX6550) on a number of cellular functions relevant toatherothrombosis. In vitro, NCX6550, but not pravastatin,inhibited dose-dependently: 1) U46619-induced human platelet aggregation in buffer andplasma; 2) collagen-stimulated platelet P-selectinexpression in human whole blood; 3) platelet adhesionto collagen-coated coverslips under high wallshear rate conditions. These effects were associatedwith an enhancement of intraplatelet cGMP andwere abolished by the guanylyl cyclase inhibitor ODQ(10uM). NCX6550 also inhibited dose-dependentlytissue factor (TF) expression in human bloodmononuclear cells exposed to LPS or PMA (85% inhibitionat 50 µM) as assessed by TF antigen and activitydeterminations and by RT-PCR. However, the inhibitionof TF by NCX6550 was not influenced by ODQnor by mevalonolactone (400 uM) or geranyl-geranyl-pyrophosphate(5 uM). Neither pravastatin nora NO-donor (SNP, 10 uM) reduced TF expressionunder our experimental conditions. Treatment withNCX6550 (48 mg/kg i.p.), but not with equimolarpravastatin, significantly reduced mortality (from80% to 37%, p

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 200455CO-068STUDY OF THE SPECIFICITY OF THE NOVEL SURAMINANALOGUE (NF449) AS A P2X1 RECEPTOR ANTAGONISTZighetti ML, 1,2 Ullmann H, 3 Lecchi A, 2 Meis S, 3Kassack MU, 3 Cattaneo M 11Unità di Ematologia e Trombosi, Ospedale San Paolo,Dipartimento di Medicina Chirurgia e Odontoiatria,Università di Milano; 2 Centro Emofilia e TrombosiAngelo Bianchi Bonomi, IRCCS Ospedale Maggiore,Dipartimento di Medicina Interna, Universitàdi Milano; 3 Pharmaceutical Institute, University ofBonn, GermanyIt has been shown that P2X1 plays an importantrole in platelet aggregation and platelet thrombusformation under high shear conditions, making it anattractive molecular target for antithrombotic intervention.In this study, we further characterized anovel suramin analogue, 4,4,4 (carbonylbis(imino-5,1,3-benzenetriyl-bis(carbonylimino)))tetrakis-benzene-1,3-disulfonicacid (NF449), a compound ofproven antagonistic activity (IC50=0.05 nM) on P2X1recombinantly expressed in Xenopus oocytes. Study#1. NF 449 was examined at P2Y1, P2Y2, and P2Y11receptors recombinantly expressed in 1321N1 astrocytomacells using as agonists 2-methylthio-ADP forP2Y1, UTP for P2Y2, and ATPgS for P2Y11, respectively.The effect of NF449 on these receptors wasestimated by measuring the inhibition of agonistinducedincrease in intracellular Ca 2+ . NF449 had noeffect at P2Y1 and P2Y2 up to a concentration of 3.2mM. P2Y11 receptors were inhibited by NF449 withan IC50 of ~180 µM. Study #2. The effect of NF449on platelet function was studied in normal humanplatelet-rich plasma (PRP) anticoagulated withPPACK, in the presence of apyrase (0.5 U/mL), to preventP2X1 desensitization. Inhibition of the P2X1-dependent platelet shape change induced by 1 and10 µM α,β,methylene-ATP was complete at NF449concentrations of 10 -5 M and 10 -4 M, respectively.Only at the highest concentration tested (10 -4 M),NF449 partially inhibited ADP (1-10 µM)-inducedplatelet aggregation, but had no effects on ADPinducedshape change or adnylyl cyclase inhibition.P2X1 desensitization with α,β,methylene-ATP hadno inhibitory effects on ADP-induced plateletresponses, indicating that the inhibitory effects ofNF449 on these platelet responses were not mediatedby P2X1 inhibition. NF449 (10 -5 and 10 -4 ) partiallyinhibited collagen (2 µg/mL)-induced plateletaggregation; similar effects were observed afterP2X1 desensitization with α,β,methylene-ATP. Inconclusion, NF449 proved a rather specific antagonistof P2X1 receptor recombinanlty expressed onastrocytoma cells. Its activity was less specific inplatelets.CO-069ANTIAGGREGATORY ACTIVITY IN HUMAN PLATELETS OFPOTENT ANTAGONISTS OF THE P2Y1 RECEPTORCattaneo M,*° Lecchi A,° Ohno M, # Joshi BV, #Besada P, # Tchilibon S, # Lombardi R,°Bischofberger N, § Harden TK, + Jacobson KAC*Hematology and Thrombosis Unit, Ospedale SanPaolo, DMCO-University of Milan, Italy. °Hemophiliaand Thrombosis Center, Dept. of Internal Medicine,IRCCS Ospedale Maggiore, University of Milan;#Molecular Recognition Section, Laboratory ofBioorganic Chemistry, NIDDK, National Institutes ofHealth, Bethesda, MD, USA, § Gilead Sciences, FosterCity, CA USA, and + Department of Pharmacology,University of North Carolina, School of Medicine,Chapel Hill, North Carolina, USAActivation of the P2Y1 nucleotide receptor inplatelets by ADP causes changes in shape and aggregation,mediated by activation of phospholipase C(PLC). Recently, MRS2500 (2-iodo-N(e)6-methyl-(N)-methanocarba-2’-deoxyadenosine-3’,5’-bisphosphate)was introduced as a highly potent and selectiveantagonist for this receptor. We have studiedthe actions of MRS2500 in human platelets andcompared these effects with the effects of twoacyclic nucleotide analogues, a bisphosphateMRS2298 and a bisphosphonate derivative MRS2496, which act as P2Y1 receptor antagonists,although less potently than MRS2500. Improvedsynthetic methods for MRS2500 and MRS2496 weredevised. The bisphosphonate is predicted to be morestable in general in biological systems than phosphateantagonists due to the non-hydrolyzable C-Pbond. MRS2500 inhibited the ADP-induced aggregationof human platelets with an IC50 value of 0.95nM. MRS2298 and MRS2496 also both inhibited theADP-induced aggregation of human platelets withIC50 values of 62.8 nM and 1.5 µM, respectively. Asimilar order of potency was observed for the threeantagonists in binding to the recombinant humanP2Y1 receptor and in inhibition of ADP-inducedshape change and ADP-induced rise in intracellularCa 2+ . No substantial antagonism of the pathwaylinked to the inhibition of cyclic AMP was observedfor the nucleotide derivatives, indicating no interactionof these three P2Y1 receptor antagonists withthe proaggregatory P2Y12 receptor, which is alsoactivated by ADP.haematologica vol. 89(suppl. n. 8):september 2004

56Oral CommunicationsCO-070NORMAL FUNCTION OF THE PLATELET P2X1 RECEPTOR INPATIENTS WITH UNEXPLAINED BLEEDING DIATHESISZighetti ML, 1,2 Podda G, 2 Lombardi R, 2Terranova L, 1,2 Cattaneo M 11Unità di Ematologia e Trombosi, Ospedale SanPaolo, Dipartimento di Medicina Chirurgia e Odontoiatria,Università di Milano; 2 Centro Emofilia eTrombosi Angelo Bianchi Bonomi, IRCCS OspedaleMaggiore, Dipartimento di Medicina Interna,Università di Milano, ItalyPlatelets express the P2X1 receptor, a ligand-gatedcation channel, which mediates a rapid ATPinducedCa 2+ influx. Due to the fast desensitizingproperty of P2X1 and because platelet studies havemainly been performed at low extracellular Ca 2+ concentrations,only recently the important role of thereceptor in platelet function at high shear has beenunravelled. Due to the above-mentioned methodologicalproblems, no systematic search for P2X1defects has so far been performed in patients withbleeding diathesis. We studied P2X1-dependentplatelet response in 3 groups of subjects: 1) 16healthy subjects; 2) 39 consecutive patients withbleeding diathesis and normal coagulation tests andplasma von Willebrand factor levels, who had beenreferred to our Center to be screened for plateletfunction disorders; 3) 15 historical patients withcongenital bleeding diathesis, in whom no knownabnormalities of the hemostatic system had beendiagnosed. P2X1-dependent platelet shape changestimulated by α,β-methylene-ATP in PPACK-anticoagulatedplatelet-rich plasma (PRP) was studied after45 min incubation at 21°C with 0.5 U/mL apyrase,added to restore P2X1 function after its desensitisationthat occurred during the preparation of PRP. Theamplitude of shape change in platelets from healthyvolunteers induced by 1 and 10 µM α,β-methylene-ATP was 9±4 and 6±1 mm, respectively. None of the15 historical patients with bleeding diathesis ofunknown cause displayed an abnormal α,β-methylene-ATPinduced platelet shape change. Of the 39consecutive patients referred to be screened forabnormalities of platelet function, 5 had PrimarySecretion Defects, 1 δ-Storage Pool Deficiency and2 Glanzmann’s Thrombasthenia, but none of themhad an abnormal platelet response to α, β-methylene-ATP.In conclusion, none of our well characterizedpatients with bleeding diathesis displayedabnormalities of the P2X1-dependent platelet shapechange induced by α,β-methylene-ATP, suggestingthat P2X1 defects are probably uncommon amongpatients with bleeding diathesis.Oral CommunicationsTHROMBOPHILIA & GENETIC POLYMORPHISMSSISET PrizeCO-071THE FACTOR VIII ASP1241GLU POLYMORPHISM AND RISK OFVENOUS THROMBOSISScanavini D,* Legnani C,° Lunghi B,* Cini M,°Mingozzi F,* Gatti S,* Grassilli S,* Palareti G,°Bernardi F**Dipartimento di Biochimica e Biologia Molecolare,Università di Ferrara; ° Dipartimento di Angiologia,Unità di Ricerca Clinica sulla Trombofilia "MarinoGolinelli”, Ospedale Universitario S. Orsola-Malpighi, Bologna, ItalyElevated levels of Factor VIII (FVIII) are a recognizedrisk factor for venous thrombosis. Althoughsubstantial genetic heritability of FVIII levels hasbeen reported, no variation in the FVIII gene has beenassociated with high FVIII levels. Moreover, in an animalmodel it has been shown that inhibition of FVI-II offers protection from venous thromboembolism.Recently the G allele of a common polymorphism inthe FVIII B domain (3951C/G, Asp1241Glu) was foundto be associated to lower FVIII activity in family studies.We investigated by MnlI restriction of exon 14PCR products whether the Asp1241Glu polymorphismwas associated with lower risk of venousthrombosis in women. Unrelated cases (n=132,median age: 36 y; range: 19-50 y) were randomlyselected among consecutive patients referred forinvestigation of thrombophilic states after experiencinga single episode of deep venous thrombosisof a lower limb during reproductive age. As controls(n=132, median age: 36 y; range: 15-51 y), weselected unrelated pre-menopausal healthy womenfrom the general population usually referred forthrombophilic screening before pill treatment. TheFVIII:C was found to be significantly higher amongthe thrombotic patients compared with the asymptomaticsubjects both for CC (p

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 200457compared with cases (16.6%), indicating thatthe1241Glu allele does not reduce thrombotic risk.This study suggests that in thrombotic disease theFVIII-lowering effects of the 1241Glu allele are counterbalancedby extragenic and/or environmentalcomponents with a major role in determining highFVIII levels.CO-072ROLE OF GLUCOSYLCERAMIDE IN ANTICOAGULANT REACTIONSAND SEARCH FOR GLUCOSYLCERAMIDE SYNTHASEPOLYMORPHISMSFaioni EM, 1 Fontana G, 1 Carpani G, 2 Cattaneo M 11Unità di Ematologia e Trombosi-DMCO; 2 CentroTrasfusionale, Az. Ospedaliera San Paolo eUniversità degli Studi di Milano, ItalyLow levels of circulating glucosylceramide, asphingolipid, have been associated with an increasedrisk of venous thromboembolism. Lipid vesicles containingglucosylceramide sustain factor Va inactivationbetter than those without. The origin of circulatingglucosylceramide is unknown. We searchedfor polymorphisms in the gene that codes for theenzyme catalyzing the conversion of ceramide to Glc,glucosylceramide synthase, which might explain thelow glucosylceramide levels found in some individualswith thrombosis. Fifty patients with a previousfirst venous thromboembolic event (median age 40,range 18-68, 20 men and 30 women) for whom DNAwas available were subjected to glucosylceramidesynthase genetic screening. Nine exons and theintron/exon boundaries of the Glc-syn gene wereamplified and sequenced. In parallel, we developedan APTT kaolin-activated assay to test the effect ofliposomes containing different lipid mixtures in sustainingactivated protein C (APC)-mediated anticoagulantreactions in plasma. Plasma from 5 normalcontrol individuals was pooled for the coagulationstudies. Liposomes containing 70% phospatydilcoline(PC), and either phosphatydilserine alone orphosphatydilserine with variable concentrations ofglucosylceramide (5, 10, 20%) were prepared by mixingwith octylglucoside followed by dialysis. Results.Three intronic and 2 exonic, presumably neutral,polymorphisms were identified in the exons andintrons of the glucosylceramide synthase gene.Coagulation studies showed that the effect of glucosylceramidewas dependent on its concentration inthe liposomes and on the APC concentration. Athigher APC concentrations (>30 nmol/L) the clottingtimes (delta above baseline) were much shorter thanat lower APC concentrations in the presence of 20%Glc in the vesicles compared to no glucosylceramide.An intermediate response was observed at the sameAPC concentrations with 10% Glc. In conclusion, nopotentially significant polymorphisms were identifiedin the glucosylceramide synthase gene (64%screening completed). Glucosylceramide enhancesanticoagulant reactions in normal plasma in an APCand concentration-dependent manner.SISET PrizeCO-073RISK OF OCCURRENCE OF DEEP VEIN THROMBOSISAND PROTEIN Z DEFICIENCYSantacroce R,° Cappucci F, Di Perna P, Sessa F,°Colaizzo D, D'Andrea G, Grandone E,Margaglione M°Unita' di Aterosclerosi e Trombosi, I.R.C.C.S. "CasaSollievo della Sofferenza", S. Giovanni Rotondo;and Genetica Medica, Università di Foggia,° ItalyProtein Z (PZ) serves as a cofactor for FX inhibitionby protein Z-dependent protease inhibitor. In vivoand in vitro studies suggest that reduced PZ levelsmay play an important role in venous and arterialthrombosis. In patients with deep vein thrombosisand in age- and sex-matched controls, we haveinvestigated whether PZ levels and PZ gene variantsare associated with deep vein thrombosis. Protein Zplasma levels were evaluated by means of anenzyme-linked immunosorbant assay. Commonand/or sporadic PZ mutations were investigated bydirect cycle sequence analysis. A wide PZ range wasfound in controls (1.36+061 mg/mL) and in patients(1.65+1.36 mg/mL), levels being similar between thetwo settings. The prevalence of PZ levels below the2.5 percentile of controls (0.44 mg/mL) was higher inpatients (8.8%) than in controls (2%; OR: 4.7, 95%C.I.: 1.0-22.9). Among PZ gene variants investigated,a significant association with PZ levels was shownfor the intron A 103a and the intron F g79a polymorphisms.Moreover, a heterozygous missensemutation was found in two patients (Arg90Pro andLeu292Phe) with low PZ levels (0.39 and 0.24 mg/mL,respectively) and in a control (Tyr59Asn) withreduced PZ levels (0.67 mg/mL). In conclusion, resultsof the present investigation confirm the wide rangeof PZ plasma values and show that genetic factorswithin the PZ locus may explain, at least in part, thisinter-individual variability. Present data suggest anassociation between low PZ plasma levels and theoccurrence of deep vein thrombosis, PZ gene variantsstrongly contributing to this relationship.haematologica vol. 89(suppl. n. 8):september 2004

58Oral CommunicationsCO-074VENOUS THROMBOTIC RISK ASSESSMENT AMONG FACTOR VLEIDEN CARRIERS UNDERGONE A THROMBOPHILICSCREENING BY DIFFERENT TYPES OF INDICATIONSCastori L, Depunzio I, Servettini I, Ferrante F,Carloni D, Iorio ASezione di Medicina Interna e Cardiovascolare -Università di Perugia, ItalyFrom a cohort of patients undergoing a thrombophilicscreening since March 1998, we identified38 FV Leiden carriers (27 F:11M). FV wildtype controlswere matched by gender, age (46±16 yrs vs 47±15yrs), date of and indication for the thrombophilicscreening. Indications for the thrombophilic tests(ATIII, PC, PS, FVL, PT20210A, ACA, LAC, Hcy) were:first occurring or recurrent venous thromboembolism(VTE, 39.4%), first occurring or recurrent superficialvenous thrombosis (SVT 18.4%), retinal vein thrombosis(5.2%), cerebral venous thrombosis (2.6%),stroke (2.6%), familiarity for venous or arterialthrombosis (respectively 23.6%; 2.6%), oral contraceptives(2.6%) and spontaneous abortion (2.6%).Unprovoked or provoked VTE (respectively 78.5% and21.5%) and unprovoked or provoked SVT (40% and60%) were separately matched. After an average2.9±1.7 years follow up (3.04±1.7 yrs in the controls),8 SVT (4 provoked, 4 unprovoked) occurred inFVL carriers compared to 1 unprovoked VTE, 4 SVT (2provoked, 2 unprovoked) and 1 stroke in the controlgroup. In both groups we measured FVIII levels andd-dimer (Hemoliance factor VIII IL, d-dimer ElisaTechno Clone GmbH). During the follow up periodthe rate of recurrent SVT was significantly higher inFVL carriers with a 2 years, at least, previous VTE/SVTas an indication to the trombophilic screening comparedto FVL carriers undergoing the screening byother types of indications (RR 8.8 CI 95% 1.2&#8211; 62.5, p=0.013). No VTE occurred in FVL carrierscompared to 1 in the control group. Among FVLcarriers, patients with recurrent SVT presented significantlyhigher d-dimer levels (644±539 vs228±354, p=0.02) and an increased trend to higherfactor VIII levels (189±56 vs 146±48). On the contrary,independently of recurrence, FV wildtypepatients with VTE/SVT as screening indication presentedsignificantly higher factor VIII levels (320±54vs 160±97, p< 0.01), but similar D-dimer levels comparedto FVL wildtypes with other indications forthrombophilic screening.CO-075FACTOR V LEVELS, APC RESISTANCE AND THROMBOSIS RISKASSOCIATED WITH THE FACTOR V ASP79HIS (409 G/C)POLYMORPHISMLunghi B, 1 Scanavini D, 1 Grandini A, 1 Busani C, 1Martinelli N, 2 Girelli D, 2 Legnani C, 3 Palareti G, 3Bernardi F 11Department of Biochemistry and Molecular Biology,Ferrara University, Italy; 2 Department of Clinicaland Experimental Medicine, Verona University, Italy;3Department of Angiology, Unità Ricerca Clinicasulla Trombofilia “Marino Golinelli”, University HospitalS.Orsola-Malpighi, Bologna, ItalyFactor V (FV) genetic variation modulating FV levelsin plasma and risk of thrombosis could provideuseful markers in thrombophilia studies. The FVAsp79His polymorphism, located in exon 3 (A1domain), has been reported either to modulate FVlevels and APC resistance (APCR) in plasma or to representa neutral polymorphism. To address thisapparent discrepancy we investigated a large cohortof subjects, characterized for FV activity levels andFV genotype. We also evaluated the relationship ofthe Asp79His change with APCR and venous thrombosisin FV Leiden carriers. Genotyping of 1000 subjectsfor the Asp79His change revealed that carriershipof the 79His allele (n=121, 118 heterozygotes,three homozygotes) was significantly (ANOVA,p=0.048) associated to FV levels (127.7±42.5) lowerthan those in non-carriers (n=879, FV:C 135.1±38.5).In the doubly heterozygous condition with FV Leiden,the Asp79His change could produce, through a partialpseudohomozygous mechanism, a relativeincrease in plasma levels of APC resistant molecules.244 carriers of FV Leiden mutation characterized forAPC ratio were genotyped for this polymorphism. Noincrease of APCR levels in doubly heterozygotes forFV Leiden and Asp79His (n=30, APCR 0.57±0.07)respect to other FV Leiden carriers (n=214, APCR0.57±0.07) was observed. To investigate the associationof FV Asp79His polymorphism with venousthrombosis we genotyped 200 unrelated carriers ofFV Leiden mutation, who suffered thromboticepisodes (n=100, TV) or were still asymptomatic forvenous thromboembolism (n=100, AS). We foundvery similar genotypic frequencies (11% TV, 12% AS),which suggests that this polymorphism does notinfluence the thrombotic risk in FV Leiden carriers.Conclusion: Although carriership of the FV 79Hisallele predicts slightly reduced (5%) levels of FVcoagulant activity in plasma, we did not detect anyeffect on APC sensitivity and on the risk of venousthrombosis.haematologica vol. 89(suppl. n. 8):september 2004

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 200459CO-076THE PROTHROMBIN G20210A MUTATION ATTENUATES THEANTICOAGULANT BUT NOT THE FIBRINOLYTIC ACTIVITY OFACTIVATED PROTEIN CBinetti BM,* Asti D,° Tripodi A,° Semeraro N,*Colucci M**Dipartimento di Scienze Biomediche, Sezione diPatologia Generale, Università di Bari, °Centro diEmofilia e Trombosi A. Bianchi Bonomi, Dipartimentodi Medicina Interna, Università di Milano, ItalyHyperprothrombinemia associated with the prothrombingene mutation G20210A is a common riskfactor for thrombosis and has been reported to causeAPC resistance. The inhibition of thrombin formationby APC, however, not only limits fibrin formation butalso stimulates fibrinolysis by reducing TAFI (thrombinactivatable fibrinolysis inhibitor) activation. Weevaluated the influence of prothrombin G20210Amutation on the anticoagulant and fibrinolytic activitiesof APC. Thirty-two heterozygous carriers and 32non carriers were studied. APC anticoagulant activitywas assessed by aPTT prolongation whereas APCfibrinolytic activity was determined by a microplateclot lysis assay. APC-induced aPTT prolongation wasmarkedly less pronounced in carriers than in non carriers,resulting in APC sensitivity ratios of 2.11±0.41and 3.15±1.37, respectively (p

60Oral Communicationswith G20210A allele and 5G/5G polimorphism ofgene PAI-I (OR= 2.3, C.I. 0.87-6, p=0.09). Our resultsshow that 4G/4G genotype is a mild independent riskfactor for VTE but it may be of clinical impact whenassociated with prothrombin mutation. Moreover,we found the lack of interaction between G1691Amutation and 4G/5G polymorphism. Factor V Leiden,as independent risk, play alone a crucial role in determiningthrombotic events. On the other hand, themechanism by which the G20210A mutation canlead to a thrombotic event is still not well understood.Probably, it needs the concomitantly presenceof an hypofibrinolytic state, induced by 4G allele, toconfer patients an higher thrombotic risk. In conclusionwe think that, to stratify the patient risk forVTE, the 4G/5G polymorphism of the gene PAI-1could to be searched in patients carrying the prothrombinmutation but it is not necessary when thefactor V Leiden is present, since the 4G/5G polymorphismof the gene PAI-1 does not significantly affectthe risk in patients carrying the G1691A mutation.Oral CommunicationsANTICOAGULANT THERAPY:CLINICAL & LABORATORY PROBLEMSCO-078EVALUATION OF BLEEDING RISK FROM THE ASSOCIATION OFANTIAGGREGANTS AND ORAL ANTICOAGULANTSFrigerio L,° Frigerio G,* Bianchi M,* Beretta A*°Centro Sorveglianza Anticoagulati,*DivisioneMedicina I, Ospedale Valduce, Como, ItalyThe association of antiaggregants and anticoagulantshas always been a difficult therapeutic choicebecause of the possibility of increasing the risk ofhemorrhage in the patients. The aim of our studywas to evaluate the incidence of major and minorbleeding events in patients treated with both thesetypes of drugs. We selected 80 patients (58 males –73%, 22 females – 27%), aged between 30 and 90yr old (mean 71, median 73), who were attendingour anticoagulant clinic. All the patients were beingtreated with oral anticoagulants and contemporaneouslywith antiaggregants. A computer programwas used to calculate the total days of therapy andthe number of days that anticoagulation was withinthe therapeutic range for each patient. A total of36881 days (corresponding to 101 yr) of treatmentwere considered; of these about 66% were passedwith the patient in the correct therapeutic range.During the follow up 6 adverse events occurred (4males and 2 females): 1 death because of cancer(1.3%, unrelated event), 1 AMI (1.3%) and 4 eventsrelated to the therapy (95% CI :0.014-0.123 – in 3patients with ischemic heart disease and in 1 patientwith peripheral arterial disease, of which 2 majorbleeding events (2.5%- severe hematoma followedby death and a sudden death) and 2 minor bleedingevents (2.5%, hematoma and epistaxis), equivalentto 4.0% of events per patient per year. The INR waswithin the therapeutic range at the time of the hemorrhagicevent. The doses of antiaggregant were: ASA160 mg for AMI event; 100 and 300 mg for the twominor bleeding events; ASA 50 mg or ticlopidine inthe two major events. Although this is a limitedseries, our results suggest that in the context of continuosand punctual monitoring of anticoagulanttherapy, the association of antiaggregants with anticoagulantsdoes not seem to lead to an increasedrisk of bleeding.haematologica vol. 89(suppl. n. 8):september 2004

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 200461FCSA PrizeCO-079INTRACRANIAL BLEEDING: RELATIONSHIPS WITHANTITHROMBOTIC TREATMENT IN 241 CEREBRALHEMORRHAGES IN REGGIO EMILIAGhirarduzzi A, Nicolini A, Iotti M, Iorio A,* Baldi G*Dpt. Area Medica 1^ - Angiologia e Centro Emostasie Trombosi, Arcispedale Santa Maria Nuova, ReggioEmilia, ItalyBackground. Anticoagulants (AC) and antiplatelets(AP) drugs are effectively used in the prevention ofthromboembolic events, with the trade-off of bleedingside effects, particularly intracranial (IB). Aim ofthe study: to determine IB incidence and to investigatethe potential effect of AC and AP. Methods. Wereviewed all the patients admitted for IB at our hospitalbetween April 1998 and September 2000. Datawere collected with a standard form. All the patientswere recalled to estimate long term mortality. Comparisonswere performed with chi-squared and t-test, as appropriate. Logistic regression analysis wasperformed to test predictors of mortality. Pharmacydata were employed to estimate the total number ofAC and AP patients. Results: we found 241 cases(107/134 female/male, mean age 61 years, 133/107spontaneous/traumatic events, 0.32/1000 /year).Twenty nine and 47 patients were given AC or AP,respectively (4.9 /1000 /year and 3.7/1000 /year). Therelative risk of IB is 11 in AP and 15 in AC treatedpatients. Mortality was 17/27 (62.9%), 26/47(55.3%) and 57/157(36.3%) in AC, AP and untreatedpatients, respectively (p = 0.015). This increasedrisk was maily confined to traumativ events (p =0.0009), without difference between AC and AP. Atthe time of the event, mean duration of AC treatmentwas 26.3 months (range 1 to 120). mean INRwas 1.94 (1.6–8.8). Overall mortality was 100/241(41.8%), 25/104 (24%) in traumatic versus 75/132(58%) in spontaneous events (p2.Warfarin was restarted the evening of the day ofsurgery in the patients who did not experience anycomplications. Results. Four patients receiving warfarin(8.2%;95%CI:0.02-0.20) developed pockethematoma [one (2.1%) in the HTR+LSHR, and three(6.1%) in the HTR+HSHR] as against six not receivingwarfarin (2.4%; 95% CI:0.01-0.05). Pocket hematomawas associated with a longer postoperativehospital stay and, in three cases, required evacuation.None of the patients showed any symptoms or signsof thromboembolic event. Conclusions. This Enoxaparin-schemaproposed may be an alternative prophylacticmethod in patients receiving anticoagulationwho require PM or ICD.haematologica vol. 89(suppl. n. 8):september 2004

62Oral CommunicationsCO-081CLOTTING PROFILE BY SONOCLOT ANALYSIS IS DIFFERENT INPATIENTS UNDERGOING OFF-PUMP AND ON-PUMPCORONARY-ARTERY BYPASS SURGERYPaniccia R,* Caciolli S,° Palmarini MFG,°Costanzo M,* Tellini I,* Coppo M,* Pretelli P,°Stefano PL,° Pepe G,* Abbate R,* Gensini GF,*Prisco D**Dipartimento Area Critica Medico-Chirurgica,Università degli Studi di Firenze; °DipartimentoCardiologico e dei Vasi, Azienda Ospedaliero-UniversitariaCareggi, Florence, ItalyThe Sonoclot analyzer, a new point-of-care device,is able to assess clotting activation and platelet functionafter blood activation by celite. This instrumentmeasures changes in blood viscoelastic properties byrecording these variations in the form of a graph.Sonoclot signatures” show a lag period (SonACT),corresponding to ACT (sec), and a primary wavewhose slope reflects the rate of fibrin formation,expressed by machine as Clot Rate (CR) and consideredas an index of clot formation. Then, an inflectionis produced as platelets are incorporated intothe fibrin mesh and a secondary upslope leads topeak, which occurs at completion of fibrin formation.Subsequent downslope is produced as plateletsinduce further clot retraction. The time to peak (minutes)(TP) reflects clot retraction, and is influenced byplatelet function (PF). Aim of this study was to investigatewhether a different hemostatic profile at Sonoclotanalysis (SIENCO Inc, USA) exists in off-pumpcoronary artery bypass (OPCAB) patients comparedwith on-pump coronary artery bypass patients [cardiopulmonarybypass (CPB) patients]. Twelve OPCABand 15 CPB patients were studied. Sonoclot analysisin OPCAB patients was performed pre and postheparinbolus (5 min), 45 min and 75 min afterheparin administration, post-protamine infusion (5min) and 24 hour after the surgery. In CPB patientsthe procedure was similar to that for OPCAB patientsexcept for extracorporeal circulation (ECC) period inwhich Sonoclot analysis was performed 30 and 60min after the ECC beginning. At baseline, in OPCABand CPB patients no difference was found for theparameters examined. After heparin, during surgeryand 24 h after surgery SonACTs were significantlyhigher (at least p

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 200463Background. Monitoring of oral anticoagulant therapyis usually performed assessing the INR on patientplasma. Recently, several point-of-care (POC) devicesfor INR determination on capillary whole blood havebeen introduced for clinical use. Objective. To comparethe accuracy of three POC devices (CoaguCheck S,Roche; ProTime, IL; Harmony, LifeScan) as comparedto standard plasma PT-INR for INR determination ina routine clinical setting. Materials and Methods. INRwas simultaneously measured in patients on stabilizedlong term anticoagulation both from venousblood (PT Diagnostica Stago on STA automated coagulometer,Roche) and from capillary blood by the threePOC devices under study. The results from the POCdevices were used in a blinded simulation study toevaluate the impact on drug dosage and follow uptime, as compared with real data produced from PT-INR. The accuracy of the POC devices versus plasmaPT-INR was evaluated by measuring the standard correlationcoefficient and the Bland and Altman procedure.Results. One-hundred-twenty patients wereenrolled and analysed. No statistical difference wasfound between the instruments for drug dosage prescription(% of cases with dose variation 15 to 26;range of D dose -2.5 to +2.5;% of cases with timevariation 10 to 15), concordance limits (95% CI -1.14to +1.28), agreement (% of results within CI, 74 to93), Mean difference (POC-PT -0.09 to 0.41). Conclusions.All the three POC devices showed a good correlationwith plasma PT-INR, and are likely to be efficientlyand safely used in clinical practice. Marginaldifferences concerning clinical and laboratory agreementas compared to plasma PT exist and could beused, besides economic considerations, in the choicebetween the devices.CO-084PHARMACOECONOMIC EVALUATION OF THE MANAGEMENT OFORAL ANTICOAGULANT TREATMENT WITH POINT-OF-CAREDEVICES IN THE ITALIAN NATIONAL HEALTH CARE SYSTEMIorio A, Minciotti A, Ferrante F, Nenci GGSezione di Medicina Interna e Cardiovascolare,Università di Perugia, ItalyBackground. POC devices allow a management oforal anticoagulation (OA) more tailored to thepatients needs than traditional laboratory methods.However, the high cost of POC-INR determinationhas to be taken into account. Aim of the study. Toevaluate direct and indirect cost of a distributedmodel of OA management (D) as compared to thecosts in a Thrombosis Centre (C). Methods. The studywas runned in a peripheral OA units network inUmbria through a questionnaire administered to 300patients and cross-analysis of the patient visits database.Pharmacoeconomic analysis has been performedby taking the payer and the societal perspective,and testing both by assuming different scenarios(service directly given or reimbursed by theNHS or paid for by the patient). Results and Conclusions.217 (72.3%) questionnaires out of 300 werereturned. The following costs (in euros) were considered:(reimbursements and direct cost, respectively):venous venipuncture = 2.6 and 0.98; venousPT = 2.6 and 0.67; short visit = 18; medical activity(one hour) = from 47.19 to 82.18; nurse activity =20.43 to 23.64; technical activity = 20.60; INR (POC)= 6.57 (5.48 + VAT 20%) travel cost per km = 0.334.The number of monthly visits per patient was 1,51 vs1,62 for (D) and (C) respectively. The time used for theactivity 2.45 vs 5.50 hours per months and the timeused by an accompanying person 3.04 vs 3.24 hoursper months for 17.5% of (D) and 57.6% of (C)patients respectively, with 14.8 vs 53.9 Km permonth. The use of the POC techniques costs 9900extra euros per 100 patients per year over venousPT-INR. However, if the NHS has to reimbourse themanagement activity, this will cost 27.500 euros per100 patients per year beyond the cost for POC-INRat home. If the patient has to pay, the cost is of 5.4euros per test, far below than the cost sustained fortravel and lost worktime.CO-085TELEMEDICINE FOR MANAGING PATIENTS ON ORALANTICOAGULANT THERAPYTesta S, 1 Alatri A, 1 Morstabilini G, 1 Denti N, 1Martellenghi E, 1 Mazzei G, 2 Negri D, 3 Pani A, 4Camaiora A, 5 Lari B 5Centro Emostasi e Trombosi, Azienda Istituti Ospitalieridi Cremona 1 ; Laboratorio Analisi, Ospedale diSospiro (CR) 2 ; Laboratorio Analisi, Ospedale di OglioPO-AO Cremona 3 ; Ospedale Geriatrico”A. Soldi” Cremona4; GTO-MMG Casalmaggiore (CR) 5 , ItalyIn the last decade the indications of oral anticoagulanttherapy have increased significantly. Severalstudies have shown that anticoagulation managementby an Anticoagulation Clinic (AC) results inbetter clinical control compared with routine medicalcare. As a consequence we observed a substantialincrease in the number of patients followed upat our AC. The patients, coming from the wholeprovince were 150 in 1991 and 2525 in 2003. Weidentified 3 problems in the organization: 1. reductionof patient comfort at AC, due to overcrowding;2. difficulty in reaching the AC for a substantial numberof patient; 3. inadequate increase of health carestaff. It was therefore necessary to reorganize theactivity, maintaing the clinical quality level reached.haematologica vol. 89(suppl. n. 8):september 2004

64Oral Communications1. Decentralize the activity in the health territorialcare units of the Cremona area, through a telemedicinesystem. 2. Improve the quality of life of thepatients living far from the AC site. 3. Reduce by30% the number of patients seen daily at the AC. 4.Maintain the same clinical quality levels. Weinstalled a centralized net supported program (intraandinternet), TAOnet®, to collect and elaborate clinicaldata, connected with peripheral districts. PT-INRis performed on Coaguchek® portable monitors. Clinicaldata are obtained from the nourse and sent inreal time to the AC. The AC sends within minutes theappropriate dose adjustment and the clinical suggestionsto the district. All the process is under qualityassessement as based upon ISO 9001-VISION2000 standards. The project started in 1-2002. Sixdistrict are available to the public and 310 patients,12% of the total, are now on treatment. Patientsexpressed their overall satisfaction, as evaluatedthrough an open questionnaire. We observed anincrease of the time spent in the therapeutic rangeas compared to the previous six months (AC= 61%,health territorial care units= 68%; p

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 200465CO-087HIGH PLASMA LEVELS OF FACTOR IX, FACTOR XI ANDHOMOCYSTEINE AND RISK OF RECURRENCE OF VENOUSTHROMBOEMBOLISMLegnani C, Cini M, Cosmi B, Boggian O,Cavallaroni K, Mattarozzi S, Guazzaloca G,Palareti GDept. Angiology & Blood Coagulation “Marino Golinelli”,University Hospital S. Orsola-Malpighi,Bologna, ItalyWe evaluated the relationship between Factor IX(FIX), Factor XI (FXI) and basal homocysteine (Homocys)levels, and risk of venous thromboembolism (VTE)recurrence. Three hundred and sixty patients, after afirst episode of venous thromboembolism (VTE),underwent clinical follow-up for a mean follow-upperiod of 18 months after oral anticoagulant (OA)withdrawal (total follow-up = 565 years). Patientswere enrolled on the day of OA discontinuation; 3-4weeks afterwards, they were given a complete thrombophilicwork-out that included: antithrombin, proteinC and protein S activities; activated protein Cresistance (APCR) with DNA analysis for R506Q FactorV mutation in all cases with an APCR normalizedratio < 0.80; DNA analysis for G20210A prothrombinmutation, Factor VIII levels, LAC. FIX and FXI levelswere measured by clotting assays; homocys levelswere measured by a fluorescence polarizationimmunoassay (FPIA). The end point was an objectivelydocumented, symptomatic recurrent VTE. RecurrentVTE developed in 31/360 (8.6%) patients (incidencerate 5.5 per 100 person-years). FIX and FXI levelswere stratified into quartiles. The risk of recurrencewas 10-fold higher in patients with FIX levelsexceeding the 75 th percentile [RR 10.88 (95%CI: 1.18-100.70)] after adjustment for all possible confoundingvariables (age, sex, duration of anticoagulation,absence/presence of thrombophilic alteration/s, FactorVIII and FXI levels). The risk of recurrence was notsignificantly increased in patients with FXI levelsabove the 75 th percentile [RR 4.63 (95%CI: 0.48-44.46)]. Patients were classified as having hyper ornormal homocys levels on the basis of age and sexrelated normal ranges. The rate of VTE recurrence wasnot significantly higher in patients with hyper-homocys[10% vs 7.7% in patients with hyper- and normalhomocys levels, respectively; RR: 1.27 (95%CI: 0.60-2.71)]. In conclusion, this study shows that high FIXlevels seem to be associated with increased risk ofVTE recurrence. On the contrary, the risk of VTE recurrencewas not increased in patients with high FXI orhomocys levels.CO-088IMPACT OF ANTIPHOSPHOLIPID ANTIBODIES ON THE RISK OFRECURRENT VENOUS THROMBOEMBOLISM AMONG PATIENTSWITH INHERITED THROMBOPHILIADe Stefano V, Rossi E, Za T, D'Orazio A, Leone GInstitute of Hematology, Catholic University, Rome,ItalyAntiphospholipid antibodies (aPL) are consideredrisk factors for first or recurrent venous thromboembolism(VTE); among them lupus anticoagulant(LA) appears to be the strongest risk factor, whereasthe risk associated with anticardiolipin (aCL),anti-β-2-glycoprotein I (aB2GPI), and antiprothrombin (aPT)is debated. Data concerning interaction of aPL withinherited thrombophilia are conflicting. We studied852 patients (M/F 357/495) referred to our ThrombosisCenter for VTE not cancer-related. All first VTEswere objectively diagnosed (576 DVT of the legs, in147 cases with pulmonary embolism –PE, 57 PE, and219 superficial vein thrombosis of the legs -SVT).First VTE occurred at a median age of 36 years (range0-84) and was spontaneous in 41.4% of the cases.Inherited thrombophilia was diagnosed in 36.7% ofthe patients (n=313, AT deficiency=8, PC deficiency=31,PS deficiency=24, FV Leiden=154, PT20210A=65, multiple defects=31). Seventy-eightpatients (9.1%) had aPL, in 38 cases associated withinherited thrombophilia (35 patients with a singleinherited trait and 3 with double inherited traits). LAwas present in 31 cases (3.6%), aCL in 13 (1.5%),and aB2GPI in 9 (1.0%); multiple combinations weredetected in the remaining 25 cases (2.9%), 23 ofthem with LA. Thus LA was the aPL most represented(overall 54 cases, 6.3%), in 27 cases associatedwith inherited thrombophilia. The median age of thefirst VTE was anyhow 34 years in patients with inheritedthrombophilia (range 0-73) and in patients withaPL (range 12-72), 35 years in carriers of both alterations(range 17-74). To investigate the impact ofaPL on the risk for recurrent VTE among patients withor without inherited thrombophilia we selected 227patients. The inclusion criteria consisted of first VTEoccurred spontaneously, antithrombotic treatmentafter the first VTE not longer than 6 months, intervaltime between withdrawal of antithrombotictreatment and referral to our Thrombosis Centerlonger than 1 year. We compared 11 pts. with aPL(group A), 97 pts. with inherited thrombophilia(group B), 13 pts. with both alterations (group C),and 119 pts. without any known thrombophilic alteration(group D, reference group). The median timebetween the first VTE and recurrence or referral toour center was 2 years (A, range 1-14), 5 years (B,range 1-39), 3.5 years (C, range 1-30), and 4 years(D, range 1-39). No significant increase in risk forhaematologica vol. 89(suppl. n. 8):september 2004

66Oral Communicationsspontaneous recurrent VTE was noticed in any groupin comparison with the reference individuals: thehazard ratio was 1.9 (95% CI 0.9-4.2) in the groupA (aPL), 1.2 (95% CI 0.8-1.8) in the group B (inheritedthrombophilia), and 0.8 (95% CI 0.2-2.8) in thegroup C (both aPL and inherited thrombophilia).Patients with aPL (group A), in spite of the observationtime shorter than that of the reference group D(median 2 years versus 4 years), showed a moderate1.9-fold increase in risk for recurrent VTE. However,the presence of aPL does not seem to increase tendencytowards VTE among patients with inheritedthrombophilia.CO-089D-DIMER AND VENOUS THROMBOTIC RESIDUE AREINDEPENDENT RISK FACTORS FOR RECURRENT VENOUSTHROMBOEMBOLISM AFTER ANTICOAGULATION WITHDRAWALCosmi B, Legnani C, Cini M, Guazzaloca G, Valdrè L,Brusi C, Palareti GDept. of Angiology & Blood Coagulation “MarinoGolinelli”, University Hospital S. Orsola-Malpighi,Bologna, ItalyIntroduction. D-dimer (D-d) and venous thromboticresidue (VTR) have been separately shown to berisk factors for recurrent VTE. Aims: to assess thepredictive value of the combination of D-d and VTRfor the development of recurrent VTE. Methods. 704consecutive symptomatic patients with a firstepisode of proximal deep vein thrombosis of the lowerlimbs were enrolled on the day of OAT withdrawal(T1). At T1 and three months afterwards (T3), D-dlevels were measured and VTR was determined bycompression ultrasonography. D-d was also measuredafter 30+10 days (T2). All the three availablemeasurements of D-d were considered to establish atrend (D-d trend): abnormal if D-d was persistentlyabove the cut-off value (75th percentile of the D-dvalues at T1 according to type of index event) at T1,T2 and T3 or if it increased above cut-off values atT2 and/or T3. Recurrences were diagnosed in case ofsymptoms of VTE by objective methods during a 2-year period. Results. Abnormal D-d trend was presentin 59.7% and VTR in 51.9% of patients. AbnormalD-d trend and VTR were associated with a multivariatehazard ratio for recurrence of 2.29 (95% CI:1.29-4.06; p=0.004) and 1.84 (95% CI: 1.15-2.95;p=0.011) respectively. When compared with patientswith normal D-d trend and absent VTR, the hazardratio for recurrence associated with abnormal D-dtrend and VTR was 6.36 (95% CI: 2.21-18.33; p=0.001) in all patients after adjustment for sex, age,OAT duration, thrombophilia, type of index event andcancer and 4.95 (95% CI: 1.45-16.89; p= 0.011) inpatients with an idiopathic DVT after adjustment forsex, age, OAT duration and thrombophilia. Conclusions:D-d and VTR are independent risk factors forrecurrent VTE.SISET PrizeCO-090RISK FACTORS AND RECURRENCE RATE OF PRIMARY DEEPVEIN THROMBOSIS OF THE UPPER EXTREMITIESBattaglioli T, Martinelli I, Bucciarelli P,Passamonti SM, Mannucci PMAngelo Bianchi Bonomi Hemophilia and ThrombosisCenter, Department of Internal Medicine and Dermatology,IRCCS Maggiore Hospital, University ofMilan, ItalyOne third of cases of upper-extremity deep veinthrombosis (DVT) are primary, i.e, they occur in theabsence of central venous catheters or cancer. Riskfactors for primary upper-extremity DVT are not wellestablished, and the recurrence rate is unknown. Westudied 115 primary upper-extremity DVT patientsand 797 healthy controls for the presence of thrombophiliadue to factor V Leiden, prothrombinG20210A, antithrombin, protein C, protein S deficiencyand hyperhomocysteinemia. Transient riskfactors for venous thromboembolism were recorded.Recurrent upper-extremity DVT was prospectivelyevaluated over a median period of 5.1 years of follow-up.The adjusted odds ratio for upper extremityDVT was 6.2 (95% CI 2.5-15.7) for factor V Leiden,5.0 (95% CI 2.0-12.2) for prothrombin G20210A and4.9 (95% CI 1.1-22.0) for the anticoagulant proteindeficiencies. Hyperhomocysteinemia and oral contraceptiveswere not associated with upper-extremityDVT. However, in women with factor V Leiden orprothrombin G20210A taking oral contraceptives, theodds ratio for upper-extremity DVT was increased upto 13.6 (95% CI 2.7-67.3). The recurrence rate was4.4% patient-years in patients with and 1.6%patient-years in those without thrombophilia. Thehazard ratio for recurrent upper-extremity DVT inpatients with thrombophilia compared to thosewithout was 2.7 (95% CI 0.7-9.8). In conclusion,inherited thrombophilia is associated with anincreased risk of upper-extremity DVT. Oral contraceptivesincrease the risk only when combined withinherited thrombophilia. The recurrence rate of primaryupper-extremity DVT is low, but tends to behigher in patients with thrombophilia than in thosewithout.haematologica vol. 89(suppl. n. 8):september 2004

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 200467CO-091RESIDUAL VENOUS OBSTRUCTION IS A SURROGATE MARKEROF AN INCREASED RISK OF RECURRENT DEEP VEINTHROMBOSISDentali F, Ageno W, Galli M, Manfredi E, Mera V,Steidl L, Venco ADepartment of Clinical Medicine, University ofInsubria, Varese, ItalyResidual venous obstruction (RVO) in patients withprevious deep vein thrombosis (DVT) of the lowerlimbs has been suggested as an independent risk factorfor recurrent venous thromboembolism (VTE).RVO could be a marker of a persistent prothromboticstate. We have compared the rate of RVO betweenpatients with DVT and a personal history of at leastone previous episode of VTE and patients with a firstepisode of DVT. All patients underwent compressionultrasonography (CUS) of the lower limbs after 1 yearfrom index DVT. In patients with recurrent DVT, theindex event was defined as the last documented DVT.RVO was arbitrarily defined as a thrombus occupying,at maximal compressibility, more than 20% ofthe vein area in the absence of compression. We haveenrolled 38 consecutive patients with recurrent DVTand 50 age matched patients with a single episodeof DVT. In the group with recurrent DVT, the indexevent was idiopathic in 57.9%, and secondary to aknown risk factor in 42.1%, 4 patients had cancer.Among patients with a single episode of DVT, in 54%the event was idiopathic and in 46% it was secondaryto a known risk factor, 5 patients had cancer.All patients with recurrent DVT were receiving oralanticoagulant treatment (OAT) after 1 year. Seventy-twopercent of patients with a single DVT had OATstopped after 3 or 6 months, in 28% the treatmentwas still ongoing. In 70.6% of patients with recurrentDVT the index event occurred in the contralateralleg or in a different segment of the omolateralleg. RVO was detected in 50% of patients with a singleepisode of DVT and in 84.2% of patients withrecurrent DVT (p=0.0009). The prevalence of RVO issignificantly higher in patients with recurrent DVTthan in patients with a single episode. Becausepatients with multiple episodes of DVT are known tobe at very high risk of further recurrences, this findingsupports the importance of RVO as a potentialmarker of a persistent prothrombotic state.SISET PrizeCO-092RESIDUAL VEIN THROMBOSIS ASSESSMENT ESTABLISHES THEOPTIMAL DURATION OF ANTICOAGULANTS IN PATIENTS WITHIDIOPATHIC OR PROVOKED DEEP VEIN THROMBOSISMalato A, Anastasio R, Milio G,* Bonifacio G,Pellegrino M,** Siragusa SCattedra di Ematologia e *Cattedra Di Angiologia,Azienda Ospedaliera Universitaria Policlinico DiPalermo, **U.O.S Di Chirurgia Vascolare, OspedaleCivico di Trapani, ItalyThe optimal intensity and duration of Oral AnticoagulantTherapy (OAT) after a first episode of DeepVein Thrombosis (DVT) is still matter of debate.Patients with unprovoked DVT are currently treatedfor up to 12 months while patients with provokedDVT are usually treated for shorter periods of time.The persistence of vein clots, Residual Vein Thrombosis(RVT) was found to be associated with an increseadrisk of recurrences. However, no prospectivestudies have been done using this parameter toselect patients who could require prolonged OAT. Themain advantage of this approach relates to the factthat patients can be treated on the basis of theirindividual risk of recurrence. In this study weprospectively investigated patients with a firstepisode of DVT and RVT was used to assess the durationof OAT. During the period March 1999 - September2002, 252 patients with DVT of the lowerlimbs (either idiopathic or provoked) were evaluatedin a randomized controlled trial; patients with canceror lupus anticoagulant were excluded. At thethird months of OAT, RVT was assessed as previouslydescribed (Siragusa et al, Thromb Haemost 1993;538:1435); briefly, RVT was considered present whenwe obtained, under a gentle pressure of the probe ofthe ultrasonography, a compressibility of the lumenvein less than 60%. Patients with RVT were randomlyassigned to continue OAT for 1 year (group A1), or tostop it (group A2). In patients without evidence ofRVT, OAT was discontinued (group B). Events, classifiedas recurrent DVT and/or Pulmonary Embolism(PE) or major and minor bleeding were evaluated; allpatients were followed-up for additional 12 monthsafter OAT discontinuation. Two-hundred and eighteenpatients completed the study; at the thirdmonths of OAT, RVT was still present in the 64.6% ofpatients. The rate of recurrent events at 12-monthsfollow-up was 7.3%, 15.6% and 1.8% in group A1,A2 and B, respectively; these differences were statisticallysignificant. The rate of major bleeding was2.4%, 1.25 and 0% in group A1, A2 and B, respectively.Considering a time to event analysis (21months), in patients without RVT the incidence ofhaematologica vol. 89(suppl. n. 8):september 2004

68Oral Communicationsrecurrent DVT remains low after OAT withdrawalwhile in patients with RVT the risk of recurrent VTEincreased after the discontinuation of OAT (Figures1-2).months. In patients with persistent vein occlusion,OAT should be continued for more than 3 monthsalthough it is not clear for how long, since prolongedOAT simply delay the risk of recurrence.CO-093DURATION AND QUALITY OF ORAL ANTICOAGULANTTREATMENT AND RISK OF SUBSEQUENT RECURRENT VENOUSTHROMBOEMBOLISMPalareti G, Legnani C, Cosmi B, Cini M,Mattarozzi S, Poggi M, Guazzaloca GDept. Angiology & Blood Coagulation “MarinoGolinelli”, University Hospital S. Orsola-Malpighi,Bologna, ItalyThis is the first prospective, randomized trialassessing the optimal duration of OAT on the individualrisk basis. This investigation shows thatpatients without RVT, after stopping OAT and nomatter the type of DVT (either idiopathic or provoked),maintain a low risk of recurrences; therefore,in these patients OAT can be safely withheld after 3After a first episode of acute VTE the risk of recurrenceis relatively high and clinical consequencesimportant. The aim of the present study was to assesswhether or not the duration of anticoagulant treatmentperformed after the first VTE episode and thequality of its laboratory control may influence therisk of recurrent VTE episodes after OAT is stopped,or of criteria that have been reported to be predictivefactors for the risk of recurrent VTE (D-dimervalues after OAT is stopped and persistence of residualvenous thrombosis -RVT). We prospectively evaluated710 patients (pts, 351 males) with a single previousVTE episode; the mean follow up was 1.62 yand the total follow up was 1148 y. Recurrent VTEduring follow up was recorded in 72 pts (10.1%). D-dimer levels, measured in 641 pts at 1 month afterOAT was stopped (n.v. < 500 ng/mL, VIDAS, bioMerrieux)were altered in 246 pts (38.4%). The persistenceof RVT at 1 month after OAT was stopped wasobserved in 336 pts (52.6% of the 639 examined).The duration of previous OAT and the quality of itslaboratory control (mean INR value, percentage oftime spent within, below, above the therapeuticrange) were calculated for each examined patientand median values and ranges evaluated in the varioussubgroup of patients. None of the investigatedOAT parameters (median duration, median INR values,time spent in- or below- or above-range) wasdifferent in pts who suffered of subsequent VTErecurrence vs those who did not, as well in pts withaltered or normal D-dimer, and in those with or withoutRVT. In conclusion, the duration of OAT after thefirst VTE episode and the quality of its laboratorycontrol do not seem to affect the risk of subsequentrecurrence and do not seem to influence the D-Dimerlevels and the persistence of RVT after OAT isstopped.haematologica vol. 89(suppl. n. 8):september 2004

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 200469Oral CommunicationsVASCULAR RISK FACTORSCO-094THROMBOPHILIC RISK FACTORS IN PATIENTS WITH SEVERECAROTID ATHEROSCLEROSIS: GENDER-RELATED DIFFERENCESMarcucci R, Sofi F, Fedi S, Cellai AP, # Lari B,Sestini I, Rogolino A, Pratesi G, Pratesi G, Dorigo W,Abbate R, Gensini GFDipartimento Area Critica Medico Chirurgica,#Dipartimento Cardiologico e dei Vasi; AziendaOspedaliero-Universitaria Careggi, Florence, ItalyArterial thromboembolism from carotid plaques isan important pathogenetic mechanism of ischemicstroke. In addition to the identification and treatmentof well-established modifiable risk factors,ongoing prevention efforts include the identificationand validation of novel biochemical and genetic factorsthat increase the risk for cerebrovascular diseaseand stroke. Aim of this study was to determine thethrombophilic risk profile of patients with severecarotid stenosis by evaluating a number of geneticand metabolic risk factors (FII G20210A, FV Leiden,MTHFR C677T polymorphisms, aCL antibodies, Lp(a)and Hcy) in addition to the traditional cardiovascularrisk factors. The study population consisted of615 patients [(410 M/205 F; median age: 73 (26-94)yrs] admitted to the Department of Vascular Surgeryof the University of Florence with severe carotidstenosis, and 615 apparently healthy subjects [(410M/205 F; median age: 73 (31-92) yrs], comparablefor age and sex. At the multivariate analysis adjustedfor age, sex and the classical cardiovascular riskfactors, independent risk factors were elevated Hcyand Lp(a) levels, the presence of anticardiolipin antibodiesand heterozygosity for FII G20210A polymorphismwhich determine a 2- to 6- fold increase in theodds of having severe carotid atherosclerosis. In thesubgroup of women, independent risk factors forsevere carotid atherosclerosis were: high levels ofhomocysteine and Lp(a) and the presence of anticardiolipinantibodies, whereas hyperhomocysteinemia,elevated Lp(a) levels, anticardiolipin antibodies,FII G20210A and MTHFR 677TT polymorphismsremained independent risk factors at the multivariateanalysis performed in the subgroup of men. Theresults of the present study demonstrate that theprevalence of the thrombophilic risk factors is definitelyincreased in patients with severe atherosclerosis.CO-095MORE CAROTID ATHEROSCLEROSIS IN HCV POSITIVE ITALIANPATIENTS?Boddi M, Chellini B, Marcucci R, Cellai AP, Fedi S,Alessandrello Liotta A, Rogolino A, Attanasio M,Gori AM, Prisco D, Abbate R, Gensini GF,Solazzo V,* Zignego AL*Dipartimento di Area Critica Medico-Chirurgico,*Dipartimento di Medicina Interna, AziendaOspedaliero-Univeristaria Careggi, Florence, ItalyThe existence of a link between HcV infection andincreased risk of atherosclerotic disease has beenrecently suggested by a Japanese cross-sectionalstudy, but not confirmed in two studies on Europeangeneral populations. However, in an Italian populationof patients affected by ischemic heart disease(CHD), HcV infection was an independent risk factorfor CHD. Aim of the study was to evaluate the associationbetween HcV infection and carotid atherosclerosisin 1123 patients consecutively referred tothe Center for evaluation of cardiovascular risk factorsof the University of Florence from 1st January to 31thDecember 2003. HcV positivity was investigated byELISA using a commercial kit. In all HcV + patients(n= 33, 3,3%) and in 86 HcV – patients, matched forage and sex (88 males, 35 females, mean age 67.6±13years) the status of carotid arteries (number ofcarotid plaque and carotid intima-media thickness)was studied by high resolution B-mode ultrasonography(Sonolayer SSA 270A equipped with a 7.5 MHztransducer). In all patients the prevalence of majorrisk factors for carotid atherosclerosis (smoking habit,hypertension, diabetes, dyslipidemia, family historyof premature coronary artery disease (CHD)), levels offibrinogen, C reactive protein (CRP) and Lp(a) lipoprotein)and main liver parenchimal function parameterswere also investigated. In the group of 123 patientsinvestigated, the prevalence of carotid lesions wassignificantly higher in HcV positive (51.3%) than inHcV negative patients (19%, p

70Oral Communicationsdata strenghten the possibility that HcV infectionfacilitates the occurrence of carotid atheroscleroticlesions, independently from the major risk factors foratherosclerosis, possibly by potentiating lipid oxidation.allele increased in the patient genotype (VV=13.8±4.9; VL=10.8±3.0; LL=7.0±0.1; p=0.001). Finally,Kaplan-Meier and log rank were used to comparesurvivals among FXIIIA genotype (Figure).CO-096FXIII-A V34L AND FXIII-B H95R GENE POLYMORPHISMS:EFFECTS ON THE EFFICACY OF THROMBOLYTIC THERAPYIN ACUTE MYOCARDIAL INFARCTIONGemmati D, Tognazzo S, Serino Ml, Catozzi L,Ferrara R,* Campo G,* Ferrari R,* Scapoli GLCenter Study Haemostasis & Thrombosis andDepartment of Cardiology,* University of Ferrara,Ferrara, ItalyThrombolysis (TBL) is the most efficacious pharmacologicaltreatment in patients with ST-elevationacute myocardial infarction (AMI). The efficacy ofTBL relies on the capability to achieve a rapid reperfusionof the infracted myocardial area, minimizingthe occlusion time of the culprit coronary artery bythrombus dissolution and by prevention of arteryreocclusion. However, approximately half of treatedpatients fails to achieve an optimal tissue reperfusion.The aim of our study was to evaluate theeffects, if any, of two common FXIII gene polymorphismson non-invasive parameters of myocardialreperfusion and on 1-year event-free survival inpatients undergoing systemic TBL. The most readilyuseful tool for assessing reperfusion is the extent ofST-segment reduction from the baseline electrocardiogram:a reduction ≥50% within the first 60-180minutes after therapy is a validate marker of successfulreperfusion. Factor XIII plays a crucial role inthrombus organization and its variants have beendescribed affecting XL-fibrin structure. Therefore, weevaluated in 318 consecutive patients with AMI andin 440 healthy controls the gene distribution of twocommon FXIII polymorphisms (FXIII-A V34L and FXI-II-B H95R). The FXIIIA-L34 allele was underrepresentedin cases (20% vs 25%; p=0.03) whereas theFXIIIB-R95 allele was underrepresented in controls(10% vs 6.2%; p=0.014). Among the whole group ofAMI, those eligible for TBL therapy were 31.4%(n=100). The effects of the thrombolytic agent used(rt-PA, Actilyse, Boehringer) were analyzed by evaluatingthe extent of reduction of ST-segment (≥50%in 90 min) and monitoring an early CPK peak appearance(≤12h). Data retrospectively stratified by FXIIIgenotypes showed a significant higher number ofcases with ST-segment reduction (≥50%) in the FXI-IIA-L34 carriers with respect to the VV subgroup(93.6% vs 70.1%; p=0.01) and an earlier achievement(h) of the CPK peak as the number of the L34The incidence for adverse cardiac thrombotic eventsat 365 days was 52.3% and 14.3% respectively for theVV and the L-carrier subgroup (p=0.001). No significativedifferences were obtained computing the FXI-IIB polymorphism data. Our results suggest a key roleof the FXIIIA-L34 allele in modulating the effect ofcoronary TBL, achieving an optimal reperfusion aftertherapy and significantly reducing the incidence ofmajor adverse cardiac events.CO-097SOLUBLE CD40 LIGAND AND STROKE IN PATIENTS WITHNONVALVULAR ATRIAL FIBRILLATIONDi Lecce VN,* Fimognari F, Ferro D, Loffredo L,Del Ben M, Milite MT,^ Sbrighi P, Alessandri C,Violi FDivisione IV Clinica Medica, Policlinico Umberto I,Università “La Sapienza” Rome, Italy, *U.O.C. MedicinaInterna, Ospedale L. Parodi-Delfino, ASL RM G,Colleferro, Italy; ^Servizio di Patologia dell’Emostatsi,Ospedale L. Parodi-Delfino, ASL RM G, Colleferro,ItalyAim. Among patients with nonvalvular atrial fibrillation(NVAF) several clinical characteristics identifypatients at high risk for stroke, but no plasmamarkers have yet been shown to predict stroke. Theobjective of this study was to assess whether solubleCD40 ligand (sCD40L), a marker of platelet activationwith proimflammatory and prothromboticactivity, is associated with ischemic stroke in patientswith NVAF. Methods and Results: One hundred andfifty-nine consecutive patients with NVAF, admittedto our division were evaluated between March 2000and July 2003; forty five NVAF patients (28.3% of159 patients) had a clinical history of previousischemic stroke. Thirty healthy volunteers matchedfor age were used as controls. Compared to controls,haematologica vol. 89(suppl. n. 8):september 2004

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 200471NVAF patients had higher sCD40L values (5.0±1.8 vs2.8±0.8 ng/mL respectively, p15 UI/mLwas present in 74/280 (26.4%) patients compared to25/280 (8.9%). Positivity of anticardiolipin antibodies,defined as levels > 20 UMPL for IgM type and/orUGPL for IgG type), was found significantly moreprevalent in patients than in healthy subjects(p15 UI/mL, ACA positivity and prothrombinvariant) and PAD. Moreover, when high levelsof Lp(a) were associated with another metabolicrisk factor (e.g. HHcy or dyslipidemia) the susceptibilityto the disease resulted to be increased. In conclusion,these results show an association betweenPAD and emerging hemostasis-related risk factors.CO-099ASSOCIATION BETWEEN –675 4G/5G POLYMORPHISM OF THEPAI-1 GENE AND OBESITY IN ITALIAN MEN: LONGITUDINALFINDINGS OF THE OLIVETTI HEART STUDYIacoviello L,* Iacone R,° Farinaro E,°° Versiero M,°Siani A,** Cappuccio F,°°° Donati MB,* Strazzullo P°*Center for High Technology Research and Educationin Biomedical Sciences, Catholic University, Campobasso,°Department of Clinical & °°ExperimentalMedicine, Department of Preventive Medical Sciences,"Federico II" University of Naples MedicalSchool, Naples; **Epidemiology & Population Genetics,Institute of Food Sciences, CNR, Avellino; Italy;°°°Department of Community Health Science, St.Georges' Hospital Medical School, London, UKBackground. Development of obesity is associatedwith extensive remodelling of adipose tissue andinvolves extracellular matrix proteolysis. PAI-1 levelshave been associated with adipose tissue remodelling.PAI-1-deficient mice on high fat diet developedmore rapidly adipose tissue than their leancounterparts while transgenic mice overexpressing astable human PAI-1 variant had virtually no intraperitonealfat. Objective. To investigate the possibleassociation of –675 4G/5G polymorphism of the PAI-1 gene, a genetic variation associated with PAI-1levels, with body mass, body fat distribution and obesity-associatedhypertension in a sample of adultmale population. Methods. The –675 4G/5G polymorphismwas evaluated in a group of 378 participantsof the Olivetti Heart Study seen at the 2001-2003 follow-up visit, who had been examined forthe first time in 1975 (all males, age range 50-81years). Complete anthropometry and blood pressure(BP) were measured with standardised procedures.Results. At the follow-up examination participantshaematologica vol. 89(suppl. n. 8):september 2004

72Oral Communicationscarrying the 5G/5G, compared to those with 4G/5Gor 4G/4G genotype, had greater values of BMI (28.3vs 27.4 or 26.8 kg/m 2 , p=0.005), hip circumference(100.3 vs 98.5 or 98.1 cm, p=0.03), arm circumference(32.2 vs 31.5 or 30.9 cm, p=0.005), leg circumference(50.0 vs 48.7 or 48.8 cm, p=0.02) and subscapularskinfold (23.4 vs 21.8 0r 20.5 mm, p=0.02).No association was detected between blood pressure,lipid or glucose levels and –675 4G/5G polymorphism.The increase in body weight since 1975(27-year average follow-up) was 5.03;0.78 kg for5G/5G vs 3.35; 0.39 kg for the other group (p=0.038).Among participants who were not overweight in1975, the 27 year-incidence of over-weight (BMI;27 kg/m 2 ) was 56.7% for 5G/5G vs 27.3% for theother participants (O.R.=3.49; 95% C.I.=1.97 to 6.21)and that of obesity (BMI; 30 kg/m 2 ) 22.6% vs 11.8%,respectively (O.R.=2.18; 95% C.I.=1.17 to 4.04). Nodifference was observed in the BP, lipids and glucosechanges over time, according to PAI-1 genotypes.Conclusions. In this sample of adult male population,the 5G/5G homozygosity was a signifi-cant predictorof overweight and obesity. Since 5G allele hasbeen associated with lower levels of PAI-1, it can behypothesised that a genetic tendency to decreasedlevels of PAI-1 is associ-ated with body weight gain.CO-100ACE DD GENOTYPE: A PREDISPOSITION FACTOR TOABDOMINAL AORTIC ANEURYSMFatini C,* Sticchi E,* Lari B,* Bolli P,* Sofi F,*Lenti M,* Gensini F,° Pratesi G,* Pulli R,* Pratesi C,*Abbate R,* Gensini GF**Dipartimento Area Critica Medico-Chirurgica,Università degli Studi di Firenze; °DipartimentoFisiopatologia Clinica, Università degli Studi diFirenze, ItalyGenetic and environmental components contributeto the etiology and progression of aortic aneurysms.To date, ACE I/D and AT1R A1166C polymorphisms ingenes encoding for Renin Angiotensin System (RAS)components have been demonstrated to be associatedwith atherosclerotic lesions and ACE D allelehas been related to angiotensin II plasma levels.Experimental studies demonstrated that angiotensinII infusion produced abdominal aortic aneurysm(AAA). In humans few data are available regardingthe role of RAS in aneurysm formation. In order todetermine the role of these two polymorphisms inthe development of AAA, we investigated 250 consecutivepatients, 217 males and 33 females (medianage 72, range 50-83), undergone to AAA electiverepair and 250 healthy controls, comparable for sexand age. ACE and AT1R polymorphisms were studiedby PCR-RFLP analysis. The genotype distribution wasin Hardy-Weinberg equilibrium for all polymorphisms.The genotype distribution and allele frequencyof ACE I/D, but not AT1R A1166C polymorphismwere significantly different between patientsand controls (ACE I/D: p=0.0002 and p

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 200473ic diseases. Results. The prevalence of the 10ins allelewas not different between Italians and Mixed-Italiansand between Belgians and Mixed-belgians, andwas higher in Italians in comparison with Belgians(p=0.0054) (see Table).Oral CommunicationsHOMOCYSTEINE &ANTIPHOSPHOLIPID ANTIBODIESFVII levels were higher in subjects without the10ins allele, in all three populations (p

74Oral Communicationsmorphism, but not in those with folic acid ≥5 nmol/L.Among all the possible combinations of the 3 analyzedpolymorphisms, the more frequent combinedgenotypes resulted 677CT/1298AC/2756AA (28.6%),677TT/1298AA/2756AA (19.6%) and 677CT/1298AA/2756AA (16.7%). Homocysteine levels were significantly(p0.05). Atthe multivariate logistic regression analysis adjustedfor age, sex, creatinine plasma levels, folate, B6and B12 plasma levels, folate and B6 intakes andMTHFR polymorphism, eNOS gene polymorphismswere not significantly associated with an increasedrisk of having high levels of homocysteine. Nevertheless,as far as subjects with lowest serum folatetertile are considered, only eNOS -786C variant influencedthe risk of being in the highest homocysteinetertiles (2 nd tertile: OR 6.4, p=0.02; 3rd tertile: OR8.3, p=0.01). Our results document a possible associationbetween eNOS T-786C polymorphism andhomocysteine plasma levels in healthy subjects withlow serum folate levels.CO-104A PROINFLAMMATORY STATE INDICATED BY HIGH IL-6LEVELS IS RELATED TO HYPERHOMOCYSTEINEMIAGori AM, Fedi S, Corsi AM,* Rogolino A,^Alessandrello Liotta A,^ Gazzini A, Lenti M, Poggi F,Rossi L, Ferrucci L,** Abbate R, Gensini GFDepartment of Medical and Surgical Critical Area,University of Florence; *Laboratory of ClinicalEpidemiology, Geriatric Department, National Instituteof Research and Care on Aging (INRCA), Florence;^Dipartimento Cardiologico e dei Vasi, AziendaOspedaliero-Universitaria Careggi, Florence,Italy; **Longitudinal Studies Section, GerontologyResearch Center, National Institute on Aging,National Institutes of Health, Baltimore, USAElevated levels of homocysteine may reflectincreasing age, male sex, renal failure, vitamin deficiencies(low folate, vitamin B12 or vitamin B6), orinherited abnormalities of enzymes involved in themethionine metabolism. Aim of this study was todetermine in a large population based samplewhether hyperhomocysteinemia is associated withhigh circulating levels of inflammatory markers.Westudied 586 men and 734 women randomly selectedfrom the people living in two sites in the surroundingof Florence, Italy. In the InCHIANTI populationhomocysteine, folic acid, vitamin B6 and B12levels were 13.7 (5.4-95.9) µmol/L, 6.3 (0.7-45.3)nmol/L, 25.3 (0.4-689.2) nmol/L and 285 (25.2-1488.1) pmol/L respectively. Dietary vitamin B6 andfolic acid intakes were strongly (p

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 200475cysteine, the pattern of inflammatory mediators, vitaminintakes and vitamin levels in a large population-basedstudy, in the InChianti Study. Interleukin-6 and interleukin-1ra, but not other markers ofinflammation, were found to be independent predictorsof homocysteine plasma levels, in addition tothe vitamins involved in methionine cycle.This work was supported by a grant from Ministerodella Salute.CO-105REDUCED IN VIVO OXIDATIVE STRESS FOLLOWING ASHORT-COURSE SUPPLEMENTATION OF5-METHYLTETRAHYDROFOLATE IN SUBJECTS WITH HOMOZYGOUSTHERMOLABILE METHYLENETETRAHYDROFOLATE REDUCTASEAND A HISTORY OF EARLY-ONSET THROMBOSISCoppola A,* D’Angelo A,^ Fermo I,° Mazzola G,^Cajani A, # Sala A, # Folco G, # Tremoli E, # Di Minno G**Regional Reference Centre for Coagulation Disease,Department of Clinical and Experimental Medicine,“Federico II” University, Naples; ^Coagulation Serviceand Thrombosis Research Unit, and °Department ofLaboratory Medicine, I.R.C.C.S. “H S. Raffaele”, Milan;#Department of Pharmacological Sciences, Universityof Milan, ItalyThe protective role of folate in vascular disease isrelated to its effects on oxidative stress. We evaluatedsome effects of a 28-day course of 15 mg of thebiologically active form of folate, D-L 5-methyltetrahydofolate(5-MTHF,Prefolic, Zambon, Italy) in 45homozygotes for the 677TT mutation of the methylenetetrahydrofolatereductase (MTHFR) gene (27 men,18 women, mean age 40.8±14.0 yrs, range 14-76, allwith a history of thrombotic episodes, 11 with fastingmild/moderate hyperhomocysteinemia). At baseline,patients’ urinary excretion of 8-iso-PGF2α, a plateletactiveproduct of arachidonic acid peroxidation andreliable marker of in vivo oxidative sress, was 304 (0.10)pg/mg creatinine (geometric mean and one SD as amultiple of the logarithmic mean, range: 101-1011),higher levels being found in subjects in the lowestquartile of plasma folate distribution (p

76Oral CommunicationsCO-107HOMOCYSTEINE PLASMA LEVELS AND MTHFR GENOTYPES INYOUNG AND OLD PATIENTS WITH RETINAL VEIN OCCLUSIONPiana A,* Camicione P,° Verrastro G,° Calabria G,°Minniti G, § Cerone R, § Calevo MG, Perrone L,•Armani U**Department of Internal Medicine, ThrombosisResearch Center and °Department of Neurosciences,Ophthalmology and Genetics, Universityof Genoa, § Department of Pediatrics II and Serviceof Statistics and Epidemiology, Gaslini Institute,Genoa, •Lab. Human Genetics, Galliera Hospital,Genoa, ItalyAim of this study is to evaluate homocysteine levelsin the fasting state and after methionine loading testand C677T MTHFR mutation in patients with retinalvein occlusion (RVO). Ninety-three consecutivepatients with a RVO were enrolled. Thirty-nine patientswere aged less than 50 years and 54 patients over 50years. Seventy-one matched controls were also included.Total plasma HCY was measured by high-pressureliquid chromatography before and after a standardizedmethionine-loading test. Hyperhomocysteinemia wasdefined as total plasma HCY level above the 95 th percentilein the control group. The odd ratio was calculatedas an estimate of the risk of RVO for individualswith hyperhomocysteinemia. MTHFR genotypes wereascertained by standard molecular methods. The meanfasting plasma homocysteine level was significantlyhigher in younger RVO group (9.8±3 µmol/µL) comparedwith the younger control group (7.3±3 µmol/L;p < 0.001). Among the elderly group, the differencebetween the fasting HCY levels in OVR patients(8.8±2.6 µmol/L) and controls (8.5±2.2 µmol/L) didnot reach statistical significance. HCY values aftermethionine loading compared with the fasting HCYlevels were not significantly different in two differentage groups. On univariate analysis, elevated fastingHCY levels conferred an increased risk of retinal veinocclusion in young OVR patients, which remained significantin a multivariate analysis (OR 1.37;CI 95%1.11-1.7; p< 0.004). Genotype distribution of the MTH-FR C677T mutation among controls and patients didnot reveal any significant difference. No significantdifferences were shown regarding plasma total HCYlevels and MTHFR genotype between CRVO and BRVOin both different age group examined. This study showa significant association between the elevated fastingplasma total homocysteine levels and retinal venousocclusive disease in young RVO patients comparedwith the control group and with older patients.CO-108HOMOCYSTEINE CAUSES THE EXPRESSION OF VASCULAR CELLADHESION MOLECULE-1 IN CULTURED ENDOTHELIAL CELLS BYA PRO-OXIDANT MECHANISM: PROTECTIVE ROLE OF DIETARYANTIOXIDANTSCarluccio MA,* Ancora MA,* Massaro M,*Carluccio M,* Distante A,* Storelli C,° De Caterina R #*CNR Institute of Clinical Physiology, Lecce and Pisa,°University of Lecce, # D’ Annunzio University, Chieti,ItalyHigh levels of homocyst(e)ine are now recognizedas an indipendent and graded risk factor for vasculardisease. Monocyte adhesion to the vascularendothelium is a crucial early event in atherogenesisand vascular cell adhesion molecule (VCAM)-1 isan important mediator of such interactions. Wetherefore evaluated whether homocysteine (Hcy)could up regulate the endothelial expression ofVCAM-1 and possible mechanisms involved. In culturedhuman umbilical vein endothelial cells (HUVEC)we found that Hcy, but not cysteine, at pathophysiologicalconcentrations (10-500 µmol/L) induced theexpression of VCAM-1 (average increase: 2 folds)and the effect became highly significant in the presenceof low concentrations of tumor necrosis factor(TNF)α (Table 1).Correspondingly Hcy, but not cysteine, increasedTNF-induced VCAM-1 mRNA at Northern analysis,as well as the activation of redox sensitive transcriptionfactors nuclear factor-cappaB (NF-κ) andactivator protein (AP)-1, as assessed by electrophoreticmobility shift assay. This proinflammatoryeffect by Hcy was accompanied by a clear overproductionof reactive oxygen species (ROS), as evaluatedby -dichlorofluoresceine diacetate at flowcytometry. Mediterranean diet antioxidants hydroxytyrosoland trans-resveratrol (10-50 µmol/L)reduced the stimulated expression of VCAM-1 andthe ROS overproduction induced by either Hcy andHcy plus TNF, pointing to a causal link between ROSoverproduction and VCAM-1 expression by Hcy. Inconclusions, our results shown that Hcy, per se andin conjunction with known proatherogenic/proinflammatoryagonists, induces the expression ofhaematologica vol. 89(suppl. n. 8):september 2004

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 200477VCAM-1 through a ROS-mediated mechanism, andsuggest the possibility that Mediterranean dietscould counterbalance some of Hcy vascular proinflammatoryeffects.CO-109PHOSPHOLIPID OXIDATION-DEPENDENT INHIBITION OFACTIVATED PROTEIN C ACTIVITY AND RISK OF THROMBOSISIN LUPUS ANTICOAGULANT PATIENTSEsmon NL, Safa O, Della Valle P, Fattorini A,Esmon CT, D’Angelo AOklahoma Medical Research Foundation, OklahomaCity, USA; Servizio di Coagulazione ed Unita’ RicercaTrombosi, HS. Raffaele, Milan, ItalyLipid oxidation enhances the anticoagulant functionof activated protein C (APC) and facilitatesdetection of its inhibition by anti-phospholipid antibodies.The anticoagulant activity of APC (0.2 µg/mLf.c.) was explored in Xa-one stage clotting assayswith non-oxidized and oxidized phospholipid (PL)after the addition of total IgG fraction (0.6 mg/mLf.c.) obtained from healthy subjects (C, n = 22),patients with atrial fibrillation on oral anticoagulanttreatment (AF, n = 21), lupus anticoagulant (LA)-freepatients with history of thrombosis on oral anticoagulanttreatment (LA-/Thr+, n = 28), patients withLA and no history of thrombosis (LA + /Thr-, n = 25)and LA patients with history of thrombosis on oralanticoagulant treatment (LA + /Thr + , n = 25). LApotency (PTT-LA, Staclot and Staclot + hexagonal PL,Stago) and aCL IgG titers at diagnosis were similarin the two groups of LA patients. With non-oxidizedPL, clotting times were prolonged to a similar extentin all groups by APC. The APC response with oxidizedPL was similar in the C and AF groups (median ratio= 3.11 and 3.24 respectively), but was lower withIgG from patients of the other groups (median ratios< 2.83, p< 0.0001). With respect to C and AF subjects,impaired PL oxidation-dependent response toAPC was observed for one patient in the LA − /Thr +group, 5 patients in the LA + /Thr − group (OR = 6.75, p= 0.07)) and 13 patients in the LA + /Thr + group (OR =29.3, p = 0.0002). By logistic regression, independentpredictors of impaired oxidation-dependentAPC response in LA patients were aCL IgG titers (OR= 1.05, p = 0.015), association with autoimmune diseases(OR = 26.3, p = 0.04) and history of thrombosis(OR = 143.0, p = 0.006). These data strongly suggestthat APC resistance with oxidized phospholipidsis a major risk factor for thrombosis in patients withlupus anticoagulants.Oral CommunicationsHEMORRHAGIC SYNDROMES:DIAGNOSIS AND CLINICAL ASPECTSCO-110VALIDATION OF A DIAGNOSTIC ALGORITHM FOR INHERITEDTHROMBOCYTOPENIAS IN 46 CONSECUTIVE PATIENTSBalduini CL, Noris P, di Bari F,* Pecci A,Di Pumpo M, Ceresa I, Arezzi N, Ambaglio C,Savoia A*Clinica Medica III, IRCCS Policlinico San Matteo-University of Pavia and *Telethon Institute ofGenetics and Medicine (TIGEM), Naples, ItalyAn algorithm to assist clinicians in the diagnosis ofinherited thrombocytopenias has been proposed bythe Italian Gruppo di Studio delle Piastrine (Haematologica2003;88:582). It includes a first phase ofclinical and simple laboratory investigations to putforward diagnostic hypotheses, followed by a secondphase of specialized investigations to make diagnosis.To validate this diagnostic algorithm, we appliedit retrospectively to 46 consecutive unrelatedpatients with inherited thrombocytopenias observedin our institution during the last 5 years. At the endof second level investigations, patient phenotypeshave been classified as follows: 11 MYH9-relateddisease (MYH9-RD); 4 homozygous Bernard-Souliersyndrome (BSS); 8 heterozygous BSS; 1 gray plateletsyndrome; 1 X-linked thrombocytopenia with thalassemia(XLTT); 1 von Willebrand disease 2B. Twoadditional patients had the clinical phenotype ofMYH9-RD (autosomal dominant macrothrombocytopenia,deafness, and kidney defect), but a normaldistribution of mutant protein (NMMHC-IIA) withinneutrophils excluded this diagnosis. Mutationscreening confirmed the diagnoses of MYH9-RD andXLTT. No mutations of MYH9 were detected in the 2patients with the clinical phenotype of MYH9-RDand normal leukocyte distribution of NMMHC-IIA.Molecular diagnostic tests in the 8 patients with thephenotype of heterozygous BSS identified theBolzano mutation in 4 cases, while no mutations ofGPIbα, GPIbβ, GPIX or GPV were observed in theremaining 4 cases. Eighteen patients did not meetthe criteria for any known inherited macrothrombocytopeniaand no further diagnoses were done byadditional investigations other than those requiredby the diagnostic algorithm. In conclusion: a) theinvestigated algorithm identified all patients with aknown form of inherited thrombocytopenia; b) thehaematologica vol. 89(suppl. n. 8):september 2004

78Oral Communicationsmost frequent inherited thrombocytopenias wereMYH9-RD and BSS; c) some patients with the phenotypeof MYH9-RD or heterozygous BSS had nomutations in the genes for MYH9 or GPIb/IX/V,respectively, thus indicating genetic heterogeneityof these disorders.CO-111THERAPY WITH HIGH-DOSE DEXAMETHASONE (HD-DXM) INPREVIOUSLY UNTREATED PATIENTS AFFECTED BY IDIOPATHICTHROMBOCYTOPENIC PURPURAMazzucconi MG,* Bernasconi S,* Fazi P,*Amendola A,* De Rossi G,° Baronci C,° Leone G,^Carbone C,' Vianelli N,°° Quattrin S,** Fioritoni G,''Gugliotta L,^^ Mandelli F* for the GIMEMACooperative ITP Study Group*Dip. Biotec. Cellulari ed Ematologia Univ. "LaSapienza", Rom; °Divisione di Ematologia, OspedalePediatrico Bambino Gesù, Rome; ^Divisione diEmatologia, Università Cattolica del Sacro Cuore,Rome; 'Sezione di Ematologia e Trapianti, SpedaliCivili, Brescia; °° Istituto di Ematologia e OncologiaMedica L.e A. Seragnoli, Università di Bologna; **U.O.Oncoematologia Ospedale Santa Maria delleGrazie, Pozzuoli; ''Div. Emat. con Trapianto, AziendaUSL, Pescara, ^^ Servizio di Ematologia, Arcispedale"Santa Maria Nuova", Reggio Emilia, ItalyWe evaluated the efficacy, safety, feasibililty ofpulsed HD-DXM, as first-line therapy, in a series ofnewly diagnosed untreated ITP patients (pts).Patients with platelet (plt) count 30×10 9 /L or>30×10 9 /L with bleeding symptoms, were enrolled.HD-DXM was given at daily doses of 40 mg for 4days for four courses (on days: 0-14-28-42).Response was evaluated at day 60 from the treatmentonset: plt count 150×10 9 /L complete response(CR); 503030’) and defective in vitroplatelet aggregation to collagen were observed in thepropositus from family 1. Normal MPV, platelet aggregationand clot retraction were detected in family 2where only one member with mild bleeding tendencyhad prolonged bleeding time (11’). A reducedplatelet surface expression of GPIa-IIa was detectedby routine flow cytometry in all patients (32-50% ofcontrol). It is known that nucleotide polymorphysmsin the GPIa gene define multiple alleles associatedwith a different density of GPIa-IIa: allele 1(807T/837T/873A/Brb), allele 2 (807C/837T/ 873G/Brb)and allele 3 (807C/837C/873G/Bra) are associatedwith increased, low and intermediate levels of GPIa-IIa, respectively. Therefore, we typed these polymorphysmsin our patients and the results of subsequentanalyses were compared with genotype-matcheddonors. Defective expression levels of GPIa-IIa wereconfirmed both by flow cytometry and SDS-PAGE withimmunoblotting (30-60% of control). Platelet adhesionto monomeric collagen type I ranged from 15 to40% of that observed for controls. In family 1, residualadhesion to decorin was 40% while in family 2residual adhesion to the CB8 peptide, obtained fromdigestion of collagen II, ranged from 30 to 55% ofhaematologica vol. 89(suppl. n. 8):september 2004

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 200479control. Searching for a molecular defect underlyingthe impaired expression of platelet GPIa-IIa, the codingregion of GPIa and GPIIa genes were analysed butno mutations have been identified. The defectiveGPIa-IIa platelet expression and the reduced celladhesion to GPIa-IIa ligands define a new inheritedthrombocytopenia whose pathogenesis is unknownsince no mutations have been identified in the genesfor GPIa and GPIIa.CO-113PLATELET AGGREGATION STUDIES: AUTOLOGOUSPLATELET-POOR PLASMA INHIBITS PLATELET AGGREGATIONWHEN ADDED TO PLATELET-RICH PLASMA TO NORMALIZEPLATELET COUNTLecchi A,° Zighetti ML,*° Lussana F,* Cattaneo M°*Unità di Ematologia e Trombosi, Ospedale SanPaolo, Dipartimento di Medicina Chirurgia eOdontoiatria, Università di Milano; °Centro Emofiliae Trombosi Angelo Bianchi Bonomi, IRCCS OspedaleMaggiore, Dipartimento di Medicina Interna,Università di Milano, ItalyPlatelet aggregation (PA) studies are important fordiagnosing patients with defects of platelet function.Due to the high variability of the test, results obtainedwith platelet-rich plasma (PRP) of the patient shouldbe compared to those of control PRP, run in parallel.Platelet counts in the two PRPs should be adjusted tothe same value, using autologous platelet-poor plasma(PPP) for proper dilution. We investigated whetheror not dilution of PRP with autologous PPP affectsthe results of PA studies. Study 1. We re-evaluated theresults obtained with 83 control PRPs in PA studiesrun with the same instrument from 1999 through2001. Twenty-six PRPs had been diluted with PPP,while 57 were used undiluted. The mean (range)platelet count was 301×10 9 /L (120-436) in diluted-PRPs and 376 (177-588) in undiluted-PRPs (p

80Oral CommunicationsCO-115STUDY OF THE ASSOCIATION OF PFA-100® CLOSURE TIMEAND THE SKIN BLEEDING TIME WITH THE SEVERITY OFBLEEDING SYMPTOMS IN PATIENTS SCREENED FORBLEEDING DIATHESISPodda GM,* Bucciarelli P,* Lussana F,° Lecchi A,*Cattaneo M°*Centro Emofilia e Trombosi Angelo Bianchi BonomiIRCCS Ospedale Maggiore, Departimento diMedicina Interna, Università di Milano; °Unità diEmatologia e Trombosi, Ospedale San Paolo, DMCO,Università di Milano, ItalyWe compared the association of PFA-100® closuretime (CT) and the Bleeding Time (BT) with the severityof bleeding history in 128 consecutive patientsreferred to our Center from June 2002 through June2003 to be screened for bleeding disorders, due tothe presence of bleeding symptoms or the casualfinding of abnormal screening tests of hemostasis. Allpatients underwent a careful medical interview andwere assigned a “bleeding score”, based on the number,type, frequency and severity (need for bloodtransfusion and/or surgical or medical intervention)of bleeding symptoms. In addition, all patientsunderwent a first-line screening, which included PT,APTT, BT and PFA-100® CT (with both the collagen-ADP and the collagen-epinephrine cartridges). Thesearch for Von Willebrand disease (VWD), plateletfunction disorders (PFD), clotting factor defects andabnormalities of fibrinolysis was performed accordingto the results of the first line screening tests andthe severity and type of bleeding history. Seven (6%)patients had type-1 VWD, 7 (6%) PFD, 29 (23%)defects of coagulation, 18 (14%) defects of the contactsystem or lupus anticoagulant, while in 67(52%) all tests gave normal results. The sensitivity ofPFA-100® for VWD was 86% (both cartridges), forPFD 71% (collagen-epinephrine) and 14% (collagen-ADP). The sensitivity of BT for VWD was 29%, forPFD 57%. After dividing the patient population infour quartiles of distribution, according to the severityof the bleeding score (null, low, intermediate andsevere), only the CT values of collagen-epinephrineshowed a progressive and significant prolongationfrom the first to the fourth quartile (p=0.04). Noassociation of BT and collagen-ADP CT with theseverity of bleeding history was found. In conclusion,PFA-100 showed a better sensitivity than BTfor VWD and PFD. CT with the collagen-epinephrinecartridge was significantly associated with the severityof the bleeding history.CO-1161-DEAMINO-8-D-ARGININE VASOPRESSIN AND PFA-100 TMIN PERCUTANEOUS ULTRASOUND GUIDED RENAL BIOPSY:A RANDOMIZED PLACEBO CONTROLLED TRIALBonifati C,* Ranieri P,° Sardone V,° Petruzzellis S,°Micelli M,° Strippoli GFM,* Campobasso N,*Schena FP,* Manno C**Istituto di Nefrologia, Università degli Studi, Bari;°Laboratorio di Coagulazione, Azienda Policlinico,Bari, ItalyBackground. Risks associated with percutaneousrenal biopsy decreased in the past two decadesbecause of technical advances. However, bleedingcomplications still occur in about 1/3 of proceduresresulting in increased costs and hospital stay. Weevaluated the pre-biopsy treatment with DDAVP onthe post-biopsy bleeding complications and the reliabilityof the in vitro closure time (CT) determinedwith the PFA-100(e)TM system (Dade-Behring, Marburg,Germany) for the evaluation of primary hemostasis.Methods. A randomized controlled trial withblinding of participants, investigators and outcomeassessors was performed in patients with normal renalfunction, IVY bleeding time and coagulation; patientswere centrally randomized to DDAVP 0.3 µg/kg s.c. 1hour prior to biopsy versus matched placebo. Beforeand after biopsy were evaluated: CT with PFA-100TM[Collagen-epinephrine (CEPI-CT) and Collagen-ADP(CAPD-CT)], FVIII:c, vWF:Ag and vWF:RCo. Post biopsybleeding complications were evaluated after 24-hours with renal ultrasound. Analysis was by intentionto treat. Results. Forty patients with no demographicdifferences were allocated to DDAVP/placebo(20/20). DDAVP vs placebo significantly reduced thenumber (6/20 versus 12/20; p=0.04) and the size ofhematomas (83±173.7 mm 2 vs 254.7±257.7 mm 2 ;p=0.02). DDAVP, but not placebo, significantly reducedCEPI-CT [137.6±33.7 to 102.3±34.4 seconds(p=0.008)] and CADP-CT [80.9±12.0 to 64.4±21.8seconds (p=0.003)] and increased FVIII:C [94.1±34.5to 196.0±111.0 IU/dl (p=0.002)], vWF:Ag [114.2±28.6to 170.3±59.0 IU/dl (p=0.004)] and vWF:Rco[111.7±26.8 to 180.3±68.1% (p=0.002)]. Correlationbetween CT and FVIII:c/vWF:Ag/vW:RCo (p

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 200481CO-117COAGULATION SCREENING FOR OTORHINOLARYNGOIATRICSURGERY: A PHARMACOECONOMIC APPRAISAL IN THESETTING OF THE ITALIAN NATIONAL HEALTH SYSTEMFerrante F, Bifarini B,* Tesoro S,* Peduto F,* Iorio ASezione di Medicina Interna e Cardiovascolare e*Servizio di Anestesia e Rianimazione Universitàdi Perugia, ItalyBackground. Tonsillectomy and adenoidectomy arethe most frequently performed paediatric surgeryinterventions in Italy. Prothrombin Time (PT) andactivated Partial Thromboplastine Time (aPTT) areused as screening tests for their supposed predictivityon post surgical bleeding, but their sensitivity andspecificity are still debated. An evaluation of the realneed of these tests is necessary. Aim of the study. Toevaluate in a prospective observational study and inthe published literature the efficiency of the coagulationscreening for paediatric ORL surgery. Methods.We considered all the patients admitted to ourhospital for planned tonsillectomy and/or adenoidectomyfrom Jan 2002 to Jul 2003 and we evaluatedthe overall cost, the cost per each screeningand the cost avoided for side effects. Contextually weconsidered studies concerning children and adolescentsundergoing coagulative screening beforeotorhinolaryngoiatric surgery in a metanalysis ofstudies. Afterwards we compared our data with dataobtained from the metanalysis and we used our costestimates to predict the cost for this bigger dataset.Results. Prospective study: out of 246 children, only5 (2.03%) resulted affected by mild coagulative disordersand none of the children submitted to theadenotonsillectomy bleeded in the perioperativephase. Considering costs, the mean cost of thescreening per patient was of 20 Euros and the meancost per newly diagnosed coagulopathy was 990Euros. Metanalysis. Out of 4695 patients, from 7 differentstudies, 283 bleeding events occurred. Thebleeding rate was 5.7 and 11.8 in patients with negativeand positive screening, respectively. By applyingto this figures the costs evaluated in the prospectivestudy, the cost for avoided bleeding event wasof 333 euros. Since no bleeding was fatal, the costfor avoided death was higher than 162,000 euros.Discussion. We concluded that coagulation screeningtests in symptomatic patient with negativebleeding history and undergoing adenotonsillectomyhave a low positive predictive value for perioperativebleeing, since they lead to a great number of falsepositives. the screening is indicated only in the presenceof a positive bleeding history.Oral CommunicationsVENOUS THROMBOEMBOLISM:EPIDEMIOLOGY AND RISK FACTORSCO-118DEEP VEIN THROMBOSIS IN ASIA AFTER MAJOR ORTHOPEDICSURGERY. AN EPIDEMIOLOGICAL STUDY WITH MANDATORYBILATERAL VENOGRAPHY AND CENTRAL INDEPENDENTADJUDICATIONPiovella F, Passera R, Barone M, Librè L, Piovella C,Wang CJ, Lu H, Lee K, Lee LH, Lee WC, Turpie AG,Gallus A, Planes A, Perdriset G, Rouillon AServizio Malattie Tromboemboliche, I.R.C.C.SPoliclinico San Matteo, Pavia, Italy; U.O. di Radiodiagnostica,I.R.C.C.S Policlinico San Matteo, Pavia,Italy; Department of Orthopedic Surgery,Chang-Gung Memorial Hospital, Kaohsiung,Taiwan; Department of Orthopedic Surgery, People sHospital, Beijing, China; Division of Pharmacy Practice,The Chinese University, Hong Kong, Hong Kong;Department of Hematology, Singapore GeneralHospital, Singapore, Singapore; Department ofPreventive Medicine, The Catholic University ofKorea, Seoul, Korea; McMaster Clinic, HamiltonGeneral Hospital, Hamilton, ON, Canada; PathologyServices, Flinders Medical Centre, Bedford Park,Australia; Service d Orthopedie, Clinique Radio-Chirurgicale du Mail, La Rochelle, France; Asia-MiddleEast Medical Direction, Sanofi-SynthelaboGroup, Gentilly, FranceIn Western countries venous thromboembolism(VTE) is a common, life-threatening complication inpatients undergoing major orthopedic procedures.Based on the results of contrast venography, theprevalence of total deep vein thrombosis (DVT) at 7 to14 days after total hip replacement (THR), total kneereplacement (TKR), and hip fracture surgery (HFS) isabout 50 to 60%. In contrast, the prevalence of postsurgicalVTE is traditionally thought to be low in Asia,and the use of thromboprophylaxis remains controversial.The objective of AIDA study was to assess theincidence of VTE in THR, TKR or HFS patients notreceiving pharmacological prophylaxis from 7 Asiancountries. The principal outcome measure was objectivelyconfirmed DVT as assessed by mandatory bilateralvenography performed 6 to 10 days after surgeryor symptomatic DVT. DVT was classified as proximal ordistal. Venograms were evaluated by a central panel(C.A.C, I.R.C.C.S. Policlinico San Matteo, Pavia)unaware of the clinical data. After giving theirhaematologica vol. 89(suppl. n. 8):september 2004

82Oral Communicationsinformed consent, consecutive patients from differentAsian ethnic subgroups, hospitalised for elective hipor knee replacement or emergency hip fracturesurgery were enrolled. Patients were then followedfor three months for symptomatic VTE. In case of confirmedDVT, the treatment modalities were accordingto local hospital practice. The study protocol wasapproved by the institutional review boards of theparticipating hospitals. Between July 2002 and March2003, 407 patients were enrolled (175 THR, 136 TKR,96 HF). Three hundreds and sixty three venographieswere centrally adjudicated (89.2% of the enrolledpopulation) and 278 (76.6%) were assessed as adequateby the Adjudication Committee. One hundredand twenty venographies were positive for DVT(43.2%); proximal thrombus (associated or not witha distal thrombus) in 30 cases (10.8%) and distal(only) thrombus in 90 cases (32.4%). Pulmonaryembolism was clinically suspected in 7 cases, andobjectively confirmed in 2 cases. This study definitelyshows that in Asia DVT is a common complicationof major orthopedic surgery of the lower limbs. In theabsence of thromboprophylaxis, venographic DVT rateof 43.2% was observed. This incidence rate is verysimilar to what observed in Western countries. Inalmost 60% of the cases, thrombosis was totallyasymptomatic. These data strongly suggest that VTEprophylaxis should be recommended in Asian patientsundergoing major orthopedic surgery of the lowerlimbs.CO-119THE LONG-TERM CLINICAL COURSE OF ACUTE DEEP VEINTHROMBOSIS OF UPPER EXTREMITIES. A PROSPECTIVECOHORT STUDYPrandoni P, Bernardi E, Marchiori A, Lensing A,*Prins M,^ Villalta S, Bagatella P, Sartor D, Piccioli A,Simioni P, Pagnan A, Girolami ADipartimento di Scienze Mediche e Chirurgiche,Università di Padova, Italy; *Center for VascularMedicine, University of Amsterdam, TheNetherlands; ^Department of Clinical Epidemiology,University of Maastricht, The NetherlandsIntroduction. In contrast with the extensive documentationon the long-term incidence of recurrentvenous thromboembolism (VTE) and post-thromboticsyndrome (PTS) after deep-vein thrombosis (DVT)of the lower extremity, the long-term clinical courseof patients with upper extremity DVT is poorlydefined. Aims. To assess the long-term incidence ofrecurrent venous thromboembolism (VTE) and that ofthe post-thrombotic syndrome (PTS) in patients withthe first episode of DVT of the upper extremity. Materialsand Methods. Fifty-three consecutive patientswith upper extremity DVT received an initial treatmentwith full-dose heparin followed by at leastthree months of oral anticoagulation. Follow-upassessments were scheduled at three and six months,and then every six months up to five years. Diagnosisof recurrent VTE was adjudicated according tostandard methods, and the PTS was assessed usinga standardized scale Results. Of the 53 patients, 3developed a recurrent thrombotic event. The cumulativeincidence of recurrent VTE after one, two andfive years was 2.0% (95% CI, 0.0 to 5.9), 4.2% (0.0to 9.9), and 7.7% (0.0 to 16.5), respectively. The PTSdeveloped in 13 patients (severe in 1). The cumulativeincidence of the PTS was 20.8% (95% CI, 9.3 to32.3) at six months, 25.1% (12.8 to 37.4) at one year,27.3% (14.6 to 40.0) at two years, and remained stableafterwards. Conclusions. Symptomatic upperextremity DVT carries a low risk of recurrent VTE.Post-thrombotic sequelae occur in almost one fourthof patients within the first two years.SISET PrizeCO-120A PROSPECTIVE REGISTRY ON LONG-TERM CLINICALOUTCOME OF VENOUS THROMBOEMBOLISM (MASTER):STUDY METHODOLOGY AND PRELIMINARY RESULTSCaponi C, Agnelli G, Rossi R, Verso M, Moia M,Palareti G, Pistelli R, Allegra C, for The MASTERInvestigatorsDivision of Internal and Cardiovascular Medicine,University of Perugia, Italy and the MASTER StudyCentersBackground. The long-term clinical course ofpatients with venous thromboembolism (VTE) hasnot been completely elucidated. Only limited informationon risk stratification for unfavorable longtermclinical outcome are available. The aim of thisstudy is to collect prospectively information on thelong-term outcome of 2000 patients with acute VTEin 30 Italian centers. Research methodology. Informationabout diagnostic methods, temporary andpermanent risk factors, methods of prophylaxis andtreatment are captured by an electronic data networkat the time of the index event. A 24-monthfollow-up includes clinical examination after 6 and12 months and after 2 years. On-going results. OnApril 20, 2004, 1477 patients have been included inthe registry. 254 patients (17.2%) have known cancersat the time of the DVT/PE index event: 143 ofthese patients are on chemo- or hormone therapy. In44 patients (2.9%) cancer was found at the DVT/PEdiagnosis. Fifty-four patients (3.6%) had a knownthrombophilia at hospital admission. 691 patients(46.8%) have one or more temporary risk factors. Ahaematologica vol. 89(suppl. n. 8):september 2004

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 200483follow-up is currently available in 897 patients(60.7%) at 6 months, 562 patients (38%) at 12months and 67 patients (4.5%) at 24 months. Duringfollow-up 146 events were recorded: 18 VTErecurrences at 6 months and 18 at 12 months; 15bleeding episodes at 6 months and 4 at 12 months;8 new-diagnosed cancer at 6 months and 4 at 12months; 2 myocardial infarction at 6 months and 2at 12 months; 2 strokes at 6 months; 21 deaths at 6months and 15 at 12 months. Conclusions: The VTEpopulation enrolled in this registry appears to be representativeof the broad spectrum of VTE patients.The long-term follow-up will allow to better understandingthe long-term clinical course of VTE and tooptimize and standardize its management.SISET PrizeCO-121PREVALENCE OF SYMPTOMATIC AND ASYMPTOMATICATHEROSCLEROSIS IN PATIENTS WITH IDIOPATHICDEEP-VEIN THROMBOSISSartor D, Petrobelli F, Hartman L, Concolato A,Bilora F, Pagnan A, Prandoni PDipartimento di Scienze Mediche e Chirurgiche,Università di Padova, ItalyBackground. Recently, an association has beendescribed between atherosclerosis and venous thrombosis,suggesting that either atherosclerosis mayinduce venous thrombosis or the two conditions mayshare common risk factors. Aim. To assess whether theprevalence of symptomatic and asymptomatic atherosclerosisin patients with idiopathic deep-vein thrombosis(DVT) is higher than that of patients with thrombosissecondary to known risk factors. Patients andMethods. Out of 180 patients over 50 years who hadsuffered an episode of DVT, alone or associated withpulmonary embolism, between 2000 and 2003, 140were retrieved who fulfilled the study criteria. Of them31 (22.1%) had a history of multiple thromboticepisodes, and 23 (16.4%) were carriers of thrombophilia.At referral, they received an interview withemphasis to previous symptomatic atherosclerosis(coronary artery disease, ischemic stroke, or artheriopathiesof the lower limbs). Then, they underwentthe bilateral ultrasound assessment of carotid arteriesto evaluate the presence of vessel wall plaques.Results. Of the 140 patients, 79 (56.4%) were labeledas having an idiopathic DVT, while in the remaining 61the thrombosis was associated with a known risk factor.The prevalence of symptomatic atherosclerosis inpatients with idiopathic DVT (27/79; 34.2%) was higherthan that observed in patients with secondary DVT(16/61; 26.2%). At least one carotid plaque wasdetected in 52 of the 79 patients (65.8%) with spontaneousthrombosis, and in 32 of the 61 (52.5%) withsecondary thrombosis. The OR for symptomatic atherosclerosisand/or carotid plaques in patients withspontaneous as compared to secondary thrombosiswas 2.0 (95% CI, 1.0 to 3.9). In a multivariate analysistaking into account age, risk factors for atherosclerosis,thrombophilia, and history of previousthrombosis the strength of the association did notchange. Conclusions. The results of this study, conductedin a wide series of patients with symptomaticDVT older than 50 years (including patients with symptomaticatherosclerosis and/or multiple thromboticepisodes), confirm the existence of an associationbetween atherosclerosis and idiopathic venous thrombosis.CO-122ANTITHROMBOTIC PROPHYLAXIS DURING PREGNANCY INWOMEN WITH PREVIOUS VENOUS THROMBOEMBOLISMMartinelli I, Battaglioli T, Mannucci PMAngelo Bianchi Bonomi Hemophilia and ThrombosisCenter, Department of Internal Medicine and Dermatology,Istituto di Ricovero e Cura a CarattereScientifico Ospedale Maggiore Policlinico, Universityof Milan, ItalyPregnancy enhances the risk of venous thromboembolism(VTE). Although previous episodes of VTEare considered risk factors for recurrence, womenwith previous VTE seem to have a low risk for recurrenceduring pregnancy. Therefore, whether or notthey may benefit from antithrombotic prophylaxis isnot established. Thrombosis during pregnancy mayalso involve the placental circulation, resulting inobstetrical complications, such as fetal loss, preeclampsiaand intrauterine growth restriction. Aimsof the study were: i) to assess the efficacy of prophylaxiswith low molecular weight heparin (LMWH)during pregnancy in women with previous VTE. ii) toassess the same regimen in preventing obstetricalcomplications. Sixty women with previous VTE whounderwent a thrombophilia screening were prospectivelyfollowed in 74 pregnancies. Twenty-twowomen without thrombophilia were followed for 28pregnancies and 38 with thrombophilia (3 proteinC-, 1 protein S-, 1 antithrombin-deficiency, 16 factorV Leiden, 10 prothrombin mutation, 1 antiphospholipidantibodies) for 46 pregnancies. Forty-sixpregnancies (32 in women with and 14 in womenwithout thrombophilia) were treated with LMWHfrom the first trimester and 28 (14 in women withand 14 in women without thrombophilia) were not.Two recurrences of VTE during pregnancy, both inwomen with thrombophilia, were observed in thehaematologica vol. 89(suppl. n. 8):september 2004

84Oral Communicationsnon-treated group and zero in the treated group (7%vs 0%). Among women with thrombophilia, therecurrence rate of VTE during pregnancy was 14% inthe non-treated and 0% in the treated group.Obstetrical complications occurred in 5 women ofthe non-treated (18%) and in 8 in the treated group(17%), independently of thrombophilia. In conclusionLMWH during pregnancy is useful in preventingrecurrent VTE in women with thrombophilia, but maynot be warranted in those without. In our selectedcohort of women with previous VTE, heparin did notreduce the rate of obstetrical complications.CO-123PROTEIN Z DEFICIENCY IS NOT ASSOCIATED WITHOCCURRENCE OF UNEXPLAINED FETAL LOSSESGrandone E, Colaizzo D, Cappucci F, Santacroce R,Sciannamè N, Vergura P, Margaglione MIRCCS Casa Sollievo della Sofferenza, Departmentof Medical Genetics, University of Foggia, ItalyProtein Z is a K-dependent glycoprotein with animportant role in the regulation of the coagulationcascade because of a serpin called protein Z-dependentprotease inhibitor (ZPI). ZPI in the presence ofprotein Z causes a fast inactivation of factor XaRecently, it has been shown that Protein Z could havea role in early fetal losses. Since March 1999 to February2003, 453 women women underwent a workupfor identifying known causes of fetal losses. At theend of the work-up, women with known causes offetal losses were excluded (n=271). Moreover,women with inherited or acquired thrombophilia(n=38), as well as those with previous thromboembolicdisease, were excluded (n=3). Thus, 141 womenwere eligible, but blood samples were available for124 of them. Mean age (±1SD) was 32±5.3 years. Allthese women had a history of recurrent (n=3) earlyunexplained fetal losses, defined as the occurrenceof fetal loss until 14 weeks of gestational age or atleast one late fetal death (>20 weeks). Protein Z valueswere 1.40±0.75 mg/mL in the whole group,1.38±0.69 mg/mL in the group with primary recurrentmiscarriages before 9 weeks, 1.24±0.56 mg/mLin the group with fetal losses between the beginningof 10 weeks and the end of 15 th week, and1.28±0.9 mg/mL in women with fetal loss >16weeks. No significant difference was observed comparingeach group with a reference group from thesame ethnic background, formed by 60 women withuneventful pregnancies (mean age 29±4.3 yrs, proteinZ values: 1.43±0.76 mg/mL). We are not able toconfirm data recently published by Gris and coll,although we employed similar selection criteria andhave analyzed data using the same categories. However,our data are in agreement with a more recentreport.CO-124RISK OF DEEP VEIN THROMBOSIS: INTERACTION BETWEENORAL CONTRACEPTIVES AND HIGH FACTOR VIII LEVELSLegnani C, Cini M, Abate C, Bettini F, Mattarozzi S,Palareti GDept. Angiology & Blood Coagulation “MarinoGolinelli”, University Hospital S. Orsola-Malpighi,Bologna, ItalyHigh levels of Factor VIII are a common risk factorfor venous thromboembolism (VTE); it was alsoshown that the combination of oral contraceptive(OC) use and high Factor VIII levels has an addictiveeffect on the VTE risk. The aim of this study was toevaluate the possible interaction between the presenceof high Factor VIII levels and OC use. Factor VIIIlevels were measured by a chromogenic assay in 174women who suffered from VTE in reproductive ageand in 484 healthy women. Subjects carrying thrombophilicalterations were excluded. The median FactorVIII levels were 1.89 (range: 0.45-4.18 IU/mL) forthe patients and 1.41 (range: 0.38-4.57 IU/mL) forthe controls (p1.95IU/mL) was 8.68 (95%CI: 4.62-16.3). Our data alsoshowed that the association between Factor VIII levelsand VTE risk has a dose-response relationship andthat the risk for venous thrombosis steadily increasesfor increasing levels of Factor VIII. Among the174 patients, 85 had experienced VTE during OC. The179 healthy women who had used OC for at least 6months in the two years before presentation but hadstopped OC at least 3 months before the blood samplingwere considered as OC users. The VTE risk of thecombination of OC use and Factor VIII levels in theupper quartile was increased about 16-fold (OR:15.6, 95% CI: 6.52-37.3). In conclusion, we confirmthat elevated Factor VIII levels are a strong risk factorfor venous thromboembolism. Our results alsoshow that there is an increased risk due to OC use inwomen with elevated Factor VIII and, as it has beenpreviously described for Factor V Leiden andG20210A mutations, both factors seem to have asynergistic effect.haematologica vol. 89(suppl. n. 8):september 2004

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 200485CO-125THROMBOTIC COMPLICATIONS AFTER INDUCTION OVULATION:ROLE OF DIFFERENT RISK FACTORSGrandone E, Colaizzo D, Vergura P, Cocomazzi N,Cappucci F, Vecchione G, Lo Bue A, Cittadini E,Margaglione MUnità di Aterosclerosi e Trombosi, IRCCS Casa Sollievodella Sofferenza, S. Giovanni Rotondo; Centrodi Biologia della Riproduzione, Palermo; Cattedra diOstetricia e Ginecologia, Istituto Materno-Infantile,Palermo; Genetica Medica, Università degli Studi diFoggia, ItalyTo calculate the magnitude of thrombotic risk duringcycles of ovulation induction and to investigatethe role of inherited and acquired thrombophilia forthese events. This is an observational study involvingoutpatients of a clinical research center. Consecutivewomen undergoing induction of ovulationfor IVF (n=305) were enrolled. Blood samples forstudying inherited and acquired thrombophilia wereobtained at least two months after the last cycle oftreatment. OR and CI were determined for markerssignificantly associated with thrombotic events.Blood samples were analysed for inherited andacquired causes of thrombophilia (antithrombin, proteinC, protein S, antiphospholipid antibodies, theFactor V Leiden and FIIA20210 mutations, the TT677MTHFR genotype, and homocysteine plasma levels).Thrombotic events were observed in 4/747 cycles ofovulation induction, with a prevalence of 0.5%, correspondingto 1.6 per 100.000 cycles/woman. Age>39 years and homocysteine plasma levels above the97.5 percentile were significantly associated withthrombotic events during IVF (OR 15.2, 95%CI: 2.0-115.0, and OR: 14.4, 95%CI: 1.5-141.3, respectively).Age >39 years and mild hyperhomocysteinemiaare strongly associated with the occurrence ofthrombotic events during IVF.Oral CommunicationsTHROMBOSIS AND CANCER:CELL AND CLINICAL STUDIESCO-126BLOCKADE OF THE RENIN-ANGIOTENSIN SYSTEM ALTERS THEPROCOGUALANT/FIBRINOLYTIC POTENTIAL OF HUMAN BREASTCARCINOMA CELLSNapoleone E, Di Santo A, Amore C, Martelli N,Donati MB,* Lorenzet R“Antonio Taticchi” Unit of Cellular and MolecularBiology of Blood Coagulation, Consorzio MarioNegri Sud, S.Maria Imbaro, and * Center for HighTechnology Research and Education in BiomedicalSciences, Catholic University, Campobasso, ItalyThe renin-angiotensin system (RAS) promotesangiogenesis and growth of neoplastic cells. Inrecent years it has been shown that angiotensinconvertingenzyme (ACE) inhibitors and blockade ofthe angiotensin II (AngII) receptor AT1 may protectagainst cancer, thus suggesting new treatmentstrategies of malignancies. Since a possible role oftissue factor (TF) and of the plasminogen/plasminogenactivator system in the metastatic process hasrepeatedly been proposed, we thought to determinewhether interference with RAS could modulate TF,tissue-plasminogen activator (t-PA) and plasminogenactivator inhibitor-1 (PAI-1) expression in themetastatic breast carcinoma cell line MDA-MB-231.To test this hypothesis, MDA-MB-231 cells were culturedand incubated with or without different ACEinhibitors (captopril and enalapril) at 37°C. At theend of incubation, conditioned medium was collectedand tested for t-PA and PAI-1 antigen levels byELISA, and cells were disrupted and tested for procoagulantactivity by a one-stage clotting assay.Both the strong TF activity constitutively expressedby the cells and the antigen levels of the fibrinolyticfactors were significantly reduced in a dosedependentmanner by the ACE inhibitors. Since flowcytometry clearly showed the presence of the AngIIreceptor AT1 on MDA-MB-231 membrane, we testedwhether blockade of AT1 could affect the procoagulant/fibrinolyticpotential of the cells. Indeed,losartan, a competitive inhibitor of AT1, reduced t-PA and PAI-1 antigen levels and TF activity at adegree similar to that exerted by ACE inhibitors. Thesame effect was observed when an anti-AT1 antibodywas used instead of losartan. To test whetherAngII was involved in the constitutive MDA-MB-231haematologica vol. 89(suppl. n. 8):september 2004

86Oral CommunicationsTF expression the cells were incubated in the presenceof an anti-AngII antibody. In this experimentalsetting, a strong inhibition of TF activity could beobserved. These results could, at least in part, explainthe efficacy of ACE inhibitors and AT1 receptorantagonists in some types of malignancy, and furthersupport their use for tumor control.CO-127LOW-MOLECULAR WEIGHT HEPARINS REDUCE THEFUNCTIONAL ADHESION OF DIFFERENT TUMOR CELLTYPES TO THE VASCULAR ENDOTHELIUMVignoli A, Marchetti M, Agosti M, Falanga ADepartment of Hematology, Ospedali Riuniti,Bergamo, ItalyAdhesion to the vascular wall is an important stepfor tumor cell extravasation and distant metastasisformation. In this study we evaluated the effect oftwo LMWHs (i.e.: dalteparin, DLT, and enoxaparin,ENX) and unfractionated heparin (UFH) on the functionaladhesion of tumor cells to human endothelialcells (EC) of two types: the microvascular cell lineHMEC-1, and the macrovascular HUVEC. After 24hincubation of EC monolayer with each heparin (10IU/mL) ± IL-1β (5 ng/mL), or the vehicle (control cells,C), the adhesion of two human tumor cell lines (i.e.the leukemic NB4 and the breast cancer MDA-MB-231 cell lines) was analyzed. Simultaneously, quantificationof the main EC surface adhesion molecules(ICAM-1, VCAM-1, E-selectin) by flow cytometry andby ELISA was performed. Tumor cell adhesion to ECmatrix produced by heparin-treated EC was also evaluated.All heparins significantly (p

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 200487CO-129THALIDOMIDE IN FRONT LINE TREATMENT IN MULTIPLEMYELOMA: SERIOUS RISK OF VENOUS THROMBO-EMBOLISMRus C, Bazzan M, Palumbo A,* Bringhen S,* Foli C,Vaccarino A, Bertola A,* Falco P,* Cavallo F,*Cangialosi C,* Boccadoro M*Gruppo Italiano Per Lo Studio Del Mieloma Multiplo(GUSMM) 1 U.O.A di Ematologia e MalattieTrombotiche, O. Evangelico Valdese, and *Divisionedi Ematologia dell'Università di Torino, AziendaOspedaliera S. Giovanni Battista, Turin, ItalyRecently thalidomide (Thal) was shown to be effectivein relapsed or refractory multiple myeloma, butthe role of this novel agent as front line treatement isstill under investigation. To address this issue, a multicentric,open, randomized trial was started in January2002 in 30 Departments of haematology in Italy.Myeloma patients at diagnosis were included, if agedmore than 65 (or less, if they refused transplantationfor personal reasons). The trial compares two treatments:oral Melphalan and Prednisone (MP) versusMP in association with low dose Thalidomide(MPThal). The treatment schedule is Melphalan (4mg/m 2 ), Prednisone (40 mg/m 2 ) p.o. for seven consecutivedays every four weeks, for a total of six courses;Thal (100 mg/die) is started on the first day of thefirst MP and continued untill evidence of a relapseddisease or any grade 3-4 toxicities. An interim evaluationhas been performed after two years, covering131 patients. The patients’ characteristics were balancedbetween the two groups (age, immunoglobulinheavy chain type, Bence Jones protein, bone marrowplasmacells, β2 microglobulin) with the exceptionof the stage of disease: stage IIA was prevalent in theMP arm and stage IIIA in the MP Thal arm (p=0.0001).No other significant difference was present in the twogroups. A high prevalence, statistically significant, ofserious thromboembolic events (6 spontaneous proximaldeep vein thrombosis, 1 superficial venousthrombosis, 1 arterial thrombosis, 3 pulmonaryembolisms) was observed in the MPThal section: 11events versus none (v< 0.0007). All clinical eventswere symptomatic and objectively diagnosed. Themajority of the events (73%) occurred during the firstthree months of treatment and was independent ofthe clinical stage. The high incidence of VTE promptedus to define an antithrombotic strategy for patientsin the MPThal group: Enoxaparin 40 mg once a day,in association with elastic stockings if the patient wasimmobilized, or elastic stockings alone when theplatelet count was under 70.000/mm 3 and Enoxaparinwas stopped. The Enoxaparin treatment was scheduledfor 3 months from starting the chemotherapy.The exact cause of this pro-thrombotic effect is atpresent unclear: at present there is no evidence of thebest anti-thrombotic regimen in this clinical setting:a prospective multi-centric and randomized study isrequired.CO-130THE FEASIBILITY OF HOME TREATMENT OF DEEP VEINTHROMBOSIS IN CANCER PATIENTSAgeno W,* Limbiati S,* Dentali F,* Grimwood R,°Steidl L,* Wells P°*Department of Clinical Medicine, University ofInsubria, Varese, Italy, and °Ottawa Health ResearchInstitute, Ottawa, CanadaOutpatient treatment of deep vein thrombosis(DVT) is a common practice in many centers. Canceris often considered an exclusion criterion for hometreatment because of an increased risk of both bleedingand recurrent DVT. We performed a retrospectivereview of clinical practice patterns to assess the rateof cancer patients who were deemed eligible for outpatienttreatment of their DVT. The charts of outpatientswith objectively documented DVT at 2 institutionswere reviewed. All patients were routinely evaluatedfor the home treatment program at both institutions.Usually, only patients with poor clinical conditions,active bleeding or high risk of bleeding, andpain requiring parenteral narcotics were admitted tothe hospital. All patients received low molecularweight heparin (LMWH) and warfarin or LMWHalone. We evaluated a total of 321 patients, meanage 60.4 years, 40.2% were males. Most frequentsites of cancer were genitourinary in 21.2%, breastin 20.5%, and gastrointestinal in 18.4%. Metastaseswere known in 52.5%. Treatment with LMWH andwarfarin was prescribed to 67.0%, LMWH alone to33%. One hundred and ninety seven patients (61.4%)were entirely treated at home. There were no differencesbetween patients treated at home and hospitalizedpatients according to gender, mean age, siteof cancer, presence of metastases, and type of treatment.After 3 months, recurrent thrombosis occurredin 6.1% of patients treated at home and in 4.8% ofhospitalised patients (p=n.s.), major bleeding in 1.0%and in 4.8%, respectively (p=0.03), and minor bleedingin 2.0% and in 4.8%, respectively (p=n.s.). Onehundred and sixty patients died (49.8%), 100 amongpatients treated at home (50.7%) and 60 amonghospitalized patients (49.6%, p=n.s.). Home treatmentof DVT is feasible for almost two thirds ofpatients with concomitant cancer. Outpatient managementof antithrombotic treatment did notincrease the rate of adverse events, even if the stageof the disease was advanced in a considerable rateof patients.CO-131haematologica vol. 89(suppl. n. 8):september 2004

88Oral Communications@RISTOS, A PROSPECTIVE STUDY ON CLINICALLY OVERTVENOUS THROMBOEMBOLISM AFTER CANCER SURGERYRossi R, 1 Agnelli G, 1 Bolis G, 2 Capussotti L, 3Scarpa RM, 4 Tonelli F, 5 Bonizzoni E, 6 Moia M, 7Parazzini F, 8 Sonaglia F, 1 Valarani B, 9Bianchini C, 10 Gussoni G, 10 on behalf ofThe @ RISTOS Study Group1Division of Internal and Cardiovascular Medicine,University of Perugia, Perugia; 2 Obstetrics andGynecology Clinic I, Univesity of Milan, Milan;3Department of Surgical Oncology, Istituto per laRicerca e la Cura del Cancro, Candiolo, Turin;4Department of Urology, “San Luigi” Hospital,Orbassano, Turin; 5 Department of Clinical Physiopathology,University of Florence, Florence; 6 Instituteof Medical Statistics and Biometry, Universityof Milan, Milan; 7 Angelo Bianchi Bonomi Hemophiliaand Thrombosis Center, IRCCS Maggiore Hospital,Milan; 8 Obstetrics and Gynecology Clinic I, Universityof Milan, Milan; 9 Hyperphar Group, Milan;10Scientific Department, Italfarmaco, Milan; ItalyCurrent epidemiology of venous thromboembolism(VTE) after cancer surgery is based on venographyclinical trials on VTE prophylaxis. However, the clinicalrelevance of asymptomatic venography-detectedDVT has been challenged and the study populationof these clinical trials is not representative of theoverall cancer surgery population. Moreover, inrecent years cancer surgery has undergone a numberof changes, hence the need for up to date dataon VTE in this clinical setting. Aim of the study wasto evaluate the incidence of clinically overt VTE in awide-spectrum of patients undergoing surgery forcancer and to identify risk factors for VTE. @RISTOSwas a prospective observational study on consecutivepatients undergoing general, urologic or gynecologicsurgery. Patients were assessed for clinicallyovert VTE occurring up to 30±5 days from surgery ormore in the case of longer hospital stay. The studyincluded 2373 patients: 1238 (52%) undergoinggeneral, 685 (29%) urologic, and 450 (19%) gynecologicsurgery. In-hospital prophylaxis was performedin 81.6% and post-discharge prophylaxis in30.7% of the patients. Fifty patients (2.1%) wereadjudicated as affected by VTE (DVT 0.42%, non fatalpulmonary embolism 0.88% and death 0.80%). Theincidence of VTE was 2.83% in general surgery, 2.0%in gynecologic surgery and 0.87% in urologic surgery.Forty percent of the events occurred later than 21days from surgery. The overall death rate was 1.72%,in 46.3% of the cases due to VTE. Five risk factorswere identified in a multivariate analysis: age above60 years (OR 2.63, 95% CI 1.21-5.71), previous VTE(OR 5.98, 2.13-16.80), advanced cancer (OR 2.68,1.37-5.24), anaesthesia lasting more than 2 hours(OR 4.50, 1.06-19.04) and bed rest longer than 3days (OR 4.37, 2.45-7.78). VTE remains a commoncomplication of cancer surgery, with a remarkableproportion of events occurring late after surgery. VTEis still the most common cause of death in thesepatients.CO-132CATHETER-RELATED COMPLICATIONS IN PATIENTS WITHHEMATOLOGICAL MALIGNANCIES: RESULTS FROM THECATHEM PROJECTMoia M,^ Cortelezzi A,° Falanga A,* Pogliani EM, §Agnelli G, # Gussoni G, $ and the CATHEMInvestigators Study GroupA. Bianchi Bonomi Hemophilia and Thrombosis Center,^Department of Hematology° IRCCS MaggioreHospital and University of Milan, Ospedali Riuniti ofBergamo*, Bicocca University of Milan, § Universityof Perugia, # Scientific Department, ItalfarmacoMilan $, ItalyFew data are available on central venous catheter(CVC)-related thrombotic complications in patientswith hematological malignancies. Thrombocytopenia,which is frequent and often severe in hematologicpatients, might decrease the risk of thrombosis,and also contraindicate the use of anticoagulants.CATHEM was a prospective, observationalstudy performed in 8 Italian Hematology Departments.Aims were i) to assess the incidence and therisk factors for symptomatic CVC-related thromboticcomplications; ii) to to evaluate the hemorrhagicrisk of prophylaxis with anticoagulants. 458 consecutiveCVC positioning in 412 patients (males 50.4%,mean age 50.9±14.7 years) were registered. Mostpatients experienced periods of thrombocytopenia(< 50,000×10 6 /L in 81.2% of cases) and many hadsevere neutropenia (< 100×10 6 /L in 59.8%). 14.2% ofpatients received antithrombotic prophylaxis (LMWHin most cases). The incidences of clinically overt complicationswere (in brackets, patients receiving antithromboticprophylaxis): CVC-related deep veinthrombosis (DVT) 1.5% (0%); DVT of lower limbs0.4% (0%); total pulmonary embolism (PE) 1.3%(0%); fatal PE 0.6% (0%), CVC-related superficialthrombophlebitis 3.9% (0.2%); CVC-occlusion/malfunctionof thrombotic origin 6.1% (1.1%); majorarterial events 1.1% (0.4%); serious bleeding 3.5%(0.4%). A composite study end-point (venous thromboembolism+ superficial thrombophlebitis + CVCocclusion / malfunction) had an overall incidence of12%. It was utilized to assess a possible predictivevalue of selected risk factors. At multivariable analysis,thrombocytopenia was associated with a trend toa reduced risk of CVC-related thrombotic complica-haematologica vol. 89(suppl. n. 8):september 2004

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 200489tions. However, 10.8% (vs 17.4%) of patients withplatelet counts > 50,000x10 6 /L had thrombotic complications.In summary, the incidence of symptomaticCVC-related thrombotic complications in patientswith hematological malignancies is not negligible.Thrombocytopenia seems to reduce, but not to abolish,these thrombotic complications. Prophylaxis withanticoagulants is not apparently related with anincreased risk of bleeding.CO-133A DOUBLE-BLIND PLACEBO-CONTROLLED RANDOMIZEDSTUDY ON ENOXAPARIN FOR THE PREVENTION OFUPPER LIMB DVT IN CANCER PATIENTS WITH CVCVerso M, Agnelli G, Bertoglio S, Ageno W,Bazzan M, Parise P, Quintavalla R, Naglieri E,Santoro A, Imberti D, Sorarù M For The ETHICSInvestigatorsSezione di Medicina Interna e Cardiovascolare,Dipartimento di Medicina Interna, Perugia; UnitàOrganica di Oncologia Chirurgica, Genova; Divisionedi Medicina Interna, Ospedale di Circolo,Varese; Divisione di Ematologia, Ospedale EvangelicoValdese, Torino; Divisione di Medicina, Gubbio;Divisione di Medicina, Parma; Unità Organica diOncologia Medica e Sperimentale, IRCCO Oncologico,Bari; Unità Organica di Oncologia, OspedaleHumanitas, Milano; Divisione di Oncoematologia,Piacenza, Divisione di Oncologia Medica, Camposampiero,ItalyEfficacy of prophylaxis with fixed dose of warfarinor low molecular weight heparin (LMWH) for upperlimb deep vein thrombosis (UL-DVT) related to centralvein catheter (CVC) has been claimed after openstudies with limited sample size. The rate of bleedingin cancer patients receiving prolonged prophylaxiswith LMWH is undefined. The aim of this studywas to evaluate the efficacy and safety of the LMWHenoxaparin in the prevention of UL-DVT in cancerpatients with CVC. Methods. Consecutive cancerpatients with CVC for chemotherapy were includedin a multicenter double-blind randomized placebocontrolledstudy performed in 11 Italian centers.Enoxaparin, 40 mg once a day, or placebo were givensubcutaneously for 6 weeks, starting 2 hoursbefore the CVC insertion. The primary endpoint ofthe study was UL-DVT, as detected by venography(CVC limb) performed at 6 weeks and/or clinicallyovert pulmonary embolism confirmed by objectivetesting. The safety endpoint was major bleeding.With a postulated incidence of DVT of 30% in theplacebo group and 15% in the enoxaparin group,300 evaluable patients were required. Results. 385patients were included in the study. 321 patientsunderwent venography (83.4%). The primary efficacyoutcome was assessed in 310 patients with adequatevenography. Enoxaparin reduced the incidenceof UL-DVT from 18.1% (28/155) to 14.2% (22/155),a relative risk reduction of 21.4% (95% CI: 0.47 to1.31, p=0.35). Two patients in the enoxaparin group(1.0%) and 6 patients in the placebo group (3.1percent)had a symptomatic venous thromboembolism.No major bleeding occurred in both treatmentgroups. Minor bleeding occurred in 6.3% of thepatients (12/191) in the enoxaparin group and 3.6%of the patients (7/194) in the placebo group. CON-CLUSIONS: Enoxaparin, at the dose of 40 mg daily,produced a 21% non significant reduction in theincidence of CVC-related DVT in cancer patients. Thisdose was safe and well tolerated: this leaves openthe option of increasing the dose of enoxaparin tooptimize its efficacy in this clinical setting.haematologica vol. 89(suppl. n. 8):september 2004

90Oral CommunicationsOral CommunicationsVON WILLEBRAND FACTOR AND DISEASECO-134IN VITRO EXPRESSION STUDY OF A NEW MUTATION (R1308L)FOUND IN A FAMILY WITH TYPE 2B VON WILLEBRAND DISEASECHARACTERIZED BY ALL SET OF MULTIMERS IN PLASMA ANDNO THROMBOCYTOPENIA AFTER DESMOPRESSINBaronciani L, Federici AB, Cozzi G, Beretta M,Canciani MT, Mannucci PMAngelo Bianchi Bonomi Hemophilia and ThrombosisCenter, IRCCS Maggiore Hospital and University ofMilan, ItalyType 2B von Willebrand disease (VWD) is characterizedby enhanced ristocetin-induced plateletagglutination (RIPA) and by loss of high molecularweigh multimers (HMWM) in plasma. A novel 2B vonWillebrand factor (VWF) variant (4173G>T, R1308L)was found in four patients with RIPA (0.3-0.4mg/mL), VWF:Ag (27-51 U/dL), VWF:RCo (23-49U/dL), bleeding time (6-11 min), all set of multimersin plasma and normal platelet count. Interestingly, acommon VWD type 2B mutation, R1308C is characterizedby loss of HMWM, low platelet count andRIPA (0.7-0.8 mg/mL), VWF:Ag (26-48 U/dL),VWF:RCo (13-39 U/dL), bleeding time (10-27 min).Mutated rVWF-R1308L and rVWF-R1308C, weretransiently expressed in Cos 7 cells, and analyzed fortheir ability to bind GpIb receptor along with rVWF-WT. Binding of rVWFs to the GpIb platelet receptorwas tested by an ELISA method (Federici et al.Haematologica 89:77, 2004), at increasing concentrationsof ristocetin (0, 0.125, 0.25 and 0.5 mg/mL),and the rVWF bound to GpIb; was revealed by anti-VWF Antibody-HRP reading O.D. 492 nm. The threerVWFs have a similar VWF:Ag and a full set of multimers.Our data demonstrated that the new mutation(R1308L) can enhance the VWF capacity to bindto GpIb receptor, but with lower efficiency comparedto the R1308C variant. This might explain why thesepatients have normal VWF multimers in plasma. Theincreased binding capacity of VWF-R1308L for GpIbdoes not cause its spontaneous interaction withplatelets, or at least, not as efficiently as VWF-R1308C does. The enhanced RIPA of 0.4 mg/mL,observed in patients with VWF-R1308L, versus 0.7mg/mL of VWF-R1308C is probably due to the presenceof all set of multimers, that strongly compensatefor the lower binding capacity to GpIb of thisnew variant.CO-135BIOCHEMICAL CHARACTERIZATION OF RECOMBINANT VONWILLEBRAND FACTOR (VWF) WITH A TYPE 2B MUTATION(P1337L) COEXPRESSED WITH A TYPE 1 VWF DEFECT (C275R):A COMPLEX MOLECULAR DEFECT IN A PATIENT PREVIOUSLYDIAGNOSED WITH TYPE 2A VON WILLEBRAND DISEASEBaronciani L, Federici AB, Beretta M, Cozzi G,Canciani MT, Mannucci PMAngelo Bianchi Bonomi Hemophilia and ThrombosisCenter, IRCCS Maggiore Hospital and University ofMilan, ItalyIn a patient with apparently type 2A VWD (meanvalues: FVIII:C = 32 U/dL, VWF:Ag = 7 U/dL, VWF:RCo= < 6 U/dL, VWF:CB = 2.0 mg/mL, loss ofhigh and intermediate molecular weight multimersin plasma and low platelet VWF) a transient thrombocytopeniaoccurred after an infusion test withdesmopressin. The propositus' brother showed a lesssevere laboratory data (mean values: FVIII:C = 36U/dL, VWF:Ag = 17 U/dL, VWF:RCo = 6 U/dL,VWF:CB=1, RIPA = 1.2 mg/mL). The two brothersresulted to be compound heterozygous for mutationP1337L and a novel candidate defect C275R. In theirchildren, mutations were associated with type 2B(P1337L) and type 1 (C275R) VWD. rVWF-P1337Land rVWF-C275R, were transiently expressed in Cos7 cells, on their own, together and with the rWVF-WT. VWFs. rVWFs were tested for VWF:Ag and multimeranalysis. Binding of rVWFs to the GpIb plateletreceptor was evaluated by an ELISA method (Federiciet al. Haematologica 89:77, 2004), at increasingconcentrations of ristocetin (0, 0.125, 0.25, 0.5, 0.8and 1 mg/mL), and the rVWF bound to GpIb wasrevealed by anti-VWF Antibody-HRP reading O.D.492 nm. Only the expression of rVWF-C275R aloneshowed a strongly reduced VWF:Ag in cells mediumand a complete absence of multimers. The remainingrVWFs (P1337L, P1337L/WT and C275R/WT)showed only a slightly reduce VWF:Ag, in comparisonto rVWF-WT, and a full set of multimers. GpIbbinding assay (Table) showed that rVWF obtained byco-expression of mutation P1337L with C275Rbehave very similarly to rVWF-P1337L. It seems thatrVWF-C275R molecules do not contribute with therVWF-P1337L to form multimers.rVWFs Ristocetin Ristocetin Ristocetin Ristocetin Ristocetin Ristocetin0 mg/ml 0.125 mg/ml 0.25 mg/ml 0.5 mg/ml 0.8 mg/ml 1 mg/mlWT 0.066 O.D. 0.071 O.D. 0.128 O.D. 0.154 O.D. 0.691 O.D. 0.924 O.D.P1337L 0.117 O.D. 0.310 O.D. 0.689 O.D. 0.984 O.D. 0.984 O.D. 1.039 O.D.P1337L/WT 0.063 O.D. 0.166 O.D. 0.409 O.D. 0.740 O.D. 0.894 O.D. 1.008 O.D.P1337L/C275R 0.084 O.D. 0.372 O.D. 0.635 O.D. 0.925 O.D. 1.105 O.D. 1.117 O.D.haematologica vol. 89(suppl. n. 8):september 2004

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 200491Therefore in the propositus and his brother mutationP1337L results to be present in all the VWF subunits,which might explain their more severe phenotypein comparison with their children that carry onlymutation P1337L.CO-136MODULATION OF THE VON WILLEBRAND FACTOR A1 DOMAINBINDING TO PLATELET GLYCOPROTEIN IB BY α-THROMBINMendolicchio GL, Celikel R, Varughese KI, Ruggeri ZMDivisions of Experimental Thrombosis and Hemostasisand of Cellular Biology, The Scripps ResearchInstitute, La Jolla, CA, USAWe used the known crystal structures of the aminoterminal domain of glycoprotein (GP) Ibα bound tothe von Willebrand factor (VWF) A1 domain or to α-thrombin to model a putative triple complex of thetwo ligands interacting with the same receptor molecule.The model indicates that intermolecular contactsmay be established between the VWF A1domain and α-thrombin bound to the receptor, andsuggests that these additional interactions could stabilizethe intrinsically low affinity binding of the VWFA1 domain. This observation delineates a potentialrole of α-thrombin in the initial stages of plateletdeposition on a thrombogenic surface through amodulation of the GP Ibα interaction with VWF. Toverify the predictions of the model, we used gel electrophoresisunder native conditions and purifiedcomponents in solution to demonstrate directly theformation of a triple complex. We then measuredthe binding of 125 I-labeled dimeric VWF A1 domainto washed human platelets in the presence of α-thrombin. There was only a minimal interaction when125I-labeled VWF A1 domain was incubated withwashed platelets, but the binding increased by morethan 3-fold when α-thrombin was added into thereaction mixture. Evaluation of the binding of 125 I-labeled VWF A1 domain to platelets by Scatchardtypeanalysis revealed the occurrence of a measurablespecific interaction only in the presence of α-thrombin. Moreover, in a platelet agglutination/aggregationmodel dependent on the interactionbetween dimeric VWF A1 domain and GPIbα, theaddition of α-thrombin substantially increased theextent of the platelet response. Our findings delineatea mechanism through which α-thrombin maystabilize platelet-surface and platelet-platelet contactsby mediating a tighter association of the VWFA1 domain to the GPIbα receptor.CO-137REGULATORY ROLE OF PLATELET GPIB-α ON THROMBIN'SHEMOSTATIC FUNCTIONSDe Cristofaro R,° De Filippis V*°Hemostasis Research Center, Catholic UniversitySchool of Medicine, Rome; *Department ofPharmaceutical Sciences and CRIBI BiotechnologyCenter, University of Padua, ItalyRecent studies indicated that platelet GpIb-α bindsto thrombin at the domain referred to as either anionbinding exosite 2 or heparin binding site (HBS), whichis involved in thrombin-FVIII interaction as well. FVIIIactivation by thrombin plays an important role in theamplification of the coagulation cascade and takesplace through specific proteolytic cleavages at Arg372,Arg740 and Arg1689. A full FVIII activation requirescleavage at Arg372. This study was aimed at investigatingthe effect of GpIb-α (1-282 fragment) bindingto thrombin HBS on FVIII activation. Similar experimentswere also carried out using a synthetic peptidemodeled on the 268-282 sequence of GpIba, and successfullyeither sulfated or phosphorylated at all tyrosineresidues present along its sequence, at position276, 278, and 279. Both GpIbα 1-282 and the GpIb268-282 peptides induced a progressive decrease (upto 70%) of FVIIIa generation, assessed by coagulationand FXa generation assays. Furthermore, SDS-PAGE/Western blot experiments showed that the specificappearance of the 44-kDa A2 domain upon cleavageof the FVIII Arg372 peptide bond was significantlydelayed in the presence of either GpIbα 1-282, orthe GpIb 268-282 peptides. Moreover, the effect ofthe latter on thrombin hydrolysis of a peptide havingthe sequence 341-376 of FVIII, was investigatedthrough reversed-phase HPLC. The kcat/Km value ofthe FVIII 341-376 peptide hydrolysis by thrombin linearlydecreased as a function of the GpIbα 268-282peptide, according to a competitive inhibition effect.Moreover, the phosphorylated GpIb peptide was ableto non competitively inhibit the thrombin amidaseactivity toward both fibrinogen and Phe-Pip-Arg-pNAsubstrate. These experiments globally suggest thatbinding of the GpIba's 268-282 region to thrombinHBS is responsible α) for the competitive inhibition ofthe Arg372-Ser373 bond’s cleavage and activation ofFVIII and b) for a non competitive inhibition of thethrombin's amidase activity toward fibrinogen. Thesefindings suggest that thrombin-GpIb interaction mayplay a regulatory role on the enzyme’s hemostaticactivities.haematologica vol. 89(suppl. n. 8):september 2004

92Oral CommunicationsCO-138EVALUATION OF A BLEEDING SCORE FOR THE DIAGNOSISOF TYPE 1 VWDTosetto A,* Castaman G,* Roberti S,* Cappelletti A,*Peake I,° Goodeve A,° Federici AB, + Batlle J,^Meyer D,** Eikenboom J,°° Schneppenheim R, ++Ingerslev J,^^ Vorlova Z,*** Lethagen S,°°° Pasi J, +++Hill F,^^^ Rodeghiero F**San Bortolo Hospital, Vicenza, Italy; °University ofSheffield, UK; + University of Milan, Italy; ^HospitalTeresa Herrera, Spain; **INSERM, France; °°LeidenUniversity Medical Center, Netherlands; ++ UniversityChildren Hospital, Hamburg, Germany; ^^UniversityHospital Skejby, Denmark; ***Institute of Hematologyand Blood Transfusion, Prague, Czech Republic;°°°University of Lund, Sweden; +++ Leicester RoyalInfirmary, UK; ^^^Birmingham Children Hospital, UKType 1 von Willebrand’s disease (VWD) is the mostprevalent hemorrhagic disorder in the population.Since no clinical or laboratory test is sufficiently specificand sensitive for the diagnosis of VWD, properclinical assessment is mandatory for the diagnosis. Toassess the sensitivity and specificity of a quantitativeclinical evaluation tool for the diagnosis of type 1VWD. A standardized bleeding questionnaire was evaluatedin 744 subjects belonging to 154 families witha diagnosis of type 1 VWD. The sample comprises 154index cases (IC), 277 affected family members (AFM)and 313 non affected family members (NAFM). Bleedingsymptoms were entered in a database and for eachsubject a bleeding score was automatically generatedby a computer program. The score is the sum of thescore attributed to each bleeding symptom reported bythe individual from birth to the moment of diagnosis.For each symptom (e.g, epistaxis, bleeding after toothextaction or surgery, menorrhagia) the score rangefrom 0 (no bleeding) to 4 (major bleeding requiringblood transfusion and/or replacement therapy). A-1score was attributed when no bleeding symptom wasreported despite at least two tooth extractions, surgeriesor deliveries. The median score was 0 in NAFM,5 in AFM, 9 in IC. A score above 2 identified 77% ofall affected individuals (AFM and IC) and excludes 88%of NAFM. There was a clear biological relationshipbetween the score and VWF:Rco (χ 2 =192.1, p

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 200493CO-140DUODENAL AND GASTRIC DIEULAFOY'S LESIONS IN A PATIENTWITH TYPE 2A VON WILLEBRAND DISEASE: A CASE REPORTMarchese M, # De Cristofaro R,* Federici AB,^Biondi A, § Petruzziello L, # Tringali A, #Mutignani M, # Spada C, # Ronconi P, §Costamagna G ##Digestive Endoscopy Unit, Department of SurgicalSciences Catholic University, Rome; *HemostasisResearch Center, Institute of Internal Medicine andGeriatrics, Catholic University School of Medicine,Rome; § General Surgery Unit, Department ofSurgical Sciences Catholic University, Rome;^Angelo Bianchi Bonomi Hemophilia ThrombosisCenter, Department of Internal Medicine andDermatology, IRCCS Maggiore Hospital andUniversity of Milan, ItalyGastro-intestinal (GI) bleeding is a relatively commoncause of spontaneous bleeding in patients withcongenital bleeding disorders and can be dramatic andlife-threatening. Vascular disorders are the most commoncause of GI bleeding and although endoscopicallysimilar, they consist of various pathological entities.Previous findings indicated that bleeding and GIangiodysplasia associated with VWD could be relatedto the absence of the higher molecular multimers ofVWF (Von Willebrand Factor) in plasma, being prevalentin congenital and acquired forms of type 2 VWD.Among vascular anomalies, angiodysplasia is the mostcommon lesion and consists of dilated veins includingcapillaries containing no dysplastic tissue whichaffects predominantly lower GI tract. Dieulafoy’slesions is another vascular disorder which are generallylocalized in the upper GI tract. These lesions arecharacterized by abnormally large and tortuous arteryrunning within the submucosa and represent an oftenunrecognized cause of spontaneous GI bleeding,potentially fatal in patients with bleeding disorder. Wedescribe here a patient with type 2A von Willebrand’sdisease (VWD-2A) characterized by a long history ofintermittent episodes of melena. He experienced twoepisodes of severe upper GI bleeding due to doublemetachronous Dieulafoy’s lesions successfully controlledwith endoscopic argon plasma coagulation. Toour knowledge, this is the first report of bleeding froma double, metachronous, Dieulafoy’s lesion in a patientaffected from VWD-2A. Our report shows not only thatemergency endoscopy could promptly identify, in twoconsecutive episodes of melena, bleeding lesions misdiagnosedat previous endoscopic explorations in aVWD-2A patient who had required daily very high doseof FVIII/VWF concentrate but also that epinephrineinjection and Argon Plasma Coagulation might representa viable and effective therapy for Dieulafoy’slesions in the long-term. In conclusion, Dieulafoy’slesions are a possible causes of recurrent and potentiallylife-threatening GI bleeding also in VWD. In thesepatients, awareness of this condition and earlyendoscopy during a bleeding episode are essential fora correct diagnosis. Previous infusion of FVIII/VWFconcentrate (or desmopressin administration in moderatetype 1 VWD) followed by endoscopic procedureinvolving epinephrine injection and Argon PlasmaCoagulator offers excellent immediate and long-termresults.SISET PrizeCO-141NO CHANGES OF ENDOGENOUS VON WILLEBRAND FACTORLEVELS DURING PREGNANCY IN A PATIENT WITH TYPE 2MVICENZA (R1205H)Dallagiovanna S, Canciani MT, Lai V, Moroni B,Baronciani L, Cozzi G, Castaldo M, Mistretta C,Coppola R, Federici ABAngelo Bianchi Bonomi Hemophilia and ThrombosisCenter, IRCCS Maggiore Hospital and University ofMilan, ItalyWomen with Von Willebrand Disease (VWD) type 1and 2 usually show increasing von Willebrand factor(VWF) levels during pregnancy, due to the constitutiverelease of VWF by the highly vascularized foeto-placentalunit. Patients with type 2M Vicenza are characterizedby very low plasma levels of VWF activitiesin the presence of supranormal multimers similar tothose found in platelet α granules and endothelialWeibel-Palade bodies. Since the original descriptionby Rodeghiero, many other families have been reportedin different countries (Germany, USA, UK) and twodistinct mutations have been identified (R1205H,M740I). However the basic mechanisms of this VWDvariant remain not completely understood despitemany attempts to study these recombinant VWFmutants after their expression in vitro. We had recentlythe opportunity to follow the pregnancy of a 37year-old woman with Type 2M Vicenza R1205H.Before pregnancy, she was exposed to an infusion trialwith desmopressin (DDAVP). During pregnancy, shehas been followed with monthly measurements ofFVIII/VWF activities and multimeric analysis of VWF.Since VWF plasma levels were < 6 U/dL, the VWF:RCowas tested by our sensitive ELISA method usingrecombinant GpIb. Basal levels of BT and FVIII/VWFactivities were the same during the last five years(mean values): BT = 6.30 minutes; FVIII:C=18 U/dLVWF:Ag=5 U/dL; VWF:RCo = 3 U/dL, with normal VWFplatelet content and supranormal multimers in plasma.She was considered responsive to DDAVP becauseFVIII:C, VWF:Ag and VWF:RCo were still relatively highat 2 hours post-DDAVP with values of 62, 36 and 32haematologica vol. 89(suppl. n. 8):september 2004

94PostersU/dL respectively. Conversely, the levels of the FVI-II/VWF activities measured at month 3, 6, 9 did notchange at all during pregnancy. These clinical datamight support the hypothesis raised by severalauthors that this type 2M VWD variant is due todefects of constitutive release of VWF.PostersVASCULAR BIOLOGYPO-001INFLAMMATORY MARKERS ARE ASSOCIATED WITH THEABDOMINAL AORTIC DIAMETERMiniati B,* Macchi C,* Marcucci R, Gori AM,Conti AA, Corsi AM,° Bandinelli S,° Ferrucci L,**Abbate R, Gensini GFDepartment of Medical and Surgical Critical Area,University of Florence, Florence, Italy; *FondazioneDon Gnocchi IRCCS, Florence, Italy; °Laboratory ofClinical Epidemiology, Geriatric Department,National Institute of Research and Care on Aging(INRCA), Florence, Italy; **Longitudinal StudiesSection, Gerontology Research Center, NationalInstitute on Aging, National Institutes of Health,Baltimore, USAThe inflammatory response plays a crucial role inthe degeneration of the elastic media, which, in turn,promotes aneurysm remodeling of the aortic wall.Degeneration of the extracellular matrix proteins,which characterizes the alteration of the elasticmedia of abdominal aortic aneurysms, is a pathologicalprocess that occurs as a consequence of complexinteractions among genetic factors, inflammatorycytokines and matrix metalloproteases. Aim ofour study was to evaluate the relationship betweeninflammatory proteins and aortic diameter. We studied346 participants of the In Chianti Study (151males and 195 females) (median age 70, 23-93 yrs).In all subjects, we evaluated C-Reactive Protein(CRP), interleukin-18 (IL-18), interleukin-6 (IL-6),interleukin 1-β (IL-1β), transforming growth factorBeta1 (TGF-β1), and tumor necrosis factor-α (TNFα)serum levels and the abdominal aortic diameterby ultrasound scanning examination. The associationbetween chronic inflammation on aortic diameterwas tested in the multivariate regression analysis,which included log-transformed aortic diameteras dependent variable and age, sex, hypertension,diabetes, coronary artery disease, body mass index,dyslipidemia, smoking habit and inflammatory markersas independent variables. We observed that thecirculating levels of IL-18 and CRP significantly associatedwith abdominal aortic diameter. At multivariatelogistic regression analysis, after adjustment fortraditional cardiovascular risk factors, subjects whohad IL-18 serum levels in the highest tertile comparedto the lowest tertile of the distribution had anhaematologica vol. 89(suppl. n. 8):september 2004

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 200495increased risk of being in the highest (> 19 mm) aorticdiameter group (OR= 4.0, 95% CI 1.3-11.7,p=0.02). High levels of CRP (top tertile vs lowest tertile)are associated, but not significantly, with anincreased risk of having high aortic diameter (OR=1.3; 95% CI 0.8-2.0 for aortic diameter >19 mm).These results strengthen the concept of a possiblelink between inflammation and aortic remodelling.PO-002EXPRESSION PROFILE OF DIFFERENT COAGULATION MARKERSIN HUMAN PLACENTAS FROM UNEVENTFUL PREGNANCIESChinni E, Colaizzo D, Bossone A, Vecchione G,Cocomazzi N, Martinelli P, Pecoraro M,Sciannamè N, Margaglione M, Grandone EIRCCS Casa Sollievo della Sofferenza, Ostetricia eGinecologia, Università Federico II Napoli, GeneticaMedica, Università degli studi di Foggia, ItalyPlacental throphoblasts express and produce coagulationcomponents, partecipating not only in hemostatsis,but also in the placental vascular developmentand differentiation. The expression of tissue factor(TF), membrane phosphatidylserine and fibrin renderthe throphoblasts pro-coagulant, thus compromisingthe risk of bleeding while exposing the placenta topro-thrombotic risks. Local inhibitory mechanisms actvia TFPI-1 and TFPI-2, thrombomodulin, annexi nV.Pregnancy complications have been associated withabnormalities in the functions of these inhibitors.Moreover, different isoforms of the thromboxane A2receptor seem to be differently expressed in mice withfetal growth restricted fetuses. The aim of our studywas to investigate whether different areas of placentaexpress different amounts of the same marker inorder to obtain information about homogeneity ofexpression. Here we present data obtained from 4 placentasof women with successful pregnancy outcome.The placentas were dissected starting from the inserctionof umbilical cord towards the peripheral zone.From each section RNA was extracted by means InvitrogenTrizol‚Reagent. Then cDNA was obtained bymeans RT-PCR (Promega Reverse trascription system),and the expression of TF, TFPI, TFPI-2, PAI-2, andthromboxane receptor α-isoform and β-isoform wasevaluated in different areas of placenta by means ofAPPLERA ABI PRISM 7700. Data were expressed aspercent of GAPDH for each sample analyzed. Data areshown in the Table. No significant difference (p>0.05)between central and peripheral areas of placentas wasobserved for all markers analysed. These preliminarydata will be useful for comparing the expression ofthese markers in normal placentas with that of placentasfrom complicated pregnancies.TF% TFPI% TFPI-2% PAI-2 TXA2R a TXA2R bMean SD Mean SD Mean SD Mean SD Mean SD Mean SDN 105 8,2 112,5 5,8 135 15,1 92 5,8 111 3,7 75 3,1A 105,3 25,6 114 2,6 136 4,7 93 1,8 86,4 4,4 76 3,6B 105,6 4,2 112,5 2,9 134 7,6 94,5 3,1 86,3 4 79 2,6Legenda: N: zone corresponding to the inserction of umbilical cord; A: zone next to N, B: marginalzone of placenta.PO-003PPAR-γ EXPRESSION AND MODULATION IN HUMANSTIMULATED FIBROBLASTSEvangelisti L, Giusti B, Bandinelli B, Attanasio M,Lapini I, Lucarini L, Cesari F, Filoni C, Ferrini S,Abbate R, Gensini GF, Pepe GDipartimento di Area Critica Medico Chirurgica,Università degli Studi di Firenze, ItalyPeroxisome proliferator-activated receptors-γ(PPAR-γ) is a nuclear transcription factor that isinvolved in inflammatory process by modulating theproduction of pro-inflammatory cytokines. Severalstudies have demonstrated that PPAR-gamma isexpressed by different human cells (adipose, endothelialand smooth muscle cells) and displays antiinflammatoryproperties. Recently, in rat sinovialfibroblasts, the expression of PPAR-γ and its LPSinducedreduction were demonstrated at both mRNAand protein levels. Data about PPAR-γ expression byhuman fibroblasts are not available. Aim of this studywas to investigate whether PPAR-γ is expressed byhuman skin and adventitial fibroblasts, and to evaluatethe modulation of PPAR-γ expression by differentinflammatory stimuli. For this purpose, we grewdermal fibroblasts from three healthy subjects andfibroblasts from the adventitia of three patientsaffected by abdominal aortic aneurysm. Fibroblastsfrom skin biopsy of controls and from adventitia ofpatients were cultured and incubated with differentinflammatory stimuli (LPS 10 µg/mL; TNF α 50 ng/mL+ IFN-γ 300 U/mL) and incubation times (0h, 6h,12h, 24h). PPAR-γ mRNA from fibroblasts wasassayed by a semi-quantitative RT-PCR method. TheRT-PCR products were quantitated after gel electrophoresisby using the GAPDH mRNA as internalcontrol. Time-course experiments with LPS and TNFα/IFN-γstimuli carried out on adventitial fibroblastsshowed an increase of PPAR-gamma expression at 6,12 and 24 hours respectively; also stimulated dermalfibroblasts displayed an increased PPAR-γ expression,but of minor intensity than adventitial fibroblasts.At the best of our knowledge, this is the firsthaematologica vol. 89(suppl. n. 8):september 2004

96Postersreport showing the presence of PPAR-γ expression byhuman dermal and adventitial fibroblasts. Our dataindicate that PPAR-gamma expression is modulatedby inflammatory stimuli and that the fibroblasts fromadventitia react more intensively.PO-004ANTITHROMBOTIC AND ANTIIFLAMMATORY PROPERTIES OFROSUVASTAIN IN CULTURED HUMAN ENDOTHELIAS CELLSMussoni L, # Brambilla M, # Banas A, # Eligini S, #Camera M,* # Tremoli E, # * Colli S ##Dipartimento di Scienze Farmacologiche,Università degli Studi di Milano, Milan, *CentroCardiologico Monzino, IRCCS, Milan, ItalyRosuvastatin (R) is a new 3-hydroxy-3-methylglutarylcoenzyme A reductase (HMG-CoA) inhibitorwith distinct physicochemical, pharmacokinetic andpharmacologic properties; it is relatively hydrophilic,with a plasma half-life of about 20 h, and, besidesplasma LDL-cholesterol level reduction, it lowerstriglycerides and increases HDL-cholesterol inpatients with mixed dyslipidemia or hypertriglyceridemia.Statins, independently of their lipid loweringeffect, modulate several mechanisms that areinvolved in the atherothrombotic process. In the presentstudy we evaluated the effect of rosuvastatin onPAI-1 and tissue factor (TF) in cultured human umbilicalvein endothelial cells (HUVEC). Moreover, thecapacity of the drug to control inflammation wasassessed through the evaluation of eNOS and Cox-2levels. Rosuvastatin, concentration-dependently (10-100 µM) reduced PAI-1 antigen, either secreted orstored, in resting HUVEC (10 µM R: -68%, p< 0.01).In contrast, TF activity induced by TNFα was scarcelyaffected, even at the highest concentration used(100 µM). eNOS levels, detected by Western blotanalysis, were increased by rosuvastatin in a concentration-dependentfashion (25 µM R: +76%,p

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 200497PO-006EFFECTS OF BEMIPARIN ON TISSUE FACTOR PATHWAYINHIBITOR RELEASE BY HUMAN UMBILICAL VEINENDOTHELIAL CELLS (HUVEC) AND ON CYTOKINEPRODUCTION BY MONONUCLEAR CELLS: IN VITRO STUDIESGori AM, Gazzini A, Evangelisti L, Attanasio M,Lapini I, Rossi L, Lucarini L, Lari B, Ferrini S,Prisco D, Abbate R, Gensini GFDepartment of Medical and Surgical Critical Area,University of Florence, Florence, ItalyBoth unfractionated heparin (UFH) and low molecularweight heparins (LMWHs) are able to release TFPIfrom the vascular endothelium. There are also evidencesof the anti-inflammatory activities of heparinand heparin-like molecules. Bemiparin is a low molecularweight heparin effective for preventing venousthromboembolism. Aim of our study was to evaluatethe effect of bemiparin on: 1) TFPI release from humanendothelial cells; 2) interleukin-6 (IL-6) and interleukin-10(IL-10) production by stimulated wholeblood. We performed ten independent experiments inwhich human endothelial cells (HUVEC), isolated fromumbilical vein (2x10 5 cells/well), were incubated for 1hour at 37°C in the presence of 0, 0.25, 0.5, 1.0U/anti-Xa/mL of bemiparin and 1 U anti-Xa/mL ofunfractionated heparin (UFH). Whole blood from 10healthy male subjects were diluted 1:10 with RPMI-HEPES buffer, stimulated with LPS (10 µg/mL) andincubated (106 cell/mL) with different concentrationsof bemiparin for 24 hours and in the supernatants IL-6 and IL-10 concentrations were determined. Incubationof HUVEC for 1 hour in the presence of 0.25, 0.5and 1.0 U anti-Xa/mL of bemiparin caused a dosedependentrelease of TFPI from HUVEC (medium:0.65±0.19 ng/10 5 cells; 0.25 U anti-Xa/mL:1.20±0.29ng/10 5 cells; 0.5 U anti-Xa/mL: 2.20±0.39 ng/10 5 cells;1 U anti-Xa/mL:2.9±0.33 ng/10 5 cells). Bemiparin at1 U anti-Xa/mL induced a release of TFPI similar, inamounts, to that of induced by UFH (1 IU anti-Xa/mL)(Bemiparin: 2.91±0.33 ng/10 5 cells UFH: 2.83±0.30ng/10 5 cells). The simultaneous addition of LPS (10µg/mL) and bemiparin at different concentrationsslightly, but not significantly inhibits IL-6 productionby stimulated whole blood (LPS: 2.75±0.99 ng/10 6cells; Bemiparin 1 U anti-Xa/mL: 2.25 ±1.13 ng/10 6cells). Increasing concentrations of bemiparin producedhigher amounts of IL-10 than in LPS stimulatedsamples (Bemiparin 0.25 U: 0.34±0.11 ng/10 6 cells;0.5 U: 0.35±0.11 ng/10 6 cells; 1 U: 0.39±0.10 ng/10 6cells, buffer controls: 0.33 ±0.09 ng/10 6 cells; UFH1U: 0.38±0.12 ng/10 6 cells). Our results demonstratethat bemiparin is able to induce TFPI release fromhuman HUVEC in a dose-dependent manner. The abilityof bemiparin of affecting the inflammatory mediatorsstimulates further studies.PostersPLATELETS: PHYSIOLOGICALAND CLINICAL ASPECTSPO-007INCREASE IN CYTOSOLIC CALCIUM AND SODIUM IONCONCENTRATION INDUCES SEROTONIN EFFLUX FROMHUMAN PLATELETSDeana R, Turetta L, Folda A, Bulato C, Pavanetto M,Donella Deana ADepartment of Biological Chemistry and Institute ofNeurosciences of Italian Research National Centre(CNR), University of Padova, ItalyThe present study aimed at elucidating the mechanism(s)of serotonin (5-HT) efflux from humanplatelets induced by thapsigargin, an inhibitor of theCa(2 + ) re-uptake into endoplasmic reticulum. Effluxof pre-loaded radiolabeled serotonin was generallydetermined by filtration techniques, whereas thecytosolic [Ca2 + ], [Na + ] and [H + ] were measured withappropriate fluorescent probes. 5-HT efflux fromcontrol or reserpine-treated platelets – where reserpineprevents 5-HT transport into the dense granules- was proportional to thapsigargin evoked cytosolic[Ca2 + ] increase. Accordingly factors as prostacyclin,aspirin and calyculin which reduced [Ca2 + ] increase,also inhibited the 5-HT efflux. Thapsigargin, whichalso caused a remarkable increase in cytosolic [Na + ],promoted less 5-HT release, in parallel to lower [Na + ]and [Ca2 + ] increase, when added to platelet suspensionscontaining low [Na + ]. The Na + ionophore monensinincreased the cytosolic [Na + ] and induced 5-HTefflux without affecting the Ca(2 + ) level. The 5-HTefflux induced by the increase of either [Ca2 + ] or[Na + ] did not depend on pH or membrane potentialchanges, whereas it decreased in the absence ofextra-cellular K+, and increased in the absence of Cl-. It is concluded that increases in [Ca2 + ] and [Na + ]independently induce serotonin efflux through theoutward directed plasma membrane serotonin transporterSERT. This event might be physiologicallyimportant at the level of capillaries or narrowedarteries where platelets are subjected to high shearstress which causes [Ca 2+ ] increase which, in turn,could induce 5-HT release.haematologica vol. 89(suppl. n. 8):september 2004

98PostersPO-008INTEGRIN α2β1 AND GLYCOPROTEIN VI DEPENDENTCALCIUM SIGNALS IN PLATELETS INTERACTING WITHACID-SOLUBLE TYPE I COLLAGEN UNDER FLOWMazzucato M, Cozzi MR, Battiston M, De Marco LCentro di Riferimento Oncologico, IRCCS, Aviano,ItalyWe studied the role of platelet integrin α2β1 andglycoprotein GPVI in stabilizing adhesion and modulatingsignal transmission of Fluo-3 AM plateletsperfused onto a surface of acid-soluble collagen typeI at wall shear rate of 250 s-1. We analyzed concurrentlythe instantaneous velocity and [Ca ++ ]i in singleplatelets interacting with collagen using a videoimagingmethod characterized by high-speed imageacquisition (25 frames/s) and high performance software(M. Mazzucato et al. Blood, 2002;100:2793-800). Perfusion of washed platelets resulted in singleplatelet adhesion that displayed a series of transient[Cav]i elevations. Two distinct peaks could bedefined on the basis of [Ca ++ ]i levels, duration, relationto motion on the surface and sensitivity to EGTA.Type α/β like Ca ++ oscillations were characterized bya rapid increase of 0.2 to 2 µM, lasting for 0.5-2.5seconds and EGTA insensitive. The discriminationbetween type α/β (typical of platelets translocatingover a surface of VWF at high shear rate) and α/β likewas obtained on the basis of dynamic behavior:platelets showing α/β like peaks were firmly adherentto collagen. Type γ like [Ca ++ ]i peaks were characterizedby a maximum level >2-3 µM lasting forseveral seconds (3-60) and stable adhesion to collagensurface and abolished by EGTA. MoAb againstintegrin α2β1 prevented platelet adhesion to collagensurface and the appearance of both type α/βlike and γ like peaks. Whereas only type γ like peak,which involve a transmembrane Ca ++ flux, was inhibitedby an anti-GPVI MoAb. Inhibitors of PI3-kinase(wortmannin and LY294002) and PLC (U73122) preventedthe appearance of both Ca ++ peaks, reducedthe arrest time from ~14 to ~3 seconds and preventedplatelet stable adhesion. Our results demonstratethat α2β1 partecipates in the initial stages ofplatelet adhesion and activation which is subsequentlyreinforced by activation through GPVI.PO-009COLLAGEN-INDUCED PLATELET ACTIVATION: NEW INSIGHTSRiondino S, Lotti L, Bronzi C, Pulcinelli FMDipartimento di Medicina Sperimentale e Patologia,Università degli Studi di Roma "La Sapienza”, ItalyShape change is platelets’ earliest response to stimuli;it is mainly dependent upon Ca 2+ /calmodulininteraction, subsequent to Ca2 + mobilization, and ismediated by MLCK. It has been recently hypothesizedthat collagen itself is not able to elicit platelet shapechange in the absence of ADP and thromboxane A2(TxA2) co-stimulation, while it is capable of inducingMLCK activation. Since we hypothesize that the morphologicalchanges of the few platelets that adhereto collagen might not be revealed by turbidimetry,aim of this study was to use electron microscopy (EM)as a tool to asses platelet shape change, in theabsence of the amplificatory feedback pathways ofADP and TxA2. For this purpose, platelets were treatedwith aspirin, AR-C66096 and MRS 2179 (P2Y12and P2Y1 ADP receptor antagonists, respectively),stimulated for 30 sec with collagen (25 µg/mL) in anaggregometer and then fixed for EM studies. Ourresults demonstrated that only those platelets thatwere more close to the insoluble collagen fibresunderwent a typical change in shape, while all otherplatelets did not modify their quiescent morphology.Turbidimetry failed to reveal such phenomenon.Moreover, since cAMP is known to inhibit plateletactivation by reducing Ca2 + mobilization in responseto all platelet agonists, except for collagen, in the presentstudy we sought to investigate whether cAMP isinvolved in the inhibition of collagen-induced plateletshape change. Platelets were thus treated with iloprost(28 nM) prior to stimulation. EM studies demonstratedthat iloprost did not modify collagen-inducedshape change, while immunoblotting studies showeda marked inhibition of collagen-induced MLC phosphorylationin the presence of enhanced cAMP levels.Since cAMP, contrariwise to what happens for allknown platelet agonists, enhances Ca2 + mobilizationin response to collagen, we can hypothesize thatcAMP exerts an inhibitory action on MLCK activation.PO-010GP91PHOX-DEPENDENT EXPRESSION OF PLATELETCD40 LIGANDPignatelli P,*° Sanguigni V,** Lenti L,* Ferro D,°Finocchi A,*** Rossi P,*** Violi F*°Divisione IV Clinica Medica°, Dipartimento diMedicina Sperimentale e Patologia*, Università diRoma "La Sapienza", Dipartimento di MedicinaInterna** e Dipartimento di Medicina Sperimentale,***Università di Roma Tor Vergata, ItalyBackground: CD40L expression on platelets ismediated by agonists but the underlying mechanismis still unclear. Methods: CD40L expression was measuredboth in platelets from healthy subjects - withand without the addition of antioxidants or a phospholipaseA2 (PLA2) inhibitor - and in platelets fromtwo patients with an inherited deficiency ofhaematologica vol. 89(suppl. n. 8):september 2004

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 200499gp91phox. Immunoprecipitation analysis was alsoperformed to determine whether normal plateletsshowed gp91phox expression. Results. Unlike catalaseand mannitol, superoxide dismutase (SOD)inhibited agonist-induced platelet CD40L expressionin healthy subjects. Immunoprecipitation analysisalso showed that platelets from healthy subjectsexpressed gp91phox. In two male patients withinherited gp91phox deficiency, collagen-,thrombinandarachidonic acid-stimulated platelets showedan almost complete absence of O2- and CD40Lexpression. Incubation of platelets from healthy subjectswith a PLA2 inhibitor almost completely preventedagonist-induced O2- and CD40L expression.Discussion. These data provide the first evidence thatplatelet CD40L expression occurs via arachidonicacid-mediated gp91phox activation.PO-011GP91PHOX-DEPENDENT PLATELET RECRUITMENTPignatelli P,*° Sanguigni V,** Lenti L,* Di Santo S,°*Ferro D,° Finocchi A,*** Rossi P,*** Violi F*°Divisione IV Clinica Medica°, Dipartimento di MedicinaSperimentale e Patologia*°, Università di Roma"La Sapienza"; Dipartimento di Medicina Interna** eDipartimento di Medicina Sperimentale***,Università di Roma Tor Vergata, ItalyBackground. Platelet recruitment at site of vascularinjury is determinant for thrombus growth. Plateletnitric oxide (NO) has an inhibitory effect on plateletrecruitment but it is unclear if oxidative stress is implicatedon its modulation. Methods. In two malepatients with hereditary deficiency of gp91phox, thecentral core of NADPH oxidase, and in 10 male healthysubjects matched for age, agonist-induced plateletaggregation and platelet recruitment were determined;in both groups agonist-induced platelet productionof 02- and NO was also measured. Results.Compared to controls, gp91phox deficient patients hadalmost complete suppression of platelet O2- and asignificant increase of platelet NO (p

100Postersthe International Society for Laboratory Hematologymethod. The correlation coefficient (r) between thetwo counting methods was 0.65. Twenty-nine patientshad a Coulter count below the treatment- thresholdof 30.000 plt/microliter; out of them, 11 had aimmunoplatelet count above this threshold. No bleedingsymptoms were observed in those patients inwhom therapy was delayed based on immunoplateletcount. Twelve patients had a Coulter count below15.000 plt/µL, a threshold possibly associated with anhigher hemorrhagic risk; out of them, 8 had aimmunoplatelet count above this threshold and 3 hada immunoplatelet count above 30.000 plt/µL. In threecases, immunoplatelet count gave slighlty lowercounts than Counter ones. In up to 11/29 (38%) ofpatients with chronic ITP, relying only to Coulter countcould result in over-treatment of patients. The disagreementbetween the two counting methods in ITPpatients is probably due to a proportion of largerplatelets, perhaps the younger ones, that are missed bythe Coulter method. Safety of avoiding therapy basedon immunoplatelet should be further evaluated.PO-014HELICOBACTER PYLORI INFECTION IN CHRONIC IMMUNETHROMBOCYTOPENIC PURPURAScandellari R,* Allemand E, Zocca N, Randi Ml,Luzzatto G, Fabris FDepartment of Medical and Surgical Sciences,Medicina Interna, University of Padua MedicalSchool, Padova, ItalyHelicobacter pylori (HP) has been suspected to beinvolved in various autoimmune disorders includingpernicious anemia and immune thrombocytopenicpurpura (ITP). Several uncontrolled studies supporteda pathogenic role of HP in ITP since they showeda 30-70% prevalence of HP infection in ITP and apartial or complete platelet recovery after bacteriumeradication. On the contrary three additional studies,one of which was prospective, failed to confirmthis association. We performed a prospective studyin order to investigate the prevalence of HP infectionin patients with ITP and the effect of its eradicationon platelet count. Since September 2003, twentysevenconsecutive adult patients admitted to ourward for ITP were enrolled in the study. Twentypatients had chronic ITP while seven had the acuteform (disease duration lower than 6 months); 18were females and 9 were males with a mean age of53 years±19 (SD) and with a mean platelet count of58±39×10 9 /L. HP infection was found in 63% ofpatients by means of the stool antigen test. HP–positiveand negative patients were comparable for age,gender, platelet count, disease duration and therapyregimens. At the present time 6 HP– positive patientsreceived triple therapy with omeprazole 20 mg twicedaily plus clarithromycin 500 mg twice daily andamoxicillin 1 gr twice daily for 7 days, and the bacteriawas eradicated in all. The patients were eligibleto the eradication if their platelet count wasmore than 20×10 9 /L, they do not need for starting ormodifying therapy of thrombocytopenia for at least3 months before. After 3 months of follow-up, aplatelet count increase more than 50% of the initialcount was observed in two patients. In conclusion,unlike a higher-than-expected prevalence of HP inpatients with ITP, our preliminary data seems doesnot support a role of the HP infection in the clinicalcourse of ITP.PO-015EFFICACY OF COMBINED APPROACH IN CHILDRENREFRACTORY IMMUNE THROMBOCYTOPENIC PURPURAParodi E, 1 Garbarini L, 1 Nobili B, 2 Matarese MR, 2Russo G, 3 Licciardello M, 3 Giordano P, 4 Nigro A, 4Amendola G, 5 Ramenghi U 1Pediatric Department, University of Torino, 1 Napoli, 2Catania, 3 Bari, 4 Nocera Inferiore Hospital 5 , ItalyIntroduction: immune thrombocytopenic purpura(ITP) in children is usually a benign, self-limitedautoimmune disorder characterized by low plateletcount and mucocutaneous bleeding. Only a little percentageof paediatric patients doesn’t respond withan increase in platelet count to conventional treatmentwith intravenous immunoglobulin (IVIG) orcorticosteroids. Patients and methods: we assessedthe effectiveness and the tolerability of the combinedadministration of IVIG and methylprednisolonefor children that didn’t respond to both drugs administeredone by one (IVIG 0.8g/kg/die for 2 days;methylprednisolone megadose pulse) and that continueto have significant symptomatic thrombocytopenia.Hypertension, hyperglycemia, overweightand positive family history for both hypertension andhyperglycemia were considered exclusion criteria.Twenty-one patients, 8 males and 13 females, witha median age at diagnosis of 6 years (range 1-15years) were eligible. Therapeutic schedule was thefollowing: i.v. methylprednisolone 20 mg/kg/die (day1, 2, 3) plus IVIG 400 mg/kg/die (day 1,2) plus gastricprotection. At day 1 (before treatment) 2, 3 clinicalevaluation, blood-pressure determination, urinalysisand blood tests (blood cell count, glycemia,LDH, AST, creatinine) were performed. Response tocombined approach was defined as positive ifplatelet count did rise above 50000/cm 3 , and good ifplatelet count did rise above 150000/cm 3 . Results:6/21 patients (29%) failed to respond to the treat-haematologica vol. 89(suppl. n. 8):september 2004

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 2004101ment, as PLTs did not rise above 50000/cmm; a positiveand a good response were detected in 4 (19%)and 11 (52%) patients respectively. In 15/21 (71%)patients no side effects were shown; one child presentedfever the day after the infusion; another onehad headache, nausea and pyrosis. 4 patient presentedmild hyperglycemia, never associated to glycosuria.Conclusion: our data show the efficacy ofcombined approach in 71% of children that hadfailed to respond to both drugs administered one byone. Moreover, the schedule resulted well tolerated.PostersVON WILLEBRAND FACTORPO-016SUCCESSFULL TREATMENT OF MINOR SURGERY IN PATIENTSWITH VON WILLEBRAND DISEASE TYPE 2MSacchi E,* Lattuada A,* Danna P,* Federici AB,°Russo U,* Rossi E**S.I.M.T. Az. Ospedaliera L. Sacco, Milano; °CentroEmofilia e Trombosi,IRCCS Osp. Maggiore,Universita' degli Studi di Milano, ItalyBackground: quantitative or qualitative defects ofVWF are associated with Von Willebrand Disease(VWD), the most common inherited bleeding disorder.Type 2M VWD is characterized by decreasedVWF-dependent platelet function in the presence ofan apparently normal VWF multimeric pattern. Inthese patients the main goal of treatment is to correctthe reduction of VWfF. In VWD type 2M the FVI-II /VWF concentrates were effective in attaining normallevels of FVIII:C postinfusion. Patients: twopatients with VWD type 2M were followed in ourhospital during surgery: one for Percutaneous TransluminalCoronary Angioplasty (PTCA) and one forcesarean delivery. The first one is a sixty years oldman and the second one is a thirty seven years oldwoman. Both patients were treated with 50 U /kg ofFVIII concentrate in the first and in the two followingdays. Before and one hour after infusion we havemeasured the FVIII:C, VWF :Ag and VWF:RCo duringthree days. Results: both patients have the typicalbasal values of 2M VWD : FVIII:C:13%, VWF:Ag: 11%,VWFRCo: 7% (man patient) FVIII:C: 16%, VWF:Ag:5%, VWF:RCo: 4% (woman patient). One our afterthe infusion the FVIII and VWF were increased: FVI-II:C 77%, VWF:Ag: 263%, VW:FRCo: 142%, (manpatient); FVIII:C: 122%, VWF:Ag: 237%, VWF:RCo:100% (woman patient). In the second and third daysthe values for both patients were FVIII:C 80%,VWF:Ag: 213%, VWF:RCo: 100%. Conclusion: Theinfusion of 50U/Kg of FVIII/VWF concentrates is safefor the prevention of surgical haemorrhage inpatients with VWD type 2M. The decision of dailyinfusion was supported by the values of the VWF:Agand VWF.RCo.haematologica vol. 89(suppl. n. 8):september 2004

102PostersPO-017GASTROINTESTINAL BLEEDING IN PATIENTS WITH VONWILLEBRAND DISEASE: HOW LONG WE CAN WAIT FORSURGERY?Siragusa S, Malato A, Anastasio R, Di Vita G,*Arcara M,* Patti R,* Bonifacio G, Mariani G**Cattedra di Ematologia, Università di Palermo,*Cattedra di Chirurgia, Università di Palermo,**Dipartimento Medicina Interna e Sanità Pubblica,Università dell'Aquila, ItalyThe association between gastro-intestinal angiodysplasiaand von Willebrand Disease (vWD) wasreported 30 years ago. The clinical course of patientswith vWD and angiodysplasia is characterized bynumerous admission to hospital for GI bleedingrequiring packed red cells transfusions or replacementtherapy with vWFactor containing concentrates.The management of these patients is problematicand a number of treatment options are available:electrocoagulation, sclerotherapy, arterialembolization, immunoglobulins, oestrogens andocteocride, with variable success rates. Althoughpotentially effective, the surgical approach may notbe suitable because of the difficulties in reachingpre-operative diagnosis. Pre or intra-operativeenteroscopy is very effective for identifying thesource of occult small-bowel bleeding; however, theare very few reports on its use. During the period2001-2003, we evaluated 2 cases of massive GIbleeding in patients with vWD Tipe I; in both cases,treatment with a vWF containing concentrates(Hemate P) was given. The total amount units ofreplacement therapy and the number of bleedingepisode are reported in Figure.Both patients were investigated by GI endoscopyand arteriography; these procedures were not able toidentify the source of bleeding. Therefore, bothpatients underwent intra-operative enteroscopy. Thisprocedure was able to identify the major source ofbleeding and related intestinal resection was performed.Peri- and post-operative management ofboth patients required Haemate P® (50 UI/Kg) for 20to 30 days; one episode of postoperative bleedingoccurred in one patient. Over a follow-up of 9months, no bleed occurred. Conclusion. Our casereport shows that vWD patients with massive GIbleeding, resection of part of the small-bowel can bean effective and definitive procedure. Reliable diagnosisof the site of bleeding can only be done byintraoperative enteroscopy.PO-018PREVALENCE AND NATURAL HISTORY OF HEPATITIS VIRUS CINFECTION IN 350 ITALIAN PATIENTS WITH VON WILLEBRANDDISEASEFederici AB,* Rumi MG,^ Santagostino E,*Soffredini R,^ Mistretta C,* de Filippi F,^ Mogni S,*Bucciarelli P,* Colombo M,^ Mannucci PM**Angelo Bianchi Bonomi Hemophilia and ThrombosisCenter, IRCCS Maggiore Hospital, and Universityof Milan, Italy; ^Angela Maria and Antonio MigliavaccaCenter for Liver Diseases, IRCCS MaggioreHospital, and University of Milan, ItalyHepatitis C is a major cause of morbidity and mortalityin patients with bleeding disorders whoreceived clotting factors concentrates before theavailability of virus-inactivated factors in the mid1980s. Compared to hemophiliacs, patients with vonWillebrand disease (VWD) have been less extensivelyexposed to large-pool concentrates because theycould be treated by desmopressin or cryoprecipitatesprepared by national blood banks. To assess theprevalence and natural history of HCV infection inVWD, 350 patients attending the ABB HemophiliaThrombosis Center in Milan were enrolled in a cohort(types 1=145, 2=184 and 3=21). The 133/350patients (37% males, with types 1=31, 2=84; 3=18)who had been firstly exposed to cryoprecipitate(34%), whole blood and/or plasma (51%), large-poolconcentrates (15%), have been prospectively followedfor five years with assessment of viral markersand liver function tests every six months. Only 2patients had serum anti-HIV. Anti-HCV was found in61 (46%, 4 treated after 1987), 49 (80%) of whomhad serum HCV-RNA, too. HCV genotypes 1, largelypresent in the Italian population of hemophiliacs(>56%), were present only in 32% of HCV-RNA-positiveVWD. Median age at infection was 24 years.Serum ALT activity was persistently or intermittentlyhigh in 33 HCV-RNA patients. Cirrhosis and HCCwere detected in 4 and 2 patients, respectively. VWDhaematologica vol. 89(suppl. n. 8):september 2004

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 2004103Italian patients showed a sporadic risk of infectionwith HIV and a lower prevalence of HCV. As expected,HCV genotypes distribution reflects the sourceof blood products and might influence the naturalhistory of HCV in VWD.PO-019RECURRENT GASTROINTESTINAL BLEEDING IN A PATIENT WITHTYPE 3 VON WILLEBRAND DISEASE AND MULTIPLE MUCOSALVASCULAR ABNORMALITIES: A ROLE FOR PROPHYLAXISWITH FACTOR VIII/VON WILLEBRAND CONCENTRATE ANDOCTREOTIDECoppola A, Cimino E, Nardone G,^ De Simone C,Conca P,* Di Minno M, Sidiropulos M, Albisinni R,Di Minno GRegional Reference Centre for Coagulation Disease,^Chair of Gastroenterology and *Sector of Hepatologyin Internal Medicine, Dep. of Clinical and ExperimentalMedicine, Federico II University, Naples, ItalyThe association between von Willebrand Disease(vWD) and mucosal vascular abnormalities (MVA) ofthe gut is well recognized, often resulting in severebleeding tendency and difficult therapeutical management.We report the case of a 57 yr-old man withtype 3 vWD and a history of recurrent melena sincethe age of 53. Endoscopic examinations revealedmultiple small MVA of the stomach and, two yearslater, of the left large bowel. Bleeding frequency andseverity progressively increased and the patientshowed chronic anemia and hemoglobin in thestools. Electro-coagulation of some actively bleedinglesions and octreotide courses did not significantlyaffect his clinical outcome: during 2002 the patientneeded transfusions (6 packed red cell units) andabout 400.000 IU of factor VIII/von Willebrand concentrates(Haemate P, Aventis Behring) on demand.The administration of Haemate P 40 IU/Kg thriceweekly was then proposed. This regimen was associatedwith improvement of the patient meanhaemoglobin levels and the cessation of clear-cutmelena and red cell transfusions. Total Haemate Punits infused were also reduced by about 15% during2003. Clinical conditions and Haemate P requirementsfurther improved when the patient was regularlycompliant to s.c. octreotide (0.1 mg b.i.d.)administration (he did not tolerate multiple dailyinjections). Prophylaxis with Haemate P is still ongoingwithout any adverse event over a 18-mo. period.Clinical course and pharmacokinetic evaluations ledto administer 40 IU/Kg each 72 hours. Possible vascularcomplications were excluded by careful clinicaland instrumental follow-up, as the patient sufferedfrom arterial hypertension and diabetes mellitus;thrombophilic abnormalities were previouslyexcluded and no signs of coagulation activation weredetected thereafter. Long-term prophylaxis withHaemate P has been showed to be safe and effectivein this patient with severe gastrointestinal bleedingdue to MVA and vWD.PO-020DOES THE COEXISTENCE OF VON WILLEBRAND FACTORDEFICIENCY AND PROTHROMBOTIC GENE POLYMORPHISMSREDUCE THE OCCURRENCE OF THROMBOEMBOLIC EVENTS?Lombardini D, Tufano A, Coppola A, Sidiropulos M,De Simone C, Cerbone AM, Di Minno GRegional Reference Centre for Coagulation Diseases;Department of Clinical and ExperimentalMedicine, "Federico II" University, Naples, ItalyA 28-year-old man was referred to our Centrebecause of a recent post-traumatic deep venousthrombosis of the leg and acute pulmonary embolism.He carried heterozygosity for Factor V Leidenmutation and homozygosity for the thermolablevariant of the 5,10-MTHFR, with moderate hyperhomocysteinemia.His mother, who developed juvenileischemic heart disease in the absence of establishedrisk factors for arterial thrombosis, had a positivefamily history for early-onset arterial thromboticevents. She exhibited, in addition to the geneticabnormalities of the son, heterozygosity for the FIIG20210A variant. Her second son was also studiedwho, at variance with his mother and his brother,did not develop thrombotic events. He exhibited heterozygosityfor the Factor V Leiden mutation,homozygosity for the thermolable variant of the5,10-MTHFR, with moderate hyperhomocysteinemia,and a mild prolonged aPTT. Factor VIII coagulantactivity and von Willebrand factor were both reduced(Factor VIII=65%, and vWF=48%), and the screeningfor Lupus Anticoagulant and Anticardiolipin antibodieswas negative. Negativity for these antibodies,as well as normal FVIII and vWF were found in hismother and brother. Nobody in the family experiencedhemorrhagic events. In a family with a highprevalence of thrombophilic gene abnormalities, thecoexistence of reduced factor VIII and vWF levels islikely to have contributed to modulate the differentphenotypic expression of the siblings.haematologica vol. 89(suppl. n. 8):september 2004

104PostersPO-021EVALUATION OF A NOVEL AUTOMATED,IMMUNOTURBIDIMETRIC ASSAY FOR VON WILLEBRANDFACTOR ACTIVITY DETECTIONVaccarino A,° Montaruli B,° Valpreda A,^ Agnes C,°Borchiellini A,^ Scirelli T,^ Saitta M,* Bazzan M°°Department of Haematology and CoagulationDisorders and *Laboratory Analysis – OspedaleEvangelico Valdese; ^Divisione Ematologiadell’ Università, Azienda Ospedaliera San GiovanniBattista; Turin, ItalyPatients with von Willebrand’s disease (vWD) mayhave normal levels of von Willebrand factor (vWF)antigen. It is therefore very important to measurenot only the antigen concentration but also the vWFactivity. The standard assay detect the ristocetincofactor activity (vWFR:Co) by measuring the abilityof patient’s plasma to induce agglutination offormaldehyde-fixed normal platelets in the presenceof a fixed amount of ristocetin. This can be measuredusing a platelet aggregometer, a slide technique or,more recently, photo-optical coagulometer. However,vWFR:Co assay standardization, due to plateletpreparation and in some cases to the agglutinationdetection, is difficult and can affect test reproducibility.We assayed the performance characteristicsof a new commercial and fully automated assayalternative to vWFR:Co for the quantification of vWFactivity (von Willebrand Factor Activity, HemosiIL, IL,USA). This new assay is a latex particle enhancedimmunoturbidimetric test which determines the vWFactivity by measuring the increase of turbidity producedby the agglutination of the latex reagent. Aspecific anti-vWF monoclonal antibody adsorbedonto latex reagent, directed against the plateletbinding site of vWF (Glycoprotein Ib receptor), reactswith the vWF of patients plasma. The degree ofagglutination is directly proportional to the vWFactivity in the sample and is determined by measuringthe decrease of transmitted light caused by theaggregates. Forty-three samples were analysed:12normal controls, 20 congenital vWD patients (3 postdesmopressin somministration) and 3 acquired vWDpatients; moreover, in order to evaluate the specificityof this new assay, we studied patients with differentcoagulation abnormalities: 1 patient treatedwith low molecular weight heparin therapy (LMWH),6 patients with Lupus Anticoagulant (LA) and 1patient with factor VIII:C deficiency. VWF activitywas assayed by vWFR:Co aggregometric method(Ristocetin Cofactor Assay, Helena) on PACKS’ 4(Helena) and by a recently developed immunoturbidimetricmethod (von Willebrand Factor Activity, IL,USA) on the automated coagulomter (ACL9000, IL).In addition we measured platelets functionality byusing PFA-100 (Dade Behring) and vWF antigen byELISA (Asserachrom vWF, Roche) and immuno-turbidimetricmethod on photo-optical coagulometer(von Willebrand Factor, IL, USA) in all patients andcontrols. Good intra and inter assay precisions wereobserved with the IL vWF activity assay using normalplasma pool: CV = 5.7% and CV = 8.6% respectively.All the congenital and acquired vWD patients aswell as normal controls and patients with coagulationabnormalities different from vWD were correctlydifferentiated by this new vWF activity assay. Allpatients treated with DDAVP showed a two/threefold increase of vWF activity with this new coagulometricassay. The method comparison plots showeda good correlation of the IL vWF activity assay withthe aggregometric vWFR:Co method (r = 0.95, PassingBablok regression y = 1.1202 × + 7.9956) and ofthe latex immunoassay for the determination of thevon Willebrand antigen with the ELISA method (r =0,96 , Passing Bablok regression y = 1.0970 × –1.6614). Interestingly one patient with congenitalvWD and contemporary LA autoantibodies was foundto be normal for IL vWF activity on the undilutedplasma, this sample after 1:100 dilution showed 28%of IL vWF activity. Our preliminary data show thatthis new automated immunoturbidimetric IL vWFactivity assay is useful for the diagnosis of congenitaland acquired vWD, its easy and rapid performancesuggest its routine application in combinationwith automated latex immunoassay for the quantitativedetermination of vWF antigen for the vWDdiagnosis. Further studies are needed about plasmaticconditions which may give interferences inthis new vWF activity assay.PO-022SENSITIVITY AND SPECIFICITY OF PFA-100 ® VERSUSBLEEDING TIME IN THE DIAGNOSIS OF TYPE 1 VONWILLEBRAND DISEASECastaman G,* Tosetto A,* Bernardi M,*Bertoncello K,* Peake I,° Goodeve A,° Federici AB, +Batlle J,^ Meyer D,** Eikenboom J,°°Schneppenheim R, ++ Ingerslev J,^^ Vorlova Z,***Lethagen S,°°° Pasi J, +++ Hill F,^^^ Rodeghiero F**San Bortolo Hospital, Vicenza, Italy; °University ofSheffield, United Kingdom; + University of Milan,Italy; ^Hospital Teresa Herrera, Spain; **INSERM,France; °°Leiden University Medical Center, Netherlands;++ University Children Hospital, Hamburg,Germany; ^^University Hospital Skejby, Denmark;***Institute of Hematology and Blood Transfusion,Prague, Czech Republic; °°°University of Lund, Sweden;+++ Leicester Royal Infirmary, UK; ^^^BirminghamChildren Hospital, UKhaematologica vol. 89(suppl. n. 8):september 2004

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 2004105The bleeding time (BT) is the only in vivo test whichexplores primary hemostasis. Its use has been considereduseful in the diagnostic work-up of VWD and tomonitor effectiveness of substitutive or desmopressintreatment. However, the test is invasive and not alwaysacceptable to the patients, especially when repeateduse is anticipated. Recently, the PFA-100® system hasbeen developed to screen for primary hemostasis sincethis test mimicks high shear stress platelet functionand the role of von Willebrand factor (VWF) in primaryhemostasis. Preliminary evaluations claim its possiblesuperiority compared to BT in the diagnosis ofVWD. To evaluate the role of this system compared tothe BT within the framework of MCMDM-1, a EUfunded multicenter study on type 1 VWD. Up to now,data were available from 96 families enrolled in 9 participatingcenters. BT was determined with either theIvy or Simplate method in all family members consideredas affected; PFA-100® closure time was determinedwith both the ADP and Epinephrine cartridge inall family members. All measurements were performedduring a baseline evaluation, in which bleeding historyand blood samples were also obtained. The referenceranges for BT were those adopted by the individualcenter; the upper limit for ADP cartridge was 120 secand for Epinephrine cartridge 177 sec (n = 53 normalcontrols). BT was available for 385 subjects, PFA-100®was available in 253 subjects (53 normal controls, 152previously diagnosed affected family members and 48normal relatives). There was a strong relationshipbetween the closure time and Ristocetin cofactor levelwith both cartridges (r2=0.58). The sensitivity andspecificity for BT was 42.7% (95% C.I. 37.8 – 47.7%)and 88.3% (95% C.I. 85.1 – 91.5%). The sensitivityand specificity for ADP cartridge in the members of theenrolled families was 82.2% (95% C.I. 77.5 – 87%)and 92.1% (95% C.I. 88.7 – 95.4%), whereas for Epinephrinecartridge was 79.5% (95% C.I. 74.6 – 84.3%)and 93.6% (95% C.I. 90.7 – 96.5%). The ratio of sensitivityvs specificity assessed by ROC curve estimationwas 0.93 for ADP and 0.92 for Epinephrine cartridges.The PFA-100® system provides a dramatic improvementof sensitivity and a slight improvement of specificitycompared to BT in the identification of patientswith type 1 VWD. The optimal use of PFA-100® for thescreening of type 1 VWD remains to be further evaluatedin terms of added benefits to patients identifiedon its basis.PO-023EVALUATION OF A NEW QUANTITATIVE, RAPID TEST (VWF-LIA)FOR THE DIAGNOSIS OF TYPE 1 VWDTosetto A,* Castaman G,* Bernardi M,*Bertoncello K,* Cappelletti A,* Peake I,° Goodeve A,°Federici AB, + Batlle J,^ Meyer D,** Eikenboom J,°°Schneppenheim R, ++ Ingerslev J,^^ Vorlova Z,***Lethagen S,°°° Pasi J, +++ Hill F,^^^ Rodeghiero F**San Bortolo Hospital, Vicenza, Italy; °University ofSheffield, United Kingdom; + University of Milano,Italy; ^Hospital Teresa Herrera, Spain; **INSERM,France; °°Leiden University Medical Center, Netherlands;++ University Children Hospital, Hamburg,Germany; ^^University Hospital Skejby, Denmark;***Institute of Hematology and Blood Transfusion,Prague, Czech Republic; °°°University of Lund,Sweden; +++ Leicester Royal Infirmary, UK;^^^Birmingham Children Hospital, UKType 1 VWD is the most prevalent hemorrhagic disorder,with an estimated prevalence ranging from0.1 to 1% depending on the criteria of patient selection.Measurement of von Willebrand factor (VWF) isa critical requirement for diagnosis, but most availablemethods are time-consuming. To evaluate thediagnostic efficiency of a new quantitative test forthe measurement of VWF antigen (VWF:Ag) in type1 VWD. VWF:Ag was measured both with an ELISAmethod (VWF Assay, Stago, Asniéres, France) in arobotic instrument (Multiprobe II, Packard, Milan)and as Latex-immunoassay (VWF LIA, InstrumentationLaboratories, Milan) in 739 subjects enrolledwithin the MCMDM-1 VWF Study, an EU fundedmulticenter study on type 1 VWD. Among these subjects,387 were healthy controls, 148 were healthyfamily members of type 1 VWD families and 204were affected members from 72 European familieswith type 1 VWD. The inter-assay CV was 7.5% and2.1% for the ELISA and LIA test, respectively. Themedian VWF:Ag level in normal subjects (either controlsand normal family members) was 111 and 105IU/dl respectively for ELISA and LIA, with a lower referencelimit (2.5 percentile) of 57 and 51 IU/dlrespectively. Based on this reference limit (hencewith a specificity fixed at 97.5%) the sensitivity fordetection of type 1 VWD affected members was69.5% and 72.5% respectively for ELISA and LIA. Areceiver-operator (ROC) analysis disclosed a marginaldifference between the two tests, LIA having aslightly higher area under the curve (0.94 vs. 0.93,p=0.03) suggesting a better diagnostic capability.Based on a 1% prevalence of VWD, however, the positivepredictive values for VWD of VWF:Ag below the2.5 percentile reference limit were very similar forboth ELISA and LIA (23.8 and 23.2%, respectively).haematologica vol. 89(suppl. n. 8):september 2004

106PostersVWF-LIA appears to be at least as sensitive as thetraditional ELISA test for the diagnosis of VWD type1. Although less sensitive than the ELISA test, theLIA assay appears to be useful for a rapid screeningof VWD in subjects with bleeding symptoms.PO-024A NEW APPROACH TO MEASURE VWF ACTIVITY AND ADAMTS13Preda l,* Lattuada A,* Rossi F,* Vaghi U,° Libera L,*Rossi E**S.I.M.T. Az. Ospedaliera L.Sacco, Milano;°Centro Trasfusionale Osp. S.Giuseppe, Milan, ItalyDeficiencies of Adamts 13, that cleaves von willebrandfactor (VWF) physiologically, reduce or abolishthe degradation of ultralarge VWF multimers thatcause the formation of intravascular thrombi inpatients with thrombotic microangiopathies (TMA).The measurements of functional VWF and Adamts13are important for the diagnosis of patients with TMA.To verify the sensitivity, accuracy of a new methodto quantify both VWF activity and Adamts13. Weanalysed a group of 60 normal controls, 10 pregnantwomen and two patients with TTP. Normal plasma (100% of Adamts13 ) was diluted with TTP patients(< 6% of Adamts13) to obtain 0, 10, 20, 40, 80 and100% of the Adamts13. A comparison was madebetween the results obtained with this method andtraditional methods. Method: one dilution in TRISbuffer and one dilution in UREA of the plasma samplesto be tested were incubated four hours at 37°C.The samples were then analysed to obtain basal andresidual VWF activity (after degradation ofAdamts13). The differences between the two valueswere directly proportional to ADAMTS13. Materials:HemosIL VWF act (IL) was used for the measurementsof VWF activity. VWF antigen ( IL) for the measurementsof VWF :Ag. Results. The coefficent ofvariation within- and between- assay was 4% and6% respectively. Normal range of Adamts13 was 75-125%. The values of Adamts13 in two patients withTTP were 3%, 5%,during normal pregnancy, themeans of Adamts13 were: 108% in the firsttrimester, 92% in the second and 89% in the thirdtrimester and in different dilution of normal plasmaand TTP plasma were 3%, 8%, 21%, 46%, 89%, 98%.Conclusion: This method is reproducible, accurate,rapid and easy to perform. This method in combinationwith automated VWF:Ag latex assay could beused for early diagnosis of VWD or TMA.PO-025AUTOMATED METHOD TO MEASURE ADAMTS13Preda L, Lattuada A, Rossi F, Crivelli S, Candolfi R,Rossi ES.I.M.T. Az.Ospedaliera L.Sacco, Milan, ItalyAdamts13 is a metalloprotease that cleaves vonWillebrand Factor (VWF) physiologically. In theabsence of this protease, ultralarge multimers ofVWF circulate in patients with thrombotic microangiopathies(TMA) and cause intravascular thrombi. Itsvalue is important for the diagnosis of patients withTTP. Aim of the study: to develop a fast and automatedmethod for measurement of Adamts13.Methods. All reagents used in this method are commercial:Von Willebrand Reagent (Dade Behringer)is reconstituted to obtain 340÷360 10⁄/microLiter ofplatelets. Ristocetin 50 mg/mL (Mascia Brunelli,Milan, Italy) is reconstituted with 0.5 mL of buffer(Diluent A ) supplied with the kit. The source of VWFused as substrate for protease was a commercial FVI-II concentrate. An automated VWF:RCo on ACL9000was used to determine Adamts13.The samples werediluted in buffer containing Urea and BaCl2 andincubated for 30 minutes a 37°C. The plasma werethen incubated over night at room temperature withsubstrate. Proteases were read automatically oncoagulometer. Patients: 60 normal controls, 10 pregnantwomen and two TTP patients. The accuracy ofthe test was determined by different dilutions ofplasma with 100% of Adamts13 and plasma with

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 2004107PostersHEMORRHAGIC SYNDROMESPO-026SCREENING FOR CONGENITAL BLEEDING DISORDERS WITHPLATELET FUNCTION ANALYZER (PFA-100) IN WOMEN WITHMENORRHAGIAGamba G, Longoni R, Gamba A, Montani N,Pedrazzoli F, Noris P, Balduini CDepartment of Internal Medicine, University ofPavia and IRCCS Policlinico San Matteo, ItalyMenorrhagia is a frequent complication in womenwith defective primary haemostasis. PFA-100 (Dade-Behring) has been reported to have a superior sensitivitythan Bleeding Time (BT) for screening vonWillebrand disease (vWd) and platelet hypofunction.We evaluated the performance of the PFA-100 forscreening congenital bleeding disorders in womenwith menorrhagia after that an acquired haemostasisdefect was excluded. Forty-seven women agedbetween 11 and 52 yrs with menorrhagia were consecutivelyenrolled. Menorrhagia was defined on thebasis of Pictorial Blood Assessment Chart and of themenstrual history. Closure Time (CT) were determinedwith PFA-100, using citrated whole blood samplesand both the collagen/epinephrine (EPI) and collagen/ADP(ADP) cartridges, within 2 hours of samplecollection. The bleeding disorders were diagnosedaccording to the conventional criteria. Seventeen outof 47 samples (36%) showed prolonged CT: 13 withboth EPI and ADP, 3 with EPI and 1 with ADP. 11women with abnormal CT presented prolonged BT,whereas 4 women with abnormal BT had normal CTwith both EPI and ADP, so a concordant resultsobtained by PFA-100 and BT were observed in 11/17patients (65%) in relation to CT and in 11/15 patients(73%) in relation to BT. Diagnosis of bleeding diseasewas performed in 19/47 women (41%): 4 vWd, 5platelet hypofunction, 8 dysfibrinogenemia, 2 FXIdeficiency. Abnormal CT was observed in all patientswith vWd, platelet hypofunction and FXI deficiency,and in 2 with dysfibrinogenemia. In these womenabnormal BT was observed in 11. In clinical practice,in selected women with menorrhagia PFA-100showed high sensitivity for screening of patientswith vWd and platelet hypofunction. Furthermoreabnormal results were observed in patients with F XIdeficiency and in some women with dysfibrinogenemia,in which a prolonged BT was also detected.PO-027PROLONGED APTT OCCASIONALLY DETECTED IN CHILDRENBEFORE ADENOTONSILLECTOMY AND BLEEDING DISORDERSGamba G, Montani N, Pedrazzoli F, Bocchi L, Longoni RDepartment of Internal Medicine, University ofPavia and IRCCS Policlinico San Matteo, Pavia, ItalyIn children congenital bleeding disorders are oftendiagnosed because of profuse bleeding occurred duringor after surgical procedures such as adenoidectomyand/or tonsillectomy. On the other hand a slightlyprolonged aPTT, observed during a preoperativescreening tests, could indicate an undiagnosed bleedingtendency. Aim of the study. To research the prevalenceof bleeding disorders in children suffering fromrecurrent tonsillitis and prolonged aPTT, occasionallydetected. Within 15 months 69 children aged between2 and 12 years were referred to our Department fromthe otorinolaryngologic specialists, to research anomaliesof haemostasis and the related hemorrhagic risk.After an overnight fast, blood samples were collectedin two different days and processed for aPTT, PT andTT detection; other tests were planned as indicated toperform an appropriate diagnosis. Prolonged aPTT wasconfirmed in only 14 children (20%). Out of the 14children with prolonged aPTT, 6 (43%) presented LupusAnticoagulant, 5 (36%) showed also a prolonged TTand Reptilase Time with normal values of fibrinogen,suggestive of a delayed fibrin formation, and 3 (21%)were affected by von Willebrand disease. In one childwith controlled normal aPTT, a prolonged PT with afactor VII deficiency was observed. 5 children showedan history of haemorrhagic symptoms and 3 had alsoa familial history positive for bleeding tendency. Conclusionsin most children the occasional result of prolongedaPTT was not confirmed after controlled bloodsample collection. In subjects with prolonged aPTT andrecurrent tonsillitis treated by antibiotics the presenceof LA was often diagnosed. High prevalence of dysfibrinogenemiaand vWd, in association to personal andfamilial history of bleeding was observed. Therefore itseems useful to carefully investigate all children withprolonged aPTT, occasionally detected before surgery.PO-028PROLONGED APTT IN PRE-OPERATIVE EVALUATION OFHEALTHY CHILDREN UNDERGOING ELECTIVE TONSILLECTOMYAND/OR ADENOIDECTOMYAgnes C,° Montaruli B,* De Vicariis A,^ Debole S,^Vaccarino A,° Foli C,° Rus C,° Saitta M,* Bazzan M°°Department of Haematology and Coagulation Disorders,*Laboratory Analysis, ^Department ofOtorhinolaryngoiatrics, Ospedale EvangelicoValdese, Turin, Italyhaematologica vol. 89(suppl. n. 8):september 2004

108PostersIn our hospital the routine pre-operative evaluationof healthy children undergoing elective tonsillectomyand/or adenoidectomy includes coagulationscreening tests (PT, prothrombin time; APTT, activatedpartial thromboplastin time). The otorhinolaryngoiatricdepartment complained with the laboratoryanalysis because of the excessive number of prolongedAPTT in children undergoing tonsillectomyand or adenoidectomy. In order to evaluate whetherthere was a problem on the APTT screening test, weanalyzed retrospectively the APTT values of otorhinolaryngoiatricchildren performed in 2003-2004(January 2003 – March 2004) by the laboratory.Between January 2003 and march 2004, we performed256 APTT in children (age < 12 years) preoperativeevaluated for otorhinolaryngoiatric surgery.59 out of 256 (23%) showed prolonged APTT (APTTratio > 1.2). In 25/59 children a coagulative analysiswas conducted: 17/59 (6,6%) of them had infectiousLA, one LA positive children was also found carrier ofvon Willebrand disease (vWD); 3 (1,2%) presentedvWD; 2 (0,8%) showed low levels of factor XII:C; 1(0,4%) low levels of factor XI:C (XI:C = 34%). 13 outof 17 LA positive children, 2 out of 4 vWD patients,3 out of 3 subjects with factor deficiencies (XII:C andXI:C) underwent surgery and presented no hemorrhagiccomplications. Only one of the 256 childreninvestigated presented hemorrhagic complicationimmediately after surgery, but in this patient thecoagulative study wasn’t performed. The number ofprolonged APTT in the 256 children investigated wasnot as higher as suspected by the otorhinolaryngoiatricclinicians (23%). Even if we conducted acoagulative investigation only in one third of thechildren with APTT ratio > 1.2, we can affirm that, asreported in literature, most of the prolonged APTTwere due to LA inhibitors (6,6%). Among the 46 childrenwith prolonged aPTT ratio who underwentsurgery only 22 subjects had a pre-operative coagulativescreening. 2 out of 22 of these patients presenteda coagulative disorder leading to a bleedingtendency and did not experienced hemorrhagic complicationsafter treatment. Only 1 children (1/46,2,2%), who was not studied experienced hemorragiccomplications. Therefore we regarded that a studyin depth of prolonged APTT was necessary and weapproved this kind of behaviour: to screen for factorVIII:Ag, VIII:C, IX:C, XI:C in order to exclude a bleedingtendency on the same tube used for pre-operativecaogulative screening tests (no need for furtherblood samples).PO-029HEREDITARY HEMORRHAGIC TELANGIECTASIA IN CHILDHOODGiordano P, Nigro A, Sabbà C,° Saracco P,*De Mattia DDipartimento di Biomedicina dell'Età Evolutivaand °Dipartimento di Medicina Interna e di MedicinaPubblica, Università degli Studi di Bari; *DivisioneUniversitaria di Ematologia Pediatrica Az. Osp.O.I.R.M. S.Anna, Turin, ItalyThe Rendu-Osler-Weber syndrome or HereditaryHemorrhagic Telangiectasia (HHT) is an autosomal disorderwith incomplete penetrance and variable expressioncharacterized by vascular dysplasia.Up to nowtwo genetic mutations have been detected: chromosomallocus 9q33-q34 in the case of the Endoglin gene(ENG) and 12q13 in the case of the Activin Like Kinase1 (ALK 1) gene mutation. Both genes encode proteinsthat can be defined as components of the receptorcomplex for Transforming Growth Factor. Clinicalmanifestations include mucocutaneous telangiectasesand arterovenous malformations in parenchymatousorgans. The clinical diagnosis is based on three of thefour suggested Shovlin criteria: epistaxis, mucocutaneoustelangiectases, family history and arterovenousmalformations. All children of family HHT- relatedboth symptomatic and without clinical signs werescreened for this study. Cutaneous telangiectases havebeen identified by the use of capillary microscopy andfibrorhinoscopy for nasal telangiectases has been performed.Pulmonary arterovenous malformations(PAVMs) were detected by the use of contrast bubbleechocardiography (BE) and/or thin section multislicecomputed tomography (CT); hepatic arterovenous malformations(HAVMs) were diagnosed by echocolourdopplerand/or abdominal CT. Angio-magnetic resonanceimaging (Angio-MRI) was used in order todetect cerebral arterovenous malformations (CAVMs).Our population included 17 children of family HHTrelated(F:10; M:7; 1-18 years of age; range:10,6±5,12), 13 (76,6%) with identified mutation and4 (23,5%) in course of identification. ALK1 wasresponsible of 9/13(69,2%) cases and ENG of 4/13(30,8%) cases. The results are reported below:EPISTAXIS 12/17 (70,6%)MUCOCUTANEOUS TELANGIECTASES 14/17 (82,3%)CAVMs 1/16 (6,25%)PAVMs 10/16 (BE+) (62%)7/11 (TAC+) (63,6%)HAVMs 10/17 (ECO+) (58,8%)6/11 (TAC+) (54,5%)In only a case with hematemesis and melena esophagogastroduodenoscopy(EGDS) revealed multipletelangiectases in gastro-intestinal trait. Our prelimi-haematologica vol. 89(suppl. n. 8):september 2004

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 2004109nary data show a high prevalence of patients withepistaxis and arterovenous malformations requiring toadopt adequate clinical-diagnostic screening and agerelatedtreatments.PO-030RECOMBINANT ACTIVATED FACTOR VII (RFVIIA) FOR THETREATMENT OF CRITICAL BLEEDING IN NON-HEMOPHILICPATIENTS. REPORT OF A SINGLE INSTITUTION EXPERIENCEMarietta M, Girardis M,* Facchini L, Busani S,*Pasetto A,* Torelli GDepartment of Oncology and Hematology, Hematology,*Department of Anesthesiology and IntensiveCare, University of Modena-Reggio Emilia, ItalyBackground: a number of case reports strongly suggestthat recombinant Factor VIIa (rFVIIa ) could be aneffective and safe adjunctive haemostatic treatmentfor severe bleeding unresponsive to conventional therapy.Unfortunately, to date no evidence-based guidelineson this issue are available. It is therefore recommendablethat every Institution develop a formal, writtenpolicy for the treatment of critical bleeding. StudyObjective: To examine our institutional experience followingthe adoption of a standardized protocol for theuse of rFVIIa in critical bleeding. Design: Retrospectivecase review. Patients and Methods: according toour Institutional protocol, non-hemophilic patientswere eligible for rFVIIa if they presented life-threateningbleeding either as regards amounts (i.e. surgicalor post-trauma bleeding) or site (i.e. intracranial bleeding)despite conventional medical and surgical treatment.Each patient received a first bolus of 120 micrograms/kgof rFVIIa, repeated 2-3 hrs later if bleedingremained critical provided standard treatment. Results:in one-year period (March 2003-2004) a total of 9patients received rFVIIa (bleeding critical for amount,7 pts.; intracranial bleeding, 2 pts). The mean dose ofrFVIIA used was 200 micrograms/kg, corresponding toa mean of 1.6 boluses/patient. Following rFVIIa infusionbleeding stopped or markedly decreased in allpatients and there was a significant reduction in transfusionrequirements for both packed cells (from 2.1 to0.59 Units/hr, p

110PostersPO-032GENETIC RISK FACTORS PREDISPOSING TO THROMBOSIS:MOLECULAR ANALYSIS IN PATIENTS WITH RETINAL VEINOCCLUSIONPiana A,* Camicione P,° Minniti G, § Cerone R, §Calevo MG, Mandich P, • Ulivi M, Bagni D,Perrone L, @ Armani U**Department of Internal Medicine, ThrombosisResearch Center and °Department of Neurosciences,Ophthalmology and Genetics; • Section ofMedical Genetics, University of Genoa, § Departmentof Pediatrics II and Service of Statistics andEpidemiology, Gaslini Institute, @ Lab. HumanGenetics, Galliera Hospital, Genoa; TIB Molbiol SLR,Genoa, ItalyRetinal vein occlusion (RVO) is the most commonretinal vascular occlusive disorder. Genetic and functionalthrombophilic abnormalities have beendescribed in different populations of RVO patients:abnormalities of anticoagulant or fibrinolytic system,Factor V Leiden and Prothrombin gene mutation,hyperhomocysteinemia and MTHFR polymorphism.However the role of these abnormalities in thepathogenesis of RVO is still unclear. Aim of the studywas to establish the role of different genetic polymorphismsand mutations in determining RVO. Ninety-threeconsecutive patients presenting a first retinalvein occlusion (RVO), including central retinalvein occlusion and branch retinal vein occlusion, duringthe last 5 years were included in the study. Thirty-ninepatients were aged less than 50 years and 54patients were over 50 years. Seventy-one controlsubjects, matched for age and sex, were also examined.The following genetic genotypes have beendetermined: Factor II G20210A, Factor V Leiden,MTHFR C677T and A1298C, PAI1 4G/5G. Factor IIG20210A, Factor V Leiden genotypes were obtainedby PCR amplification and gel electrophoresis, accordingpublished methods. MTHFR C677T and A1298C,PAI1 4G/5G were performed with a single-tubemethod for genotyping the polymorphisms that combinesboth rapid-cycle PCR with real time monitoringof the amplification process and generation ofallele-specific fluorescent probe melting profiles onthe Light Cycler. Total plasma homocysteine (HCY)was measured by high-pressure liquid chromatography(HPLC) with fluorescence detection. Hyperhomocysteinemiawas defined as total plasma HCY levelabove the 95 th percentile in the control group. Theeffect of each polymorphism, singularly or in association,has been investigated. Moreover the MTHFRC677T and A1298C polymorphisms have been correlatedwith homocysteine plasma levels. Preliminaryresults do not demonstrate a major role of thesegenetic variants in the pathogenesis of RVO.PO-033ASSOCIATION BETWEEN THE MATRIX METALLOPROTEINASE 35A/6A POLYMORPHISM AND RISK ON INCIDENT CEREBROAND CARDIO-VASCULAR DISEASEAlbiero E, Simioni M, Madeo D, Tosetto A,Rodeghiero FHemophilia and Thrombosis Center, HematologyDepartment, S. Bortolo Hospital, Vicenza, ItalyThe 5A/6A polymorphism in the matrix-metalloproteinase3 gene is a recently described polymorphismthat has been associated with an increased riskof atherosclerotic cardiovascular disease in retrospectivestudies. We aimed at evaluating if this polymorphismrepresents an independent risk factor forcerebro and cardiovascular diesease in a prospecitivestudy. We analyzed 53 cases of incident AMI (n=39)or stroke (n=14) occurred during the follow up period1996-2000 in the VITA Project cohort and fulfillingthe WHO MONICA criteria. The cases werematched with 106 controls, controlling for age, sexand time of follow-up in a nested cases-control study.Analysis of the 5A/6A polymorphism was carried outby PCR amplification and subsequent analysis of heterozygosityby DHPLC analysis in the original samplesor in mixtures from known 5A or 6A homozygotes.The male:female ratio was 2.8:1, with a mean age of55 years. The 5A and 6A allele were in Hardy-Weinbergequilibrium in the studied subjects, with an allelefrequency for 6A of 46% in cases and 40% in controls.Compared to 5A5A homozygotes, the crude estimateof relative risk was 0.5 (95% CI 0.23-1.1) in 5A/6Aheterozygotes and 2.7 (95% CI 0.95–7.7) in 6A/6Ahomozygotes, consistent with an autosomal recessiveeffect. Logistic regression showed a relative risk of4.3 (95% CI 1.6-11.6) in 6A/6A homozygotes comparedto 5A/5A and 5A/6A subjects, after adjustementfor smoking status, sistolic blood pressure, and cholesterollevel. There was no sustantial differencebetween risk estimates in cerebro or cardiovascularend-points. The MMP3 5A/6A polymorphism representsa new, possibly strong genetic risk factor forincident cardio or cerebrovascular disease.PO-034INHERITED THROMBOPHILIC GENE MUTATIONS DO NOT HELPINENTIFY PATIENTS WITH EARLY-ONSET ISCHEMIC STROKECoppola A, Tufano A, Madonna P, Albisinni R,Cirillo F, Varricchione N, Cerbone AM, Orefice G,*Di Minno GRegional Reference Centre for Coagulation Disease,Dep. of Clinical and Experimental Medicine, and *Dep.of Neurological Sciences, Federico II University,Naples, Italyhaematologica vol. 89(suppl. n. 8):september 2004

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 2004111The mechanisms of ischemic stroke in young adultsare poorly understood. Over the last years, a role ofgenetic factors predisposing to thrombophilia andfor moderate hyperhomocysteinemia in this settinghas been largely debated. We evaluated in 221 consecutivepatients (111 men, 110 women; mean age39.7±11.6 yrs) with a history of early-onset ischemicstroke (mean age at first event, 35.4±11.3 yrs; range6 mo.-50 yrs) the prevalence of factor V (FV) Leiden,prothrombin (FII) G20210A, and C677T 5,10-methylenetetrahydrofolatereductase (MTHFR) gene mutationsand fasting serum total homocysteine levels(tHcy). Three hundred twenty-nine healthy subjects(167 men, 162 women; age 36±13.2 years) served ascontrols. Fasting tHcy levels differed significantlybetween patients and controls (15.8±21.7 vs.10.7±6.2 µmol/L, p=0.006). In contrast, the 677TTMTHFR genotype, although more frequent inpatients, was not statistically different in the twogroups (26.2% vs. 17.2%). Frequencies of FV Leidenand of FII G20210A mutations were also comparablein patients and controls (6.8% vs. 5.8% and 7.2%vs. 5.2%, respectively). When patients with othercoexisting established cardiovascular risk factors(cigarette smoking, arterial hypertension, hypercholesterolemia,diabetes mellitus, obesity/overweight,oral contraceptives) were excluded, only 1 patientwas found to carry the FII G20210A variant among26 (11.8%) patients with apparently idiopathicevents. The latter showed a similar frequency of the677TT MTHFR genotype (23.1%), but lower tHcy levels(8.5±3.0 µmol/L, p

112PostersPO-036CATASTROPHIC VASCULAR OCCLUSION SYNDROME IN APATIENT WITH MILD HYPERHOMOCYSTEINEMIA ANDABDOMINAL AORTIC ANEURYSMBenevolo G, Vendramin C, Fra GP, Gaidano GUDA Ematologia & Clinica Medica Generale,Dipartimento Scienze Mediche, Università delPiemonte Orientale "Amedeo Avogadro", AziendaMaggiore della Carità, Novara, ItalyHyperhomocysteinemia is a recognised risk factorfor venous thromboembolism and is associated withan increased risk of occlusive arterial disease. Highplasma levels of total homocysteine result from theinteraction between genetic and acquired determinants;moreover, some authors have reported a relationshipbetween hyperhomocysteinemia andabdominal aortic aneurysm (AAA). We report thecase of a 69-year old man who was diagnosed ashaving mild hyperhomocysteinemia (17.4 mmol/L)with heterozygous 677C→T substitution in MTHFRgene and AAA (4.2 cm) with thrombi four monthsbefore his current admission to our Division. Thepatient was being treated with oral anticoagulantstherapy (OAT) for wandering phlebitis and with vitaminsfor hyperhomocysteinemia. On admission, hepresented with sudden chest pain, dyspnea and feverwith elevated D-dimer levels, normal levels of homocysteineand INR 2.76 (target 2.5). A lung perfusionscan revealed pulmonary embolism whereas a colordopplerultrasonography resulted negative for deepvein thrombosis. Thrombophilic screening showedabsence of antiphospholipid antibodies and normalityof AT, PC and PS levels. FV Leiden and abnormalprothrombin were absent. OAT was replaced by subcutaneouslow-molecular-weight heparin (LMWH)with rapid improvement of lung perfusion and thepatient was discharged with OAT (INR target 3.00).During subsequent OAT, at day 21 after discharge, thepatient developed a femoral vein thrombosis despitean INR=2.90 and homocysteine=8.9 umol/L. On newadmission, he was managed with unfractionatedheparin (UFH) followed by LMWH. After three dayshe developed an extensive stroke with hemiparesis.The clinical course was complicated by peripheralarterial ischemia treated with embolectomy. Despiteheparin therapy, the patient died after developingcoma. Blood cell counts remained normal throughoutthe clinical course. An autopsy with histologicalexamination did not reveal occult cancer disease butshowed a widespread venous and arterial thromboticocclusion. In patients with AAA, the use of supplementalvitamins aimed at lowering plasma homocysteinemay not be sufficient to modify vasculardisease progression.PO-037HOMOCYSTEINEMIA AS RISK FACTOR FOR CARDIO-EMBOLICSTROKE IN PATIENTS WITH NONVALVULAR ATRIALFIBRILLATIONLoffredo L, * D'Angelo A, **Fimognari FL,Cangemi R,* Sampietro F, * Mazzola G, Di Lecce VN,Buttiglieri R, Violi FUniversity "La Sapienza", Rome, Italy, *Servizio diCoagulazione ed Unita’ Ricerca Trombosi, ScientificInstitute H.S. Raffaele, Milan, Italy, **U.O.C. MedicinaInterna, Ospedale L. Parodi-Delfino, ASL RM G,Colleferro, ItalyAtrial fibrillation (AF) is complicated by a high rateof ischemic stroke. Previous studies have shown thatincreased circulating total homocysteine (tHcy) is anindependent predictor of stroke, but it is unclearwhether it does predict stroke also in patients withatrial fibrillation. The objective of this study was toevaluate if increased tHcy is an independent predictorof cardio-embolic stroke in patients with nonvalvularAF. We studied 163 consecutive patients (77males and 86 females; mean age 72.3±8.8 years)with chronic (n=118) or paroxysmal (n=45) AF ofnonvalvular origin. Among them, ischemic stroke wasdocumented by MNR or CT imaging in 40 patients(16 males and 24 females mean age 74.8±8.8 years).Fasting tHcy levels were determined by HPLC aftercleavage and reduction with sodium borohydride followedby derivatization with SBD-F (ammonium-7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate). Multivariateanalysis showed that age (OR:1.063 for each1 year increase of age; 95% C.I.: 1.009-1.121,p=0.034), total homocysteine (OR:1.052; for each 1µmol/l increase, 95% C.I.: 1.005-1.102; p=0.025) andfibrinogen (OR:1.008 for each 1 mg/dL increase; 95%C.I.: 1.002-1.013; p=0.009) were independent predictorsof ischemic stroke. In patients with nonvalvularatrial fibrillation, increased fasting tHcy levelsare an independent predictor of cardio-embolicstroke, suggesting that changes in methioninemetabolism may predispose to thromboembolism inthis setting.PO-038C677T AND A1298C MTHFR, A2756G MTR AND 844INS68CBS GENE POLYMORPHISMS IN ATRIAL FIBRILLATIONGiusti B, Poli D,° Gensini F, # Sestini I, Poggi F,Rossi L, Lapini I, Lucarini L, Giuello A, Pepe G,Abbate R, Gensini GFDepartment of Medical and Surgical Critical Area,University of Florence; °Dipartimento Cardiologico edei Vasi, Azienda Ospedaliera Careggi, Florence;#Department of Clinical Pathophysiology, Universityof Florence, Italyhaematologica vol. 89(suppl. n. 8):september 2004

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 2004113Elevated plasma total homocysteine (tHcy) concentrationsare a risk factor for cerebrovascular disease.Recent data indicate that hyperhomocysteinemia is arisk factor for atrial fibrillation (AF). Hyperhomocysteinemiamay be associated with the presence of polymorphismsin genes codifying for enzymes involved inthe Hcy metabolism. Aim of this study was to evaluatethe prevalence of C677T and A1298C of 5, 10-methylenetetrahydrofolate reductase (MTHFR),A2756G of methionine synthase (MTR) and 844ins68of cystathionine &#61538; synthase (CBS) gene polymorphismsin relation to homocysteine (Hcy) in AFpatients. We studied 245 patients with AF and 245matched healthy control subjects. For polymorphismdetection, DNA was amplified by PCR and genotypewas determined by microarray technology by usingelectronic chips (Nanogen technology). Hcy plasmalevels were significantly higher in AF patients than incontrols (14.8, 5.4-54.1 versus 11.5, 1.6-52.6 umol/L,p=0.001). Hcy plasma levels were significantly higherin AF patients with history of ischemic event(s)than in AF patients without history (15.4, 5.4-54.1versus 13.9, 6.7-38.6 µmol/L, p

114Postersprostanoide intravenously without any reduction ofthe lesion. We investigated the parameters or thromboticdiseases like Antithrombin, Protein C, ProteinFree S, APC-r, Lupus anticoagulant, homocystinemiaand we studied in PCR the genetic mutation of FactorV Leiden, Prothrombin and MTHFR. We didn’t findany positive evidence, neither on functional coagulationparameters, nor on Factor V Leiden and Prothrombingenetic mutations. We observed as onlypathological result, high homocysteine level andgene MTHFR mutation. We have been pushed, afterthis results, to start folic acid, B12 vitamin and B6vitamin therapy, observing it for three months. Sincethe lesion has been observed to reduce within threemonths, we suggest using a folic acid, B12 vitaminand B6 vitamin therapy as a support to cure the nonhealingulcers.levels in amniotic fluid were 1.05±0.7 µmol/L, witha 95% C.I. 0.98-1.11. In amniotic fluids of malefetuses the value was µmol/L, while it was µmol/Lin amniotic fluids from female fetuses; this differencewas not statistically significant (p= 0.001). In amultivariate analysis, homocysteine levels in amnioticfluids were not significantly related to maternalage (Spearman correlation, p= 0.84), or gestationalage based on menstrual history (Spearman correlation,p= 0.6), while they were significantly related tofemur lenght (p=0.009) and BPD (p=0.012). We concludethat homocysteine levels in amniotic fluid aresignificantly related to the fetal growth. The knowledgeof normal homocysteine values at the time ofmid-trimester amniocentesis could be useful indetecting fetal abnormalities resulting in higherhomocysteine levels in the amniotic fluid.PO-041HOMOCYSTEINE LEVELS IN AMNIOTIC FLUID: NORMALVALUES IN UNEVENTFUL PREGNANCIES AND CORRELATIONWITH FETAL GROWTHColaizzo D, Grandone E, Vecchione G, Cappucci F,Tiscia G, Monaco M, Sciannamè N, Croce AI,Notarangelo A, Cocomazzi N, Margaglione MIRCCS Casa Sollievo della Sofferenza, S. GiovanniRotondo, ItalyIt has been reported that amniotic fluid obtainedfrom pregnancies with fetuses suffering from NTD(Neural Tube Defects) shows high homocysteine levels.Nevertheless, data about normal values of homocysteinein amniotic fluid are sparse and inconsistentand no evidence is available in Mediterranean Countries,where dietary vitamin content differs from thatin Northern Europe or North America. Our goal wasto establish reference values for amniotic fluid fromnormal pregnancies in our geographical area. Amnioticfluid was obtained from 384 healthy femalesundergoing mid-trimester amniocentesis. No chromosomalabnormalities were found in fetal cells, andno fetal anomalies were found by transabdominalsonography at 21 weeks. Samples of amniotic fluidwere taken from each mother, placed on ice, centrifugedat 4°C 3000 rpm within 1h and the supernatantwas collected and stored at -80°C until testing.In addition, biparietal diameter and femur lenghtwere measured at the time of mid-trimester amniocentesisto verify the correspondence of fetal biometryand gestational age on the basis of last mestrualperiod. Mean age (±1SD) of women included in thestudy (n= 384) was 37.1±3.6 years. Amniotic fluidwas drawn at a gestational age of 17.46±1.35 weeks.In 198 cases, fetuses were male, and in 190 females.Four were twin pregnancies. Overall, homocysteinePO-042MTHFR C677T POLYMORPHISM IS ASSOCIATED WITH LOWLEVELS OF FOLIC ACID IN THE INCHIANTI STUDYGori AM, Corsi AM *,Alessandrello Liotta A,°Falciani M, Mannini L,° Brogi D, Bandinelli B,Fedi S, Fatini C, Ferrucci L,** Abbate R, Gensini GFDepartment of Medical and Surgical Critical Area,University of Florence, Italy; °Dipartimento Cardiologicoe dei Vasi; *Laboratory of Clinical Epidemiology,Geriatric Department, National Institute ofResearch and Care on Aging (INRCA), Florence, Italy;**Longitudinal Studies Section, Gerontology ResearchCenter, National Institute on Aging, National Institutesof Health, Baltimore, USAImpaired folate status is associated with increasedrisk of several conditions such as cardiovascular diseaseand stroke. Methylenetetrahydrofolate reductase(MTHFR) is a regulating enzyme in folatedependenthomocysteine remethylation. Individualsthat are homozygous for MTHFR polymorphismC677T and in whom the folate intake is inadequatehave lower blood folate and higher plama homocysteinethan those with the CC genotype. The aim ofthis study was to determine the relationship betweenfolic acid plasma levels and vitamin status (vitamainB6, B12 and folic acid) and MTHFR genotypes in apopulation-based study (586 men and 734 women)which sampled people living in two sites in the surroundingof Florence (Italy), the InChianti Study. Inthe InChianti population the dietary intakes of folicacid, vitamin B6 and vitamin B12 did not significantlydiffer among MTHFR genotypes. General linearmodel analyses, after adjustment for age, sex,vitamin B6, B12 and folic acid intakes and creatininelevels demonstrated that serum folic acid levels wererelated to MTHFR C677 genotype: folate levels werehaematologica vol. 89(suppl. n. 8):september 2004

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 2004115significantly (p

116Posters5 mg folic acid daily, plus 1000 mcg cyanocobalaminmonthly). After three years the patient appears symptom-free,with normal levels of Hcy (mean 12mmol/L). Coronary angiography and 2-D B-modeultrasound scans of the legs showed complete regressionof atherosclerotic lesions. On suspending vitaminB6 and B12 Hcy levels promptly rose again. Thepatient is now only taking pyridoxine and folic acidand his Hcy is normal. Conclusions. The severe clinicalphenotype and the very high Hcy levels can beexplained by the multiple heterozygous mutations ofthe enzymes involved in Hcy metabolism (MTHFRC677T and A1298C plus CBS G919A). Monomers ofthe CBS protein with the G919A mutation are degradedrapidly intracellularly and will therefore not be amajor constituent of the active tetrameric protein.The drop in CBS activity justifies the clinical benefitof pyridoxine replacement to induce trans-sulfurationpathways.PO-045INTERACTION OF COLINE/BETAINE METABOLYSM ANDHOMOCYSTEINE PLASMA LEVELSGori AM, Sofi F, Carboni E, Cellai AP,^Alessandrello Liotta A,^ Giuliani G, Cesari F,Vecchio S, Casini A,* Surrenti C,* Moneti G, #Abbate R, Gensini GFDepartment of Medical and Surgical Critical Care,University of Florence; ^Dipartimento Cardiologico edei Vasi, Azienda Ospedaliero-Universitaria Careggi,Florence; *Department of Clinical Pathophysiology,University of Florence; # Department of Pharmacology,University of Florence, ItalyBetaine is an alternate methyl donor for homocysteineremethylation, derived from choline oxidation;βine homocysteine methyltrasferase (BHMT)transfer the methyl group from betaine to homocysteine,resulting in demethylated betaine (dimethylglycine,DMG) and methionine. The influence ofbetaine supplementation on circulating homocysteineconcentration has been studied mainly in clinicalsettings. Betaine treatment combined with folicacid normalized fasting plasma homocysteine concentrationsin patients with severe inborn hyperhomocysteinemia.Little published information is availableconcerning the effect of betaine on plasmahomocysteine concentration in subjects with normalto mild elevated homocysteine levels. Aim of thestudy was to examine the interaction betweenhomocysteine and choline/betaine metabolism in agroup of healthy subjects with normal vitamin status(folic acid, vitamin B6 and B12). We analyzedcholine, betaine and DMG in 72 healthy subjects(32F; 40M; median age: 58 years). Determination ofcholine, betaine and DMG was obtained by a highthroughputmethod based on normal-phase chromatography-tandemmass spectrometry. Homocysteineand choline plasma levels were found to besignificantly related (R=0.33; p13 µmol/L) respect to those (11.3±3.1 µM) withnormal values of homocysteine. These preliminarydata show the possible interaction between cholineand homocysteine levels.This work was supported by a Grant of the Ministerodell’Istruzione, Università e Ricerca, ItalyPO-046COMPARISON OF A NEW ENZYMIC ASSAY AND HPLC METHODTO DETERMINE TOTAL PLASMA HOMOCYSTEINEFermo I,* Mazzola G, Sampietro F, D'Angelo A*Laboratory of Chromatografic and SeparativeTechniques and Coagulation Service and ThrombosisResearch Unit, IRCCS H S.Raffaele, Milan, ItalyOver the last two decades a number of studies haveshown that moderate and persistent hyperhomocysteinemiais implicated in the development of atherosclerosisand thrombosis. Several methods have beendeveloped for measuring total plasma homocysteine(tHcy), most of them based on HPLC analysis and fluorescencedetection. Very recently, a totally automatedenzymic assay for tHcy determination (Carolina,Liquid Chemistries Corp, CA, USA), performed on a biochemicalanalyzer capable of dispensing threereagents and of measuring absorbance at 340 nm, hasbeen introduced. Our aim was to compare the performanceof this new procedure and an establishedhome-made HPLC method based on SBD-F derivatizationand the use of an internal standard. Since inour Institute tHcy levels are also routinely determinedafter the methionine loading test, we performed thisstudy in the range: 4.0-60.0 µmol/L. Sodium citrateplasma samples were taken from 96 fasting donors(55 women and 45 men; age: 49±18.7 yr, mean±SD)and frozen at -80°C until assay. The enzymic assay testis based, after reduction to free HCY, on the followingbiochemical reactions, catalyzed by CBS, CBL and LDenzymes, respectively.1) serine+Hcy (arrow) L-cystathionine;2) L-cystathionine (arrow) hcy+pyruvate+NH3haematologica vol. 89(suppl. n. 8):september 2004

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 20041173) pyruvate+NADH (arrow) lactate+NADThe chromatographic procedure included reductionof disulfide bonds by NaBH4, use of cysteamineas internal standard, derivatization with ammonium-7-fluorobenzo-2-oxa-diazole-4-sulphonate(SBD-F), and fluorescence detection after separationby reversed-phase HPLC. Medians and ranges of tHcyplasma levels (n=96) observed were: 16.1 (3.9-61.8)and 17.5 (4.9-58.3) µmol/L for HPLC and enzymickit, respectively. Linear regression analysis yielded:enzymic assay = 1.044 (± 0.546) + 1.011(± 0.024)HPLC (±SE), r = 0.975, Sx/y = 2.85 (p

118Postersrelated to an acute inflammatory response. Thesenoninvasive indexes may help in further examiningT1DM pathophysiology and monitoring pharmacologicalinterventions to interfere with disease developmentand progression.PO-048THE ROLE OF CRP IN THE OUTCOME OF CARDIOVASCULAREVENTS IN TYPE-2 DIABETIC PATIENTS: THE GENDIABE STUDYZito F,* Di Castelnuovo A,* Ciccarone E,* #Capani F, # Volpi R, § Donati MB,* Iacoviello L,*on behalf of the GENDIABE Investigators*Laboratory of Genetic and EnvironmentalEpidemiology, Center for High Technology Researchand Education in Biomedical Sciences, CatholicUniversity of Campobasso, Italy; # Department ofMedicine and Aging, University of Chieti, Italy;§3 rd Laboratory, Spedali di Brescia, ItalyBackground. CRP is an acute phase protein producedby the liver in response to inflammatory stimulileading to high IL6 levels. CRP is high in subjectswith cardiovascular disease (CVD) and predicts therisk of cardiovascular events in both stable andunstable angina. It is also found high in subjects withtype-2 diabetes. CRP levels are influenced by severalfactors such as gender, obesity, socio-economicclass, alcohol. A recently developed high-sensitivitymethod to detect CRP (hsCRP) is presently the mostlargely used in CVD assessment. Aim and Methods.We aimed at investigating the association of hsCRPlevels with macrovascular events (AMI, stroke andunstable angina) in the GENDIABE population oftype-2 diabetics. GENDIABE is a case-control studyconducted in Central Italy (Diabetology Unit ofPescara) between 1997 and 2000. 837 diabeticpatients were consecutively included into the study:301 were cases (with CVD events) and 536 controls(without CVD). We measured hsCRP (immunoturbidimetricassay) with other biochemical markers such asblood glucose, total cholesterol, tryglycerides, glycosilatedhaemoglobin (HbA1). We also consideredthe duration of diabetes as a prognostic indicator.Results. Cases were 301 (36%) and controls were536 (64%), as expected by the prevalence of CVD intype-2 diabetes. Higher hsCRP (p=0.03), male gender(p=0.001), higher age (p=0.0006), longer duration ofdiabetes (p

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 2004119observe differences in long-term mortality (4,2% inCES group vs 4,6 in ATS group, p=ns) and long-termre-hospitalization (16,3% in CES group vs 14,7% inATS group) between the two analysed group. Conclusions:Our study, with the limitations of the retrospectivestudies, shows that elevated CRP at hospitaladmission represents a marker of poor prognosisin elderly patients with ischemic stroke. Elderlypatients with CES seem to have higher levels of CRPassociated to more severe short-term prognosis.PO-050INFLAMMATORY MARKERS ARE INCREASED IN CAROTIDATHEROSCLEROTIC PLAQUES AND IN PLASMA FROM PATIENTSINFECTED WITH CHLAMYDIA PNEUMONIAEGresele P, 1 Falcinelli E, 1 Corazzi T, 1 Giordano G, 2Colucci M, 3 Sidoni A, 4 Sensini A, 5 Marroni M 61Departments of Internal Medicine, 2 VascularSurgey, 4 Experimental Medicine and BiochemicalScience, 5 Microbiology, and 6 Infectious Diseases,University of Perugia; 3 Department of BiomedicalScience, University of Bari, ItalyChronic infection with Chlamydia pneumoniaehave been implicated as potential etiologic agents ofatherosclerosis. The aim of our study was to evaluatethe association between Chlamydia pneumoniaeinfection and inflammatory activation in carotidatherosclerotic plaques and in plasma of patientsundergoing endoarterectomy. Carotid plaques wereobtained from 36 patients: 18 seropositive toChlamydia pneumoniae (IgG >1:16), and 18 seronegativeto Chlamydia pneumoniae. Carotid plaque tissuewas weighted, minced and extracted. Matrixmetalloproteinases (MMPs by ELISA and zymography),COX-2 (by Western blotting), Tissue Factor (TFby ELISA) and Thrombomodulin (TM by ELISA) wereexpressed in significantly higher amounts in plaquesfrom seropositive patients (MMP-2: 45.3±6 ng/mg vs17.2±3.5, p

120Postersexplanation of the increased number of clinically relevantthrombotic episodes observed in acutely illpatients. Early detection of the ketonemia or pharmacologicprophylaxis of the prothrombotic statecould constitute a step forward in diabetis care.PO-052PLASMINOGEN ACTIVATORS IN VERNALKERATOCONJUNCTIVITIS PATIENTSLeonardi A, 1 Sartori MT, 2 Saggiorato G, 2 Pellati D, 2Brun P, 3 De Dominicis C, 1 Morello M, 2 Spiezia L, 21Department of Neuroscience, Ophthalmology Unit;2Clinical Medicine, Department of Medical andSurgical Sciences; 3 Department of Histology andMicrobiology; University of Padova, ItalyThe serine protease, urokinase (uPA) is a potentchemoattractant for leukocytes and may be involvedin the pathogenesis of severe forms of allergic conjunctivitissuch as vernal keratoconjunctivitis (VKC).Tear and peripheral blood samples were obtainedfrom 20 active VKC patients and 19 normal subjectsas a control group. Levels of plasminogen activity,uPA, tissue plasminogen activator (tPA) and theirinhibitor, plasminogen activator inhibitor type 1 (PAI-1) antigen were measured in tears and plasma ofVKC patients with and without severe corneal damage.Tissue expression of tPA, uPA and urokinasereceptor (uPAR) were evaluated by immunohistochemistryand immunocytochemistry. uPA, tPA andPAI-1 were measured in supernatants of conjunctivalfibroblasts cultures. Four VKC patients presentedcorneal ulcers, and 6 a mild keratopathy. Tear levelsof uPA (3.49±3.5 vs 0.8±1.3 ng/mL, p=0.0004) andtPA (8.68±12 vs 1.9±1.7 ng/mL, p=0.0002) were significantlyincreased in VKC patients compared tocontrols. PAI-1 tear levels were not detectable. Tearplasminogen activity levels were significantlyincreased in VKC compared to controls (4.7±3.4%vs. 0.65±0.8%, p=0.003). No correlations were foundbetween tear levels of fibrinolytic parameters andclinical score, whereas only tPA correlated withcorneal involvement. uPA levels were correlated withthe number of inflammatory cells in tears. Increasedexpression of plasminogen activators was found inVCK tissues compared to normal conjunctiva. Byimmunocytochemistry, eosinophils and neutrophilsshowed either intra-cytoplasmic or membrane uPApositive staining. Increased fibrinolytic expressionand imbalance between plasminogen activators andPAI-1 may be involved in the pathogenesis of severeallergic conjunctivitis.PO-053ASYMMETRIC DIMETHYL ARGININE (ADMA), HOMOCYSTEINEAND SEMICARBAZIDE-SENSITIVE AMINE OXIDASE ACTIVITY(SSAO): NEW MARKERS OF OXIDATIVE STRESS IN ACUTECORONARY SYNDROMESMarcucci R, Raimondi L,* Sofi F, Baldi M,Giuliani M, Vecchio S, Cellai AP, # Casini A°,Mugelli A,* Surrenti C,° Abbate R, Gensini GFDipartimento Area Critica Medico Chirurgica; *Dipartimento di Farmacologia; ° Dipartimento diFisiopatologia Clinica; # Dipartimento Cardiologico edei vasi, Azienda Ospedaliero-Universitaria Careggi,Florence, ItalyHigh levels of Homocysteine (Hcy) are a wellestablishedrisk factor for atherosclerotic disease.One of the mechanism evoked to support its pathogeneticrole in atherosclerosis is the induction of anoxidative stress, but in vivo data are lacking. Ex-vivoreports documented an inhibitory effect of high levelsof Hcy on the enzyme dymethyl arginine dymethylaminohydrolysis(DDAH) which converts theAsymmetric dimethyl arginine (ADMA) in citrullin.Elevated levels of ADMA, the most potent endogenousinhibitor of the nitric oxide synthase activity,are markers of endothelial dysfunction and are foundelevated in patients with atherosclerosis. The semicarbazide-sensitiveamine oxidase (SSAO) is a tissue-boundamine oxidase activity with adhesionproperties highly expressed in the vasculature. Forunknown reasons in the metabolic syndrome and incardiovascular diseases, elevated levels are found buttheir pathological significance remains to establish.One hypothesis is that this enzymatic activity participatein generating endothelial dysfunctionthrough the production of cytotoxic aldehydes andhydrogen peroxide. The aim of this study was to evaluateHcy and ADMA levels and SSAO activity inpatients with acute coronary syndromes (ACS). 75patients (age 62.6; 11.6; 70 M/5 F) with ACS (51 AMI,24 UA) referred to the Cardiologic Care Unit of theUniversity of Florence and 75 controls (age 61; 9.8;69 M/6 F) were enrolled. SSAO activity, ADMA andHcy levels were significantly higher in patients thanin controls (31.6;4.6 pmol/mg/h vs 0-2 pmol/mg/h;1851.58;254.65 pmol/mL vs 520; 360 pmol/mL and22.7;18.4 µmol/L vs 12.3;10.5 µmol/L). SSAO activitysignificantly correlated with Hcy (r=0.57; p =0.001) and Hcy levels significantly correlated withADMA (r=0.49; p

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 2004121generated by hyperhomocysteinemia. The pathwaysby which ADMA and SSAO are involved in the oxidativedamage caused by hyperHcy seem to be independent.This work was supported by a grant from Ministerodella SalutePO-054CHRONIC ENDOTHELIAL CELL DAMAGE/PROLIFERATION ANDINCREASED NEOANGIOGENESIS BY ENDOTHELIAL GROWTHFACTOR (VEGF), PLATELET-DERIVED GROWTH FACTOR (PDGF)AND INTERLEUKIN-6 (IL-6) SERUM ABNORMAL RELEASE INSICKLE CELL DISEASEMusso R, Cultrera D,* Chiarenza A,* Meo A,**Burgio N, Cipolla N, D'arpa S, Giustolisi R*Haemophilia and Thrombosis Reference RegionalCenter; *Institute of Hematology, University ofCatania; **Department of Pediatrics, Universityof Messina, ItalyAbnormal circulating endothelial cells (EC)(SamuelO Sowemino-Coker et al. American J Hematology 31;1989), as well as vascular endothelium chronic damagewith abnormal cytokines release (Musso R. et al.6 th Intern. Conference on Thalassemia and Haemoglobinopathies,Malta, 1997) have previously beenreported in sickle cell disease (SCD). We here reportthat vascular endothelial growth factor (VEGF), themost important growth and survival factor forendothelium and platelet EC mitogen, is increased inSCD, thus suggesting an important role in the regulationof the vasculogenesis. We further carry evidencethat human platelet derived growth factor AB(PDGF), the major mitogenic factor, is also raised inthe serum of SCD patients. 18 SCD patients (10females and 8 males, age ranging 22-59 yrs,&#946;+ thalassemia/S trait, 7 previously splenectomized,without renal nor liver dysfunctions), bothin steady state and during painful episodes (n=19)were studied. 15 healthy subjects, sex and age comparable,served as controls. Human VEGF by ELISAmethod (R&D Systems, Italy) in conjunction withvWF (ELISA procedure, Dade Behring Institute, Italy)as index of EC function and human PDGF-FAB (ELISA,R&D Systems, Italy), as indicator of platelet releaseand of chemotaxis for neutrophils and fibroblast inconjunction with Interleukin-6 (IL-6) (ELISA, R&DSystems, Italy) were assayed. Our results are belowreported (see Table below). Our data showed thatincreased VEGF circulating levels are present in SCDalso in the steady state. Human PDGF-FAB, vWF andD-dimer were parallely elevated. Among the severaleffects induced by VEGF, it is reported that thiscytokine increases micro-vascular permeability,vasodilatation, EC proliferation and cell migration,abnormal platelet release with elevated plateletcount and influences the neo-angiogenesis and vasculogenesis.VEGF also promotes extravasation ofplasma fibrinogen, leading to fibrin deposition andpromotes the migration of macrophages and fibroblastsand EC proliferation. By considering that abnormalplasma levels of vWF and PDGF with elevatedcirculating D-dimer as index of thrombin activationand fibrin deposition in vivo, in conjunction withraised amounts of IL-6, well known cytokine-mediatorof coagulation process, were present in SCDpatients, we suggest that this observation stronglyconfirms their proneness to constantly increasedthrombogenic risk.VEGF vWF PDGF-FAB D-dimer IL-6pg/mL % pg/mL pg/mL pg/mLPatients ss pc ss pc ss pc ss pc ss pcn=18 70± 97± 115± 285± 23± 58± 485± 641± 33± 49.6±13.9* 4.3** 38* 69** 7.1* 2** 69* 93** 21.7* 30.3**Controlsn=15 57±7.1 95±18 13.6±6.4 210±80 22±9.4PO-055CHANGES IN COAGULATION VARIABLES FOLLOWING A6-MO R-HGH REPLACEMENT IN GROWTH HORMONE DEFICIENTADULTSValle D, 1 Cirillo F, 2 Merola B, 3 Beck-Peccoz P, 4Attanasio AF, 1 Lombardi G, 3 Di Minno G, 2,5 onbehalf of Eli-Lilly B9 REWGDED Italian Study Group1Eli Lilly, Florence, Italy, 2 Department of Clinical andExperimental Medicine, “Federico II” University,Naples, Italy; 3 Department of Endocrinology,University of Naples; University of Milan; 4 Instituteof Endocrine Sciences, Ospedale Maggiore IRCCS,and 5 laboratory of Thrombosis and haemostasis,Ospedale “Casa sollievo della Sofferenza” IRCCS,S. Giovanni Rotondo, ItalyIn the frame of a large multicenter, internationalstudy, in a subset of 71 patients (28F, 43M) withgrowth hormone deficiency randomized to receivetwo different recombinant human GH (r-HGH) regimens(low dose, n=37, age:42.4±16.2 yr; BMI:26.7±5.3 Kg/m 2 ), 3 µg/Kg/d for three mo followed by6 µg/Kg/d; (high dose, n=34, age:38.3±15.7 yr; BMI:26.7±4.9 Kg/m 2 ) 6 µg/Kg/day for three mo followedby 12 µg/Kg/d, we have measured changes in fibrinogen,PAI-1 activity, t-PA antigen, protein C, and proteinS levels. Patients were on thyroid, adrenal andhaematologica vol. 89(suppl. n. 8):september 2004

122Postersgonadal hormone replacement therapy. Coagulationparameters were measured at baseline and after 6mo-treatment. In all patients, baseline fibrinogenconcentrations were in the upper normal levels (200-400 mg/dL) and significantly correlated (r=0.32,p65 were considered(n=11; 347.00±83.896 vs. 315.00±74.59,p=0.024). No change was seen in PAI-1 and TPAthroughout the 6-mo treatment and the baseline concentrationscorrelated with age (r=0.30,p

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 2004123mebrane there are also several classes of porin,which control cell permeability by forning crossmembranechannels. Because responses to the bloodinvasion of Gram negative bacteria include also disseminatedintravascular coagulation (DIC) and septicshock, we studieid the role of porins fromSamonella Typhimurium on human blood coagulationin vitro. Materials and Methods. Porins werepurified to homogeneity from Salmonella typhimuriumSH5014 (from the collection of the Istituto diMicrobiologia, Seconda Università di Napoli) accordingto the method of Nurminen as described byTufano et al; Blood from ten young non-smokerssubjects was obtained in order to have platelet freeplasma and blood serum. Beside PT, aPTT, CT of recalcifedplasma (RT), serum residual prothrombin test(SRPT), AT III serum and plasma levels, serum TAT weanalysed in vitro effects of porins and/or AT III on asimplified coagulation system and thrombin esteraseactivity was observed using Cromozyn. Results. Thepresence of porins did not modify PT, aPTT, SRPT,plasma and serum AT III levels, plasma and serumTAT concentrations, while markedly modified CT andthrombin esterase activity as showed in the Tablebelow.Thrombin cromozym A 405 Proteolysis %Nothing 0.050±0.002 0Porin (0.15 µM) 0.086±0.005 + 72LPS (0.15 µM) 0.048±0.003 0AT III (1.50 µM) 0.005±0.001 − 90Porin + AT III 0.142±0.03 + 184Discussion. Using appropriate experiments weobtained data showing the main target of porin-procoagulanteffect. Moreover a possible binding ofporin directly to thrombin may be suggested also invivo on the basis of data we reported; so, the implicationsof these findings in pathogenesis of the DICoccurring during the Gram negative sepsis should behypothised and might explain also the poor resultsobtained on DIC. septic shock from therapy of basedon anti-LPS antibodies.PostersNUTRITON & VASCULAR RISKPO-058HEMOSTATIC EFFECTS OF DIETS CONTAINING OLIVEOIL OR SOY OIL IN HYPERTENSIVE PATIENTSTrifiletti A, Scamardi R, Lasco A, Pizzoleo MA,Morabito N, Atterritano M, Gaudio A, Sottile L,Frisina NDepartment of Internal Medicine, University ofMessina, ItalyThe influence of dietary fat on blood pressure andon the hemostatic system has been investigated inseveral studies. We evaluated, in the present study, theeffects of two diets, one rich in monounsaturated fattyacids(MUFA) and the other rich in polyunsaturatedfatty acids(PUFA), on hemostatic parameters andblood pressure levels of hypertensive patients. Fortyfourhypertensive patients with objective organicchange were assigned randomly to extra-virgin oliveoil(MUFA) or soy oil(PUFA) diet for 3 months. At baselineand after 3 months blood pressure, body massindex(BMI), plasma levels of cholesterol, trygglicerides,tissue-type plasminogen activator(t-PA),plasminogen activator inhibitor(PAI-1), D-dimer(DD)and prothrombin fragment 1+2(F1+2) were evaluated.xAfter diet in each grup there were no significantdifferences in change of systolic blood pressure, BMI,total cholesterol, tryglicerides and DD. With both dietsa significant reduction of diastolic blood pressure, t-PA, PAI-1 and F1+2 was found. xMoreover Pai-1 andF1+2 were significantly lower at the end of the MUFAdiet compared with the PUFA diet. In conclusion, ourfinding seems indicate that, in hypertensive patients,dietary reduction in saturated fat using extra- virginolive oil affects diastolic blood pressure and hemostaticsystem, significantly decreasing some importanthemostatic parameters.PO-059POLYUNSATURATED OMEGA-3 FATTY ACIDS REDUCE THEPLASMA LEVELS OF N-EPSILON-(CARBOXYMETHYL) LYSINEADDUCTS OF PROTEINS IN PATIENTS WITH MYOCARDIALINFARCTIONBasta G,* Del Turco S,* Lazzerini G,* Papa A,*Schmidt EB, # Christensen JH, § De Caterina R*°*CNR Institute of Clinical Physiology, Pisa, Italy;#Department of Preventive Cardiology andhaematologica vol. 89(suppl. n. 8):september 2004

124Posters§Department of Nephrology, Aalborg Hospital, ÅrhusUniversity, Denmark; °"G. d'Annunzio" University,Chieti, Italy(Carboxymethyl)lysine (CML), an advanced glycationend product formed on proteins by combinednonenzymatic reactions of glycation and oxidation(glycoxidation), may be considered a marker ofoxidative stress as well as a risk factor for atherosclerosisand diabetes, and is produced in conditionsof inflammation. Since several studies have indicatedthat n-3 polyunsaturated fatty acids (PUFA) haveprotective effects on cardiovascular disease, weinvestigated the effect of dietary n-3 PUFA on plasmalevels of CML in survivors of myocardial infarctionForty patients (none diabetic) were randomlyallocated to receive either 5.2 g of n-3 PUFA daily(n=20 patients, 65.4±5.48 years) or olive oil (n=20controls 61±8 years) for 12 weeks. CML plasma levelswere analysed by a specifically developed ELISAbefore and after n-3 PUFA supplementation. Results.Baseline plasma CML concentrations were significantlyhigher in the patient group vs control group10.18 (4.40-43.12)g/mL (median 10-90 percentiles)vs 6.08 (1.76-22.60) g/mL; p=0.027). The plasma levelsof CML were significantly decreased in the patientgroup following to PUFA supplementation (from10.18 to 5.57 g/mL; p=0.04) but not in the groupthat received olive oil (from 6.08 to 4.26 g/mLp=0.11). Further, in the control group decreased totalcholesterol (p

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 2004125etarian diet (0.3 g/kg/day) supplemented with a mixtureof essential amino acids and ketoacids (VSD)were investigated for lipids and apolipoprotein parameters(traditional CVRF) as well as for oxidativestress (oxidized LDL, antibodies against OxLDL andthiobarbituric acid reactive substances -TBARS-),total Homocysteine, Lipoprotein (a), Albumin andCRP (Non traditional CVRF) including vitamins A, E,B12 and folic acid. And they were compared with 31patients (20 M, 11 F) aged 65 yrs (range 29-82 yrs)on conventional low protein diet (CLPD: 0.6 g/kg/day)with a similar renal function (median sCr: 5.2 mg/dL).Patients on VSD showed increased HDL-Cholesterollevels with a significant reduction of LDL-Cholesteroland an increased apoA1/apoB ratio. Among nontraditional CVRF, a mild but significant reduction ofOxLDL (p

126PostersPostersWOMEN'S HEALTH ISSUESIN THROMBOSIS & HEMOSTASISPO-063RISK OF DEEP VEIN THROMBOSIS: INTERACTION BETWEENCOAGULATION FACTOR IX AND XI LEVELS AND ORALCONTRACEPTIVE USECini M, Legnani C, Frascaro M, Lo Manto G, Oca G,Poggi M, Palareti GDept. Angiology & Blood Coagulation “MarinoGolinelli”, University Hospital S.Orsola-Malpighi,Bologna, ItalyHigh Factor IX (FIX) and Factor XI (FXI) levels areassociated with an increased risk of venous thromboembolism(VTE); it has also been suggested thatoral contraceptive (OC) use, in women with high FIXor FXI levels, may further increase the VTE risk. Wemeasured FIX and FXI levels by clotting assays in 173women who suffered from VTE in reproductive ageand in 456 healthy women. In both groups subjectswere excluded in case of: Antithrombin, Protein C orProtein S deficiency, Factor V Leiden or G20210A prothrombinmutation, Lupus Anticoagulant phenomenon(LAC). FIX and FXI levels were stratified intodeciles; the ORs for FIX and FXI levels above the 90thpercentile (adjusted for age, levels of fibrinogen, FactorII, V, VIII, XII, and Factor XI or IX for Factor IX orFactor XI, respectively) were 3.86 (95%CI: 1.18-12.59) and 8.93 (95%CI: 2.48-32.12), respectively.Between all circumstantial triggering risk factors forVTE, OC use was the most frequent. Among thepatients, 84 (57.9%) used OCs at the time of VTE.Among the controls, the 168 women who had usedOCs for at least 6 months in the two years beforepresentation but had stopped the treatment at least3 months before the time of blood sampling wereconsidered as OC users. The risk for VTE associatedwith OC use was 2.36 (95% CI: 1.61-3.45). The VTErisk for women using OCs with FIX or FXI levels above90th percentile was 2.98 (95% CI: 0.41-21.59) and26.11 (95% CI: 2.99-227.83), respectively. In conclusion,high levels of FIX or FXI significantly increasethe risk for VTE. Our data suggest that FXI may constitutea strong risk factor for VTE and that the jointpresence of elevated FXI levels and OC use has asuper additive effect on the risk of VTE.PO-064THROMBOPHILIA IN WOMEN WITH UNEXPLAINED FETALLOSSESBonifacio G, Grimaudo S, Malato A, Anastasio R,Siragusa SCattedra di Ematologia, Azienda OspedalieraUniversitaria Policlinico di Palermo, ItalyBackground. Recent investigations highlight thepotential role of thrombophilia for determining fetalloss. However, the results so far published furnishdiscordant results. The purpose of this study was toinvestigate the possible role of inherited andacquired thrombophilia in women with unexplainedabortions and intrauterine fetal death. Methods: Westudied 49 women with a first episode of unexplainedlate fetal loss (fetal death after 20 weeks ormore of gestation) and 48 women who had had oneor more normal pregnancies and no late fetal losses.All the women were tested for the common testsfor acquired and congenital thrombophilia. RESULTS.The results are shown in the table.Patients Controls Odds Ratio p(95% Confidence Intervals) valueNo. of patients 49 48 n.s.Mean age, y. 28.6±7.4 27.8±7.8 n.s.ThrombophiliaLAC 30% 2.1% 4.9 (1.7-8.19)

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 2004127PO-065RISK OF RECURRENT VENOUS THROMBOEMBOLISM DURINGPREGNANCY AND PUERPERIUMRossi E, Za T, D'Orazio A, Leone G, De Stefano VInstitute of Hematology, Catholic University, Rome,ItalyThe opportunity of administering antithromboticprophylaxis to pregnant women with previous historyof venous thromboembolism (VTE) has been debated(N Engl J Med 2000;343:1439). We studied a retrospectivecohort of 483 women with a first previousVTE occurred during fertile age (15 to 45 years)and referred to our Thrombosis Center for laboratoryevaluation. The previous clinical history was takenat the admission blinded to the laboratory results.The median age at the first thrombotic event was 31years. All the events were objectively diagnosed.Women were stratified according to the triggeringcircumstances of the first event: 167 had first VTEduring pregnancy and puerperium (PP), 91 duringoral contraceptive (OC) use, 112 during transient riskcircumstances (surgery, trauma, bed rest) (TR), and113 spontaneously with no recognizable risk factor(SP). The rate of recurrent VTE was 34.5% in the SPgroup (3.5% of recurrences associated with pregnancy-puerperium),35.3% in the PP group (32.2% ofrecurrences associated with pregnancy-puerperium),19.7% in the OC group (11.1% of recurrences associatedwith pregnancy-puerperium), and 30.3% inthe TR group (5.9% of recurrences associated withpregnancy-puerperium). After the first VTE 99women completed 168 pregnancies (in 27 casesreceiving heparin prophylaxis and in 2 cases receivingaspirine); out of the 139 pregnancies not prophylaxed,52 occurred in 36 women with thrombophilia.Eleven pregnancies (9.6% of deliveries) and16 post-partum periods (14.0% of deliveries) withoutprophylaxis were complicated by VTE, 12 of themin women with inherited thrombophilia. (FVL=10,PS=1, PT 20210A=1); 19 of the recurrent events(70.3%) occurred among women of the PP group.Among the women of the PP group the VTE rate inthe subsequent unprophylaxed pregnancies (n=64)was 10.9% antepartum and 18.7%, postpartum),with a relative risk (RR) 2.8-fold increased (95% CI1.3-5.9) in comparison with the women of the othergroups (n pregnancies=75). The women of the PPgroup without any known thrombophilic trait(n=112) had during subsequent unprophylaxed pregnancies(n=46) a 10.9% VTE rate antepartum and17.4% postpartum; women with previous spontaneousVTE and/or inherited thrombophilia with previousVTE, both conditions suggesting prophylaxisduring pregnancy and puerperium, showed a riskquite similar (n pregnancies=53, 11.3% VTE rateantepartum and 13.2% postpartum, RR 1.1, 95% CI0.6-2.2). The postpartum VTE rate was 9.5% amongthe women of the OC group (n pregnancies=21) and8.7% among the women of the TF group (n pregnancies=23); no VTE occurred antepartum in suchlatter groups. In conclusion the risk of recurrenceamong pregnant women with previous VTE occurredduring pregnancy and puerperium is not negligible,irrespective of the presence of inherited thrombophilia.Prophylaxis is warranted through all thepregnancy as well as puerperium.PO-066THROMBOEMBOLISM IN PREGNANCY AND PUERPERIUM:RISK FACTORS AND TREATMENT IN GROUP OF 94 PATIENTSDonvito V, Maina A, Arrotta M, Cicogna L,Di Prima SServizio di Medicina Interna. Azienda OspedalieraOIRM-Sant' Anna, Turin, ItalyObjectives. We retrospectively rewiewed a group of94 patients with pulmonary embolism (PE) or deepvenous thrombosis (DVT) that occurred during gestationor after delivery to evaluate risk factors and efficacy/safetyof the conventional treatment. Cases. Wetreated and followed-up 94 patients from 1994 untilApril 2004: 59 of them had thromboembolism duringtheir pregnancy; the others 35 events occurred in thepost-partum period. Venous thromboembolism (VTE)occurred with similar frequency in each of the threetrimesters: 35% in the first, 38% in the second and25% in the third trimester.Pulmonary embolism wasmore frequent after delivery than during pregnancy(42% vs 22%). The risk of VTE in puerperium wasfound to be related to the route of delivery: 30/35occurred after caesarean section.We have carried outthrombophilia screening in 70 out of 94 patients (from1997 to 2004). Acquired or inherited thrombophiliawas found in 24 patients (34%). Treatment. All thepatients were treated with UF Heparin until 1998,than with subcutaneous low molecular weightheparin (LMWH) after 1998. They continued the treatmentfrom the time of diagnosis until delivery. In thepost-partum period they continued heparin orswitched to coumarin for at least six weeks. Caval filterwas positioned in two patients: one because ofrelapse of pulmonary embolism despite heparin treatment.The other one because of DVT onset near thetime of delivery. We didn’t find any heparin-inducedthrombocitopenia(HIT). One patient complained oflumbar bone pain and was found to have a significantreduction in bone density. No vertebral fractures weredetected. Four patients had bleeding complicationafter delivery and two of them required blood transfusion.Bleeding complications were not related tohaematologica vol. 89(suppl. n. 8):september 2004

128Postersexcess of the anticoagulation effect. No heparinrelatedfetal complications occurred. Conclusions.Pregnancy-related VTE occur evenly throughout gestationand peak in post-partum period, mostly inpatient delivered by caesarean section. Inherited oracquired thrombophilia is detected in about one thirdof the cases. LMWH is the treatment of choice inpregnant women. Careful evaluation is needed inpost-partum period.PO-067RISK OF VENOUS THROMBOEMBOLISM AMONG WOMENWITH A PREVIOUS HISTORY OF OBSTETRIC COMPLICATIONSDe Stefano V, Rossi E, Ferrazzani S,* De Carolis S,*Za T, D'Orazio A, Caruso A,* Leone GInstitute of Hematology and *Department ofObstetrics and Gynecology, Catholic University,Rome, ItalyInherited thrombophilia and antiphospholipid antibodies(aPL) are established risk factors for venousthromboembolism (VTE); they are recognized also asrisk factors for obstetric complications (preeclampsia,recurrent miscarriage, intrauterine fetal death).Occlusive changes of placental vessels leading tounderperfusion are hypothesized as the link betweenthrombophilia and obstetric complications. Yet therisk for VTE or obstetric complications seems to beshared by the same individuals only in a part of thecarriers of thrombophilia. In order to investigate therisk for VTE among women with obstetric complicationswe investigated 225 women with a history of2 or more uneplained fetal losses before the 20thweek of gestation (group A), 88 women with a historyof at least 1 fetal death after the 20th week ofgestation (in 45 cases associated also with at least1 miscarriage) (group B), and 180 women with a historyof preeclampsia (group C). A history of VTE waspresent in 26 women of the group A (11.5%), 16women of the group B (18.1%), and 16 women of thegroup C (8.9%). Thrombophilia, defined as the presenceof AT, PC, PS deficiency, factor V Leiden, PT20210A, and aPL as a single trait or in combination,was detected in 41.3% of the group A, 51.1% of thegroup B, and 26.6% of the group C. The carriershipof thrombophilia did not produce any increase in therisk of VTE among the women with recurrent miscarriage(relative risk 1.1, 95% CI 0.7-1.7) and thewomen with fetal death (RR 1.3, 95% CI 0.8-2.0) aswell as in the whole cohort of women with fetal losses(RR 1.2, 95% CI 0.9-1.6); this was the case alsofor the women with a history of preeclampsia (RR0.9, 95% 0.4-2.2). Such results did not substantiallychange after adjustement for the presence of aPLneither considering as outcome of interest only VTEoccurred during pregnancy and puerperium (14 casesin the groups A + B, 7 cases in the group C). Overall,174 pregnancies (of 1002) were completed bythe women of the groups A + B, and 314 pregnancies(of 425) were completed by the women of thegroup C. The VTE rate was estimated 4.6% antepartumand 3.4% postpartum among the women withfetal losses; the VTE rate among women withpreeclampsia was 0.6% antepartum and 1.6% postpartum.Therefore it can be suggested a higher riskof VTE during subsequent pregnancies amongwomen with a history of obstetric complications;underestimation of such risk could depend on thelow rate of pregnancy completion.PO-068D-DIMER AS FIRST MARKER OF THROMBOPHILIA IN WOMENAFFECTED BY UNEXPLAINED PRIMARY OR SECONDARYINFERTILIYDi Micco P,* D'Uva M,° Strina I,° Granata G,*Bonamassa B,* Mollo A,° Amato V,° de Marino C,°Niglio A,* De Placido G°*IV Divisione di Medicina Interna e PatologieEpato-bilio-metaboliche Avanzate, SecondaUniversità di Napoli, Napoli; °DipartimentoUniversitario di Scienze Ostetriche Ginecologiche eMedicina della Riproduzione, Area Funzionale diMedicina della Riproduzione ed Endoscopia Ginecologica,Universita degli Studi di Napoli 'Federico II',Napoli, ItalyBackground. D-dimer is considered a marker ofhypercoagulable state besides of endogenous fibrinolysis,so increased d-dimer is detectable in patientsaffected by thrombosis. Yet, several studies showedthat also infertility, in particular secondary infertilitydue to recurrent fetal losses, have been oftenrelated to thrombotic events, in particular in womencarrying thrombotic risk factors such as inheritedthrombophilia (MTHFRC677T, PTHRA20210G, FactorV Leiden polimorphisms and/or inherited protein C,protein S, AT III deficiency) or acquired thrombophilia(primary antiphospholipid syndrome, acquired proteinC, protein S, AT III deficiency, drugs inducedthrombophilia). However, because its high predictivenegative value in case of suspected thrombosis,increased d-dimer have been often associated tosubclinical thrombophilia. The aim of this study is toinvestigate the role of d-dimer as first marker ofthrombophilia in women affected by unexplainedinfertility and subsequently to search the cause ofincreased d-dimer, such as inherited and/or acquiredthrombophilia. Patients and Methods. We selected55 patients with unexplained primary or secondaryinfertility. We excluded patients affected by hydros-haematologica vol. 89(suppl. n. 8):september 2004

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 2004129alpinx, uterine fibroids, uterine malformations,endocrinological and immunological diseases, lutealinsuficiency, cytogenetical alterations. All remaining28 patients were tested for d-dimer. Following d-dimer examinations all patients were screened forMTHFRC677T, PTHRA20210G, Factor V Leidenpolimorphisms, protein C, protein S, AT III, anticardiolipinIgM and IgG, lupus anticoagulant. Moreoverall patients showing increased d-dimer levels weretested also for β-HCG in order to exclude an earlypregnancy, a well known condition associated toincreased d-dimer levels. Results. Inherited/acquiredthrombophilia was detected in 13/16 patients withincreased d-dimer and in 6/12 patients with normald-dimer. Early pregnancy was identified only in 1patient with increased d-dimer levels. Discussion. D-dimer is a well known marker of hypercoagulablestate, in particular its high predictive negative valuein case of suspected thrombosis has been recognizedby several reports. Yet, increased d-dimer has beenidentified also for subclinical thrombophilia besidesfor vascular thrombosis. Our data, in fact, showedan interesting role of d-dimer to identify womenaffected by unexplained primary or secondary infertilityand thrombophilia: 82% of women affected bythrombophilia vs 50% in women without increasedd-dimer. Is there a role for d-dimer in these subjectsfor its predictive positive value? Of course furtherdata on large based population are needed to confirmour results.PO-069A SCORE-BASED ASSESSMENT TOOL OF THE THROMBOTICRISK IN WOMEN IN MENOPAUSE TO BE TREATED WITHHORMONE REPLACEMENT THERAPYConsiglio P, De Michele S, Salvia M, Calabrò G,Aiosa A, Di Maio C, Faranna C, Trapani MR,Vitale G, Piraino L,* Menozzi IA.R.N.A.S Ospedale Civico, Palermo, LaboratorioAnalisi; *Divisione di CardiologiaHormone replacement therapy (HRT) in menopauseis associated with a significantly higher thromboticrisk, as reported in the current literature on thethromboembolism predisposing factors. In agreementwith the gynaecologists of our hospital, weprepared an anamnestic-diagnostic questionnaire forthe thromoboembolic risk assessment, to be admisteredbefore performing specialistic laboratory tests.The features of the possibly HRT-treated women inmenopause that we considered in our questionnaireare: thrombosis family and personal history, age,obesity, smoking habits, hypertension, diabetes mellitus,hypercholesterolemia, malignancy, psychotropicdrugs and obstetric pathology. We assigned a scoreranging from 0 to 5 to each factor according to therisk level. The scale has been designed on case-controland cohort studies from the current literature.Therefore patients are splitted in two groups: lowrisk women with a total score 5 and average or highrisk women with a total score > 5. Women with averageor high risk will undergo neither HRT nor specialisticlaboratory tests. Women with low risk will bescreened for thrombophilia, including basic coagulativetests, physiological inhibitors, omocysteinemia,antiphospholipid antibodies and genetic mutationsfor Factor V, Factor II and MTHFR. A positiveresult in the thrombophilic study excludes the treatmentwith HRT. Only women with risk 5 are fit for theHRT. Both low risk groups, either using HRT or not,will have been monitoring for 2 years in a prospecticstudy to consider the possible appearance ofthromboembolic events. xConclusions: the use of thisquestionnaire allows a better selection of thepatients for HRT and a reduction of the necessaryspecialistic tests. The recent introduction of thequestionnaire does not permit to present a statisticalanalysis.PO-070SHORT-TERM EFFECTS OF TRANSDERMAL HORMONEREPLACEMENT THERAPY ON HAEMOSTASIS ANDINFLAMMATIONPapa ML,* Capasso F,* Pudore L,* Torre S,* Russo V,*Papa R,° Bartoli MG,° Bordone S,° Insidioso M,°Di Maggio F,° Cerasuolo L,° Tesorone M,°De Lucia D^*Laboratorio di Emostasi e Trombosi, Ospedale S.Giovanni Bosco, ASL1, Napoli; °ConsultoriFamigliari, Dipartimento Materno-Infantile, ASL1,Napoli; ^Istituto di Patologia Generale ed Oncologia,II Università, NapoliAn increased risk of cardiac heart disease(CHD) andvenous thromboembolism(VTE) during combinedHormone Replacement Therapy(HRT),found in secondaryCHD prevention trials, has also been confirmedin primary prevention trials. We have observedthe effects of transdermal HRT on haemostasis andinflammation variables in healthy women during thefirst year of treatment. 100 healthy postmenopausalwomen(mean age±SD=52.5±10.7) were studied: 50transdermal estradiol plus progesterone for 1-yearusers and 50 non-users. 100 healthy premenopausalwomen were chosen as control group. We assayedthe procoagulant proteins: factor VII(VII:C), factorVIII(VIII:C), factor IX(IX:C), factor XI(XI:C) and factorXII(XII:C); the natural anticoagulant proteins:antithrombin(ATIII), protein C (PC), protein S (PS),freeand total TFPI and the resistance to activated proteinhaematologica vol. 89(suppl. n. 8):september 2004

130PostersC (APC- Resistance). The fibrinolytic proteins : t-PA,PAI-1 and TAFI were also measured. The factors (IX:C:90± 21%, 91.9± 18.4%, 90±20.7%; XI:C: 90±17.2%,99±29.1%, 82.1±18.1%;XII:C: 92.8±27.1%,89.9±23.5%, 93.1±20.3% respectively) did not significantlychange among the three groups. The userslevels of VIII:C and VII:C were similar to non-userseven if higher compared to premenopausal group (126± 58%, 120±59%, 85±15%, p=0.001;113±23%, 103±19%, 90±16%, p=0.001).The valuesof APC-R were the same in two populations ofwomen (1.02±0.7, 1.02±0.8, 1.1±25, p=0.02). Novariations were observed in free and total TFPI in theHRT group even though its slightly increased withposmenopausal status (9.21±1.65, 9.19±2.55,5.9±1.60ng/mL, p=0.001; 64.4±14.2, 71.1±17. 4,57.9±12.4ng/mL, p

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 2004131PostersTHROMBOSIS & CANCERPO-072PROCOAGULANT STATE IN CANCERMoniello G, Silvestri A, Tirelli P, De Lucia D,*Mazo S, Passariello N, Gambardella ADepartment of Geriatric Medicine and MetabolicDisease, Second University of Naples: *Institute ofGeneral Pathology and Oncology, Second Universityof Naples, ItalyIn cancer, mostly in metastatic cancer, are commoncomplications as thrombosis and disseminatedintravascular coagulation. Our purpose was to investigatethe coagulation parameters in cancer. We haveenrolled 39 cancer patients (20 male, 19 female, 55+ 4), subdvided in two groups. Group A consisted of18 patients with metastatic cancer (11 M, 7 F, age57±3 years): 6 with non-small cell Lung cancer, 3with pancreatic cancer, 3 with colon-rectal cancer,2 with Gastric cancer, 2 with Head-neck cancer and2 with Breast cancer. Instead, in group B there were21 patients who underwent a surgical treatment (12F, 9 M, age 54±4 years)s: 8 with breast cancer, 5 withcolon-rectal cancer, 3 with non-cmall cell lung cancer,3 with Head-Neck cancer and 2 with kidney cancer.Inboth groups we measured PT, APTT, fibrinogen,AT III, PC, PS, APC resistance, fibrinolytic system andactivation peptides (D-dimer, prothrombin fragment1+2/F1+2).The results were compared with healtycontrol group C (21 female, 17 male, age 53±4). Ingroup C we have found lower levels of fibrinogen, D-dimer and F1+2 than group A (p< 0,01) and group B(p

132Postersgene substitution may be involved in gastrointestinalcancer pathogenesis. We performed a prospectivecase-control study to analyze the prevalence of thetwo commonest thrombophilic mutations, factor VLeiden and prothrombin G20210A polymorphism, inpatients with colorectal cancer. Methods: 152patients with colorectal carcinoma and 125 healthysubjects, colonoscopically selected, comparable forage and sex, were investigated. The factor V Leidenand the prothrombin mutation were detected bypolymerase-chain-reaction-based technique. Theprevalence of the two prothrombotic polymorphismswas not significantly different between the groups.Conclusions: The study suggest that in colorectalcarcinoma acquired rather than genetic risk factorsare involved in the thrombophilic cancer state andthat prothrombin G20210A mutation doesn't seem tobe a cofactor in colorectal cancer pathogenesis.PO-075ACUTE PHASE RESPONSE IN CANCER PATIENTS WITH NONMETASTATIC SOLID TUMORSBattistelli S, Stefanoni M, Lorenzi B, Cappelli R*Dipartimento di Chirurgia e Specialità Chirurgiche;*Dipartimento di Medicina Interna, Cardiovascolaree Geriatrica, Università degli Studi di Siena, ItalyIt is well known that neoplastic growth is associatedwith acute phase response, and that the levelof this response, in patients with advanced cancer, isa strong predictor of survival. We performed aprospective study to investigate the pattern of acutephase response in a group of cancer patients withnon metastatic solid tumors, eligible for curativesurgery, tested for several acute phase reactants.Methods: Eighty one consecutive patients with gastrointestinalor pelvic solid cancer, all included in thestages T1-3, N0-2, M0, and 58 healthy control subjects,comparable for age and sex, were enrolled inthe study. All patients were evaluated for fibrinogen,C4b-binding protein (C4BP), total protein S and factorVIII plasma levels. Results: Compared to the controlgroup, in cancer group, mean levels of fibrinogen(both antigen and functional), C4BP, total proteinS and factor VIII were significantly higher(p

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 2004133PO-077COAGULATION DERANGEMENTS IN ORAL AND MAXILLOFACIALSURGERY IN PATIENTS WITH A DOCUMENTED CANCER:A FOLLOW-UP STUDY AFTER SURGERYDe Lucia D, 1 Perricone F, 1 Sessa M, 1 Misso M, 1Adamo S, 1 Tagliaferri A, 1 De Francesco F, 1Pignalosa O, 1 Russo T, 1 Galante M, 1 Bernacchi M, 1Capasso F, 2 Pudore L, 2 Torre S, 2 Russo V, 2 Papa ML 21Clinical Pathology, Laboratory of Haemostasis andThrombosis; II University of Naples; 2 Haemophiliaand Thrombosis Center, San Giovanni BoscoHospital; Naples, ItalyHypercoagulability is normally due to derangementsin the blood coagulation cascade, both geneticand acquired, which may predispose to abnormalclotting activation or deficiency in natural inhibitors’control. Thrombophilia is usually caused by aberrantcoagulation and by cell activation, but it is also relatedto such specific factors as oral contraceptives,pregnancy, puerperium, trauma, prolonged immobilization,cancer, and surgery. The aim of our studywas to determine the mechanism by which cancercould cause or favour the development of a thrombo-embolicstate in patients who had undergonesurgery. Fifteen patients with a documented diagnosisof cancer and 30 healthy subjects, blood donorsat our University matched for sex, age, body massindex, height, and social background were enrolled.The protocol was approved by an Institutional ReviewBoard, and patients gave informed consent. Plasmasamples for determination of PT, APTT, Fibrinogen(Fg), coagulation factors (II, VII, VIII, IX, X, XI, XII),coagulation inhibitors (ATIII, PC, PS, and APC resistance),fibrinolysis (PLG, t-PA, PAI-1), antiphospholipidantibodies (ACA β 2GPI IgG and IgM, LupusAnticoagulant), and activation peptides (D-Dimersand F1+2) were executed immediately after entryand then daily one day before and two days aftersurgery. All parameters were carried out according toInternational Laboratory Techniques. Normal plasmalevels for PT, APTT, natural clotting inhibitors, andcoagulation factors were documented in all patients.During the observational surgery period, no changesin all of these parameters were observed. High basalvalues of Fg, PAI-1, F VIII, F1+2, and D-Dimers werefound in all patients compared with healthy subjects.The plasma levels rapidly increased beforesurgery reaching the highest values after surgery.Moderate titles of IgG anticardiolipin antibodies b2-glycoprotein type I were found in eight of 15patients. Analysis of our data suggests a state ofhypercoagulability in cancer disease associated withmaxillofacial surgery. The significant increase ofplasma fibrinogen levels and the activation peptidesshow a hypercoagulability state in this setting associatedwith endothelial cell activation and dysfunction,with thrombin formation and fibrin deposition.Analysis of our data demonstrates a hypercoagulablestate during the follow-up period of maxillofacialsurgery associated with cancer disease. Ourreport gave considerable information on the clinicalsituation when surgeons cause patients to becomethrombophilic by removing their cancer. The mechanismthat causes these coagulation derangementsis still unknown.PO-078LOW-MOLECULAR-WEIGHT HEPARIN AND CANCER SURVIVAL:POOLED ANALYSIS OF 1726 PATIENTS TREATED FOR THREEMONTHSGuercini F, Conti S, Iorio ASezione di Medicina Interna e Cardiovascolare, Universitàdi Perugia, ItalyBackground: Heparin has been the main subjectof intensive investigation and clinical use because ofits therapeutic anticoagulant properties. It alsoexhibits many other biological activities, and it wasclaimed to show a beneficial effect on cancerspreading. Searching for papers investigating theeffect of low-molecular weight heparin (LMWH) oncancer mortality we found two trials and three metaanalyseswhich showed a reduction in the mortalityrate in cancer patients. However, the biological rationaleof this effect for a short treatment with LMWHremains unclear. We wanted to check if similarresults were achieved in cancer patients treated withLMWH for a longer period of time. Aim of the study:To evaluate the effect on cancer mortality of a threemonthtreatment with LMWH by performing a metaanalysisof published studies comparing LMWH andoral anticoagulants (OA) in the prevention of recurrentvenous thromboembolism (VTE). Materials andMethods: Computerized searches of MEDLINE andEMBASE were performed; clinical trials were alsolocated through colleagues and hand-scanning ofmeeting proceedings. Elegibility of the studies andextraction of data were performed by two authorsusing a standard form. The meta-analysis was performedassuming a fixed-effect model. Results: Eightstudies were identified that fulfilled our predefinedcriteria, for a total of 1726 patients. After six monthsof follow up, 304 patients died of cancer, 149 in theLMWH group and 155 in the OA control group (OR0.95 95% CI 0.72 – 1.24; z=0.41; p=0.69) Consideringcancer mortality in 948 cancer patients the figureswere 162/472 in the LMWH group and 169/476in the oral anticoagulant control group (OR 0.95 95%CI 0.72 – 1.25; z=0.38; p=0.71). Conclusions: In 1726patients randomized to receive a three month coursehaematologica vol. 89(suppl. n. 8):september 2004

134Postersof LMWH or OA for the treatment of VTE, no effecton cancer mortality at six months was found.PO-079VASCULAR ENDOTHELIAL GROWTH FACTOR CONCENTRATIONSIN PLASMA-ACTIVATED PLATELETS RICH FRACTIONS OFHEALTHY CONTROLS AND COLORECTAL CANCER PATIENTS:POSSIBLE BIOLOGICAL AND CLINICAL SIGNIFICANCERanieri G,* Coviello M,* Patruno R,* Valerio P,°Martino D,° Catalano V,** Scotto F,**De Ceglie A,** Quaranta M,* Pellecchia A**Department of Experimental Oncology, NationalCancer Institute of Bari, Italy; °Division of Surgery,Di Venere Regional Hospital of Bari, Italy;**Department of Services and Diagnosis, NationalCancer Institute of Bari, ItalyVascular endothelial growth factor (VEGF) is knownto play a key role in tumour angiogenesis. Our pilotpublished data suggest that plasma-activatedplatelets rich (P-APR) rather than other blood plasmacompartments (i.e. S, plasma, plasma-platelets poor)is the more suitable blood fraction for measuringVEGF in a miscellaneous series of gastrointestinal cancerpatients. The aims of this confirmatory study wasto assess VEGF in P-APR blood compartments of 30healthy control subjects (HCS) and a homogeneousseries of 62 CRCP, prospectively collected, to evaluateits possible clinical-biological significance. Venousblood was dispensed into a into a polystyrene tubescontaining sodium citrate for plasma (SC) (3.1 mg/mLw/v final concentration) (Becton Dickinson VacutainerSystems, Plymouth, UK) and into sodium citrate,theophylline, adenosine, dipyridamole tubes for plasma(CTAD) (Becton Dickinson Hemogard VacutainerSystems, Plymouth, UK). SC and CTAD samples wereboth centrifuged at 180 × g × 10 min. The supernatant,SC and CTAD-plasma respectively, was carefullyremoved from the centre portion of the liquidphase using a polyethylene Pasteur pipette obtaininga platelet-rich plasma. After subjecting the plateletrichplasma to platelet count (Automated HaematologyAnalyser SE-9000 RET/SYSMEX), it was treatedwith thrombin (Hemoliance Q.F.A. Thrombin Bovine)(80 mL/mL) and incubated for 30 min at room temperature.After centrifugation at 1500 × g × 15 min,the aggregated platelet sediment was eliminated andthe supernatant, P-APR, was recuperated. P-APR VEGFlevels were examined using the Quantikine HumanVEGF-enzyme-linked immuno-absorbent assay(ELISA) (R&D Systems Inc, Minneapolis, MN, USA). Thebest differentiation between HCS and CRCP in VEGFlevel was seen for P-APR-VEGF level in CTAD (medianvalue: 255 picogrammi/mL versus 142 picogrammi/mL;p=0.000 by Mann-Whitney U test). We suggestthat P-APRCTAD fraction, obtained according towell standardised conditions, could represent the suitableblood compartment for the assessment of VEGFas marker of malignant intestinal transformation.PO-080PATTERN OF COAGULATION/FIBRINOLYTIC SYSTEM PROTEINSIN MONOCYTES, ENDOTHELIAL AND CANCER CELLS: EFFECTOF PACLITAXELNapoleone E, Di Santo A, Amore C, Donati MB,*Lorenzet R“Antonio Taticchi” Unit of Cellular and MolecularBiology of Blood Coagulation, Consorzio MarioNegri Sud, S.Maria Imbaro, and *Center for HighTechnology Research and Education in BiomedicalSciences, Catholic University, Campobasso, ItalyPaclitaxel is a microtubule-stabilizing compoundwith potent antitumor activity that has been clinicallyused in a wide variety of malignancies. Sincetissue factor (TF) is often expressed by tumor-associatedendothelial and inflammatory cells, as well asby cancer cells themselves, and it is considered ahallmark of cancer progression, we decided to investigatewhether paclitaxel could modulate TF expressionin stimulated human mononuclear cells (MN),umbilical vein endothelial cells (HUVEC), and theconstitutively TF- expressing metastatic breast carcinomacell line MDA-MB-231. Since a role of theplasminogen/plasminogen activator system in themetastatic process has also been proposed, wethought to determine whether paclitaxel could modulatetissue-plasminogen activator (t-PA) and plasminogenactivator inhibitor-1 (PAI-1) expression inthe same cells. To test this hypothesis, the cells werecultured and incubated with or without paclitaxelat 37°C. At the end of incubation, conditioned mediumwas collected and tested for t-PA and PAI-1 antigenlevels by ELISA, and cells were disrupted andtested for procoagulant activity by a one-stage clottingassay. Both the strong TF activity constitutivelyexpressed by MDA-MB-231 and the TF induced byLPS and IL-1β in MN and HUVEC were significantlyreduced by paclitaxel at nanomolar concentrations.Paclitaxel caused a 50% inhibition of t-PA and PAI-1 release from MDA-MB-231, while no modulationof t-PA levels could be observed in MN and HUVEC.In addition, paclitaxel strongly downregulated PAI-1 levels in LPS- and IL-1β-stimulated HUVEC. Sincepaclitaxel has been shown to induce expression ofinflammatory genes in monocytes and tumor cells,albeit at concentrations much higher than thoseused in this study, we tested whether paclitaxel couldinfluence IL-1β and IL-6 release from our cells. Neitherthe constitutive expression of these cytokines byhaematologica vol. 89(suppl. n. 8):september 2004

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 2004135MDA-MB-231 nor the LPS-induced release from MNand HUVEC were affected. Our data support thehypothesis that the anti-tumor effects of paclitaxelmay, in part, be mediated by the capacity of this drugto modulate the procoagulant/fibrinolytic potentialof cancer and host cells.PostersTHROMBOSIS &MYELOPROLIFERATIVE SYNDROMESPO-081EVALUATION OF THROMBOPHILIC ALTERATIONS IN PATIENTSWITH MULTIPLE MYELOMA TREATED WITH THALIDOMIDECini M, Valdrè L, Legnani C, Zamagni E,*Cosmi B, Cellini C,* Cavo M,* Palareti GU.O. di Angiologia e Malattie della Coagulazione“Marino Golinelli”, *Istituto di Ematologia eOncologia Medica “Seragnoli”, PoliclinicoS. Orsola-Malpighi, Università di Bologna, ItalyThalidomide is an active treatment of multiplemyeloma (MM). Among major toxicities, deep venousthrombosis (DVT) has been observed in approximately25% of patients receiving thalidomide and combinedchemotherapy. A complete thrombophilic study[Antithrombin (AT), Protein C (PC) and Protein S (PS)activities, activated protein C resistance (APCR) withDNA analysis for Factor V Leiden mutation in all caseswith an APCR normalized ratio < 0.80, DNA analysisfor the G20210A prothrombin mutation, tests fordiagnosing Lupus Anticoagulant (LAC)] was performedin patients with newly diagnosed MM beforestarting thalidomide therapy, to evaluate if theincreased DVT risk was associated with the presenceof thrombophilic alterations. Patients were also reexaminedafter four months therapy. 175 patientswere enrolled (M/F 92/83, median age 58 years) andreceived at least 1 month of therapy. Thrombophilicscreening was normal in 137 (78.3%) patients; 4(2.3%) and 5 (2.9%) patients resulted carriers of FVLeiden or prothrombin mutation, respectively. Beforestarting thalidomide therapy, 13 (7.4%) patientsshowed an altered APCR not associated with FV Leidenmutation, 1 (0.6%) had a positive LAC and 15(8.6%) showed low levels of AT, PC or PS. 8/13patients with altered APCR, 1/1 patient with LAC and4/15 patients with AT, PC or PS reduction were reexaminedafter four months therapy and none of theprevious alterations persisted. Since symptomaticDVT was diagnosed in 5 (26%) out of the first 19patients, all the others received a fixed low-dosewarfarin (1.25 mg/day) as DVT prophylaxis. Amongthe 156 patients receiving thalidomide-dexamethasoneplus warfarin DVT prophylaxis, 14 (9%) experienceda thromboembolic event. Among the 19patients with DVT 2 had thrombophilic alterations(FV Leiden, prothrombin mutation). In conclusion,haematologica vol. 89(suppl. n. 8):september 2004

136Postersthe increased DVT risk associated with thalidomidedexamethasonetherapy in MM seems to be onlyweakly related to the presence of thrombophilicalterations.PO-082FIBRINOLYTIC CHANGES AS EARLY MARKERS OF HEPATICVENO-OCCLUSIVE DISEASE AFTER BONE MARROWTRANSPLANTATION IN PEDIATRIC PATIENTSSartori MT, 1 Spiezia L, 1 Messina C, 2 Cesaro S, 2Saggiorato G, 1 Paris M, 2 Pellati D, 1 Varvarikis C, 1Cella G 11Clinical Medicine II, Department of Medical andSurgical Sciences; 2 Pediatric Hematology andOncology Unit; University of Padua, ItalyHepatic veno-occlusive disease (VOD) is a severecomplication after bone marrow transplantation(BMT). The diagnosis, based on clinical criteria, isoften late. Recent studies in adults patients receivingBMT have identified in the plasminogen activatorinhibitor-1 (PAI-1) increase a possible marker ofVOD. To confirm this finding, the fibrinolytic (PAI-1,t-PA, D-dimer, plasminogen), coagulation andendothelial activity (PT, PTT, fibrinogen, antithrombin,P-selectin, NO), and liver function parameters (bilirubin,ALT) were assayed before and weekly for 1month after BMT in 54 consecutive pediatricpatients. A total of 61 BMT were performed. VODwas diagnosed according to clinical criteria in 6patients (9.84%), namely 3 of 48 allogeneic BMT(6.25%) and 3 of 7 autologous BMT (42.8%). VODwas classified as mild in 3 (50%), moderate in 1(16.6%) and severe in 2 (33.3%) cases. In non-VODpatients, all studied parameters remained unchangedafter BMT, and no difference was seen respect topre-BMT. In contrast, after BMT a precocious andstatistically significant increase in mean values ofPAI-1 antigen (p=0.000013) and activity(p=0.00001), t-PA antigen (p=0.00001) and D-dimer(p= 0.0062), as well as a significant decrease in meanvalues of plasminogen (p=0.0094) and PT (p= 0.002)were found between non-VOD and VOD patientsalready one week before clinical diagnosis of VOD. Asignificant difference in the same parameters and inantithrombin levels was detected between non-VODand VOD patients after 1, 2 and 3 weeks from theonset of VOD. In contrast, the increase in bilirubinlevels was significant only later. There was no differenceas for other parameters. In conclusion, thevariation of fibrinolytic tests after BMT may be helpfulto early detect and confirm the clinical diagnosisof VOD in paediatric BMT patients.PO-083HYPERCOAGULABILITY IN CHILDREN WITH ACUTELYMPHOBLASTIC LEUKEMIA UNDER INDUCTION TREAMENTWITH L-ASPARAGINASE: ROLE OF SOLUBLE P-SELECTINAND PLATALET ACTIVATIONSaracco P,* Farinasso L,* Del Vecchio GC,°Crescenzio N,* Iavarone A, % Russo G, £Molinari AC, § Bertorello N,* Bo M,* Luciano C, %Nigro A,° Giordano P°Div. di Onco-Ematologia Pediatrica,Dip. di Scienze Pediatriche e dell'Adolescenza, Universitàdegli Studi, Turin;* Div. di Onco-Ematologia,Ospedale "G. Gaslini", Genova; § Dip. di Biomedicinadell'Età Evolutiva, Università di Bari;° Dip. di Pediatria,Università di Catania; £ Dip. di Chimica Clinica,Az. Osp. O.I.R.M.- S. Anna, Turin, ItalySoluble P-selectin (sP-sel) has been proposed notonly as a marker of platelet and endothelial activation,but also as a direct inducer of a pro-coagulant state.During induction for acute lymphoblastic leukemia(ALL) there is a well known thrombophilic state, withL-Asparaginase being the major responsible bydecreasing biosynthesis of protein C, S and mainlyanti-thrombin. It has been postulated that L-Aspmight also exert an agonist effect on platelet activation.Study: sP-sel and platelet activation have beenanalyzed during induction in 97 consecutive ALL children(aged 1-16) treated with AIEOP(AssociazioneItaliana Ematologia e Oncologia Pediatrica) protocol(ALL-2000) at 4 different Institutions, and in 24 agematched controls. Blood samples were drawn at diagnosis,at day +24, +36 ( after 5 and 8 L-asp doses), and+52. In a 51 patients cohort from a single centre,platelet activation was directly measured by FlowCytometer at same time intervals. Plasma sP-sel wasmeasured with ELISA immunoassay (R&D System);platelet activation was studied by flow-cytometryusing Actiplate-FITC PROTOCOL (Tau Technologies BV).Data were analyzed with Mann-Withney test andMultivariate Tests. Results. Plasma sP-Sel levels werehigher at diagnosis compared to controls (p=0.004),and significantly increased at day +36 and + 52(p=0.001 and p=0.005,respectively). Expression ofplatelet activation by flow cytometer analysis showedan increasing trend in leukaemic patients vs controlsat all time intervals, though no significant differencewas noted between diagnosis and days +24, +36,+52.Mean platelet counts were higher at day + 36 and+52. Our data indicate sP-sel as a marker of pro-coagulantstate in patients with ALL during induction, notnecessarily due to direct platelet activation. Furtherstudies are needed to confirm this role and to identifyother predictive markers of thrombotic risk in orderto establish an optimal antitrombotic prophylaxis inthese patientshaematologica vol. 89(suppl. n. 8):september 2004

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 2004137PO-084DIAGNOSIS AND MONITORING OF CENTRAL VENOUS-LINERELATED THROMBOSIS IN CHILDREN WITH ACUTELYMPHOBLASTIC LEUKEMIAFarinasso l,° Gaijno TM,* Bertorello N,° Garbarini l,°Campagnoli MF,° Santoro BM,* Valori A,*Barisone E,° Cordero Di Montezemolo L,°Saracco P°°Dipartimento di Scienze Pediatriche, Università diTorino; *Ospedale Infantile Regina Margherita,Turin, ItalyVenous thrombosis (VT) in the upper venous systemin children with central venous lines (CVL) may resultin collateral intrathoracic veins. The reported longterm outcome of symptomatic and asymptomaticCVL-related VT are post-phlebitic syndrome, recurrentVT and rupture. Bilateral venography is the goldstandard to investigate intrathoracic veins, as ultrasound(US) may detect VT mainly in cervical veins. Inchildren a non invasive and valid alternative tovenography may be spiral computed tomography(CT). Children with acute lymphoblastic leukemia(ALL) have a significant risk of CVL-related VT duringinduction treatment with L-asparaginase. Aim ofthis prospective study was to detect and monitoroutcome of symptomatic and asymptomatic CVLrelatedVT, by using spiral CT (Siemens Somaton Plusand Magic View software) and ultrasound (US), in acohort of 57 children (aged 14 months-15 yrs) withALL and with newly inserted CVL. Patients weretreated according to AIEOP ALL 95 and 2000 protocols(15 and 42 respectively). In ALL 2000 protocolspatients were randomized for prednisone or dexamethazoneduring induction. CVL were inserted within15 days from diagnosis by venous cut-down techniquein 46 cases (in 47% via right jugular vein) andpercutaneous (subclavean vein) in 11 patients.Patients were screened for Leiden, 20210, MTHFR,Lp(a), basal homocysteine, LAC. US and spiral CT wereperformed at completion of induction (after 8 dosesof L-asparaginase) and after CVL removal. The first CTscan detected moderate VT-occlusion (jugular andintrathoracic veins) in 52%, only 5 being symptomatic.In 65% CT scan showed normalization afterCVL removal, howewer a serious complication wasreported in 1 patient (3%), with enclosure of CVLwithin vascular wall hindering final removal. Incidenceof early asymptomatic CVL-related VT in childrenwith ALL is significant and spiral CT is sensitivein detecting occlusion and monitoring outcome.PO-085MEDIASTINAL B LARGE CELL LYMPHOMA AND INTERNALJUGULAR VEIN THROMBOSIS: WHICH PATHOGENESIS?Paolini R, Cuppini S, Ramazzina E, Zamboni SDepartment of Medicine, Rovigo General Hospital,ItalyIntroduction: A link between thrombosis and solidtumors is well documented as well as betweenthombosis and chemoradiotherapy for some of them.Less data are available about thrombosis, non-Hodgkin’s lymphomas and chemotherapy for suchdiseases. Moreover, the prevalence of superior venacava thrombosis in the primitive mediastinal B celllymphoma with sclerosis (PMBL) has been reportedto be very high (70%) and its pathogenesis multifactorial.Aim of the study: To verify the higher prevalenceof mediastinal vein thrombosis in PMBLpatients, with respect to other mediastinal lymphomas.To clarify the single role of jugular veincompression or invasion of the venous wall by PMBLmass, or a possible chemo-radiotherapy effect. Toclarify utility of screening for thrombotic diathesis inthese patients. To establish the opportunity of a standardantithrombotic prophylaxis in PMBL patients inorder to prevent the fearful venous mediastinic syndrome.Methods: We observed internal jugular veinthrombosis in all three patients treated for PMBL inour Department in 2003. In contrast, no thromboticevents were registered in eight patients affected bymediastinal mass due to Hodgkin’s disease observedin the same period, similar for age and sex to thePMBL group. The first patient affected by PMBL wasa 44-year-old woman who presented mediastinalsyndrome.All the biochemical values were normal.CT scan performed at presentation evidenced a 7 cmlymph nodal enlargement of the antero-superiormediastinum, with superior vena cava compression.Diagnosis of PMBL was made by mediastinoscopy.Chemotherapy was suddenly started, according tothe weekly VACOP-B schedule. After four weeks, thepatient experienced a superficial thrombophlebitisof the left arm. LMWH at therapeutic dosage wasgiven and a new CT scan of the thorax documentedthe disappearance of the mediastinal mass but thepresence of a right internal jugular vein thrombosisextending to both brachiocephalic trunks and distallyto the azygos vein. Routine coagulative tests, evaluationfor an acquired or congenital abnormality,search for the presence of anti-heparin antibodieswere negative. Full heparinization was continuedduring the subsequent mantle-field radiotherapy. CTscan performed during follow-up showed fibroticocclusion of both brachiocephalic trunks. The secondpatient was a 40-years-old woman arrived withanterior mediastinal mass of 8 cm.and a concomitanthaematologica vol. 89(suppl. n. 8):september 2004

138Postersinternal jugular vein thrombosis showed by a CT scanof the thorax. PMBL diagnosis was made with mediastinoscopy,and radiologic control after four weeksof VACOP-B chemotherapy showed regression ofmediastinal mass and thrombosis, in the absence ofanticoagulation. The third patient was a 22-yearsoldwoman who experienced a clinically evidentjugular vein thrombosis 6 months before the radiologicevidence of an anterior mediastinal mass (PMBLdiagnosis was made by mediastinoscopy).The mediastinalmass together with thrombosis disappearedafter weekly chemotherapy, in the absence of anticoagulation.Conclusions: The high incidence of themediastinic venous thrombotic complication ofPMBL patients and its heavy clinical impact, deservesa retrospective analysis of a more consistent numberof cases. Maybe a search for plasmatic tumoralthrombogenic substances could be useful in newpatients, to correctly assess the risk in each patient.PO-086PREVALENCE OF THROMBOEMBOLIC EVENTS ANDTHROMBOPHILIAS IN PATIENTS WITH ACUTE LEUKEMIAGrandone E, Melillo L, Colaizzo D, Cappucci F,Chinni E, Margaglione MIRCCS Casa Sollievo della Sofferenza, S. GiovanniRotondo, and Medical Genetics, University of Foggia,ItalyThrombotic events (TE) are poorly investigatedcomplications of adults with acute leukemia (AL).Aim of this study was to determine the prevalence ofTE and to detect any association of TE with the presenceof congenital or acquired prothrombotic factorsin patients affected by AL. From December 1999 toJune 2003 we evaluated the prevalence of geneticand acquired risk factors of thrombosis in a consecutiveseries of 72 adult patients with AL at diagnosis.Thirty-one patients had acute lymphoblasticleukemia (ALL) and 41 acute myeloblastic leukemia(AML) ( AML-M3 in 9 cases). Median age was 39years (range 16- 62). They were treated accordingto the established GIMEMA protocols. No patientsreceived heparin or any other anticoagulants. FactorV G1691A mutation (FV-L), prothrombin G200210A(FII20210) gene variant, MTHFR C677T variant, proteinC, protein S and antithrombin (AT) deficiencyand the presence of Lupus Anticoagulant (LA) andanticardiolipin antibodies (aCL) were evaluated. FV-L was observed in 1 patient (1.4%), FIIA20210 mutationin 2 patients (2.8%) and MTHFR C677T variantin 12 (16.6%) patients. The prevalence of thesemutations was not statistically different from thatobserved in the general population from the sameethnic background. Twenty-six (36.1%) patients hadhyperhomocysteinemia (>95% above reference values),partially related to the presence of TT MTHFRhomozygosity. As far as natural anticoagulants areconcerned, 6 patients (9.5%) showed low levels ofprotein S, while no patient showed protein C or ATdeficiency. Low titer of aCL was found in 2 (3.1%)cases. Six patients (8.3%) ( 2 ALL, 2 AML and 2 AML-M3) developed TE, 5 out of them in the first monthof follow up. Two out of six patients ( 33.3%) showedthe association of FV-L or FIIA20210 mutations andhyperhomocysteinemia. The OR for TE in patientswith double defects was 11.0 (95%CI 1.4-85.8, Fisherexact test, p= 0.048). The prevalence of TE isexceedingly high in adults with AL. Patients withdouble thrombophilic defects appear to be at highrisk for the development of early TE.PO-087CENTRAL VENOUS CATHETER RELATED THROMBOSIS INPATIENTS WITH HEMATOLOGIC MALIGNANCIES WITHOUTPHARMACOLOGICAL PROPHYLAXIS: RETROSPECTIVEANALYSISSilingardi M, Bonini A,* Galimberti D, D’incà M,Nicolini A, Negri EA, Casali A, Trenti C, Iotti M,Gugliotta L,* Iori IU.O Medicina I^, Centro Emostasi e Trombosi, *U.O.Ematologia, Az. Ospedaliera S. Maria Nuova, ReggioEmilia, ItalyThe reported incidence of symptomatic centralvenous catheter (CVC) related deep venous thrombosis(DVT) in adult cancer patients varies from 0.3to 28.3% in different series. The issue of antithromboticprophylaxis in these patients is still a matter ofdebate. These data are obtained mainly from nonhematologicalsolid cancers. Few data are availablefor haematological patients. We retrospectively analyzedthe clinical records of 128 consecutive patientswith haematological malignancies. Between January1998 and December 2003, 218 CVCs wereimplanted in 128 patients (56 females, 72 males:mean age 47, range 15-70) consecutively admittedto the Haematology Unit for acute lymphoblasticleukemia (8 ), acute myeloid leukemia (32), Hodgkin’slymphoma ( 14), non- Hodgkin’s lymphoma (29),multiple myeloma (34), myelodiplastic syndrome (1),primary amyloidosis (1), severe neutropenia (1). AllCVCs were centrally inserted under aseptic conditions.No pharmacological prophylaxis was administered,according to our Institution’s policy. Symptomaticcatheter related deep venous thrombosis (DVT)was recorded. Superficial thrombophlebitis was notconsidered. The median duration of the CVCs was 29days for a total of 6322 catheter/days. Seven symptomaticepisodes of catheter-related upper extrem-haematologica vol. 89(suppl. n. 8):september 2004

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 2004139ity DVT were registered (4 subclavian, 3 subclavianand jugular vein) in 128 patients (5.4%; 1.1events/1000 catheter days). The majority of theevents (5/7) occurred in patients with acute myeloidleukemia. No signs/symptoms of pulmonaryembolism (PE) were reported. Mean platelet and leucocytecount at the time of diagnosis were 16×10 9 /Land 0.228×10 9 /L respectively. CVC related infectionwas documented in 28% (2/7) of episodes. Upperextremity DVTs were treated with low-molecularweight-heparin(6/7) and warfarin (1/7). No majorhaemorrages were reported. CVC was removed inone case. The incidence of CVC-related thrombosis inthrombocytopenic patients with haematologicalmalignancies is comparable with that reported fornon-thrombocytopenic cancer patients. We didn’tfind any association with PE. CVC related infectionplays a major role in thrombosis determination. Evenif the number of DVT we observed was small,LMWHwas a safe antithrombotic treatment in hematologicthrombocytopenic patients.Defibrotide (DF) has proven effective and safe inresolving severe hepatic veno-occlusive disease(VOD), a life-threatening complication of hematopoieticstem cell transplantation. To evaluate the mechanism(s)of DF effect, we conducted a study: 1. invivo in VOD patients given DF, and 2. in vitro inmicrovascular endothelial cells (HMEC-1) treatedwith DF. In vivo, we assessed the pattern of clottingand fibrinolytic plasma markers in six patients withhaematological malignancies, receiving escalatingdoses DF (10-40 mg/kg/d, for 6-19 days) for severepost-transplant VOD. Plasma markers were measuredbefore treatment (d-0), after 6 days of DF administration(d-6), and at 6 days after stopping DF (d-6A).At d-6, the overall plasma fibrinolytic activity(euglobulin lysis area, ELA) was 94% increased vsbaseline, in parallel to a 96% increase in plasma t-PA, and a minor increase in PAI-1 levels (46%). Ond-6A, ELA significantly decreased (p

140PostersPostersANTIPLATELET THERAPYPO-089HIGH RATE OF NON RESPONDERS TO ASPIRIN AND/ORCLOPIDOGREL TREATMENT IN THE FIRST DAYS FOLLOWINGPERCUTANEOUS CORONARY INTERVENTIONS: AN EX VIVOSTUDYPaniccia R, Tellini I, Costanzo M, Valente S,Lombardi A, Giglioli C, Lazzeri C, Prisco D,Gensini GF, Abbate RDip. Area Critica Medico-Chirurgica, Universitàdegli Studi di Firenze; Dip. del Cuore e dei Vasi,Azienda Ospedaliero-Universitaria Careggi,Florence, ItalyAspirin (ASA) therapy following both acutemyocardial infarction (AMI) and acute coronary syndromes(ACS) has been shown to reduce the relativerisk of recurrent vascular events and death. However,the inhibition of platelet function by ASA is notuniform among patients and this may be relevantfor the occurrence of new ischemic events. Recently,PFA-100 (Dade Behring, Germany) closure time(CT) has been suggested as a possible tool to identifyASA-resistant patients. Aim of this study was toinvestigate the response to ASA+clopidogrel treatmentin 60 patients with AMI or ACS after PCI, byassessing PFA-100 CTs by collagen+epinephrine(CT/EPI) and collagen+ADP (CT/ADP) cartridges. Allpatients were treated with 325 mg ASA once a dayand clopidogrel - 300 mg bolus immediately beforethe procedure followed by 75 mg/day. Citrated bloodsamples were taken 5-36 hrs after the procedure and(in 42/60 patients) also 4-5 days after the PCI. Acontrol group of 101 healthy subjects, who had nottaken any antiplatelet agent for 15 days before bloodsampling, was also studied. ASA and clopidogrelresponders were defined respectively as patients witha CT/EPI and a CT/ADP over the 95th percentile valuein the control group (CT/EPI >203 s and CT/ADP>139 s). Five to 36 hrs after PCI, 14/60 patients(23%) had normal CT/EPI values and 42/60 patients(70%) had normal CT/ADP values. Eighteen out of 60patients (31%) showed both CTs prolonged, whereas13/60 patients (22%) had both CTs in the normalrange. Four to 5 days after PCI, 16/42 patients (38%)had normal CT/EPI values. Ten of these 16 patientshad had prolonged CT/EPI values 5-36 hrs after theprocedure. Thirty-five out of 42 patients (83%) hadnormal CT/ADP values. Eight of these 35 patients hadhad prolonged CT/ADP values 5-36 hrs after the procedure.In conclusion, short-term antiplatelet treatmentwith combination of ASA and clopidogrel doesnot prolong PFA-100 CTs in a relevant proportion ofAMI and ACS patients. If this lack of ex vivo responsivenessto ASA and/or clopidogrel is related to anincreased risk of medium–term cardiovascular complicationsshould be assessed by further studies.PO-090ASPIRIN RESISTANCE IN PATIENTS UNDERGONE CAROTIDENDOARTERIECTOMY AND RE-STENOSIS: PRELIMINARY DATARuzzon E, Randi Ml, Tezza F, Toldo C, Gallo S,Fabris FDept. of Medical and Surgical Sciences, Universityof Padua Medical School, ItalyAspirin antiplatelet effect is not uniform in allpatients. Aspirin resistance occurs in about 30% ofpatients with thromboembolic vascular eventsdespite therapeutic doses of aspirin. We report thedata of 27 patients (6 females and 21 males, meanage 66±9.5 years) who underwent endoarteriectomybecause carotid symptomatic stenosis and treatedwith Aspirin 100 mg/day for at least 6 months. Thepatients were divided in two groups A) patients whoexperienced a re-stenosis B) patients without restenosis.We performed aggregation study with Bornmethod in all patients during Aspirin treatment andin 6 normal subjects matched for sex and age treatedwith low-dose Aspirin for at least 15 days. Aggregationwas considered to be reduced if < 40% withADP and

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 2004141tions. The remaining patient acknowledges not totake Aspirin before the previous test. We concludethat 1) about 50% of patients with carotid re-stenosishad normal ADP aggregation in spite of Aspirintreatment; 2) more patients than controls had normalADP aggregation under Aspirin, as previouslyreported in diabetic patients. This may be due to ashorter treatment period in control group 3) aspirinresistance seems not to be due to altered gastricabsorption.PO-091DIRECT AND IRREVERSIBLE INHIBITION OF COX-1 BYNITROASPIRIN (NCX-4016) AND ITS METABOLITESCorazzi T, Leone M, Gresele PDepartment of Internal Medicine, Section ofInternal and Cardiovascular Medicine, Universityof Perugia, ItalyCyclooxygenases (COXs) are inhibited by aspirinand other non-steroidal anti-inflammatory drugs(NSAID). Nitroaspirin (NCX-4016), which is compoundedby acetylsalicylic acid, a spacer molecule(3-hydroxybenzyl alcohol), and nitric oxide, hasattracted considerable attention for its lower gastrointestinaltoxicity and for a wider range ofantithrombotic activities. However it is not knownwhether nitroaspirin needs to liberate acetylsalicylicacid in vivo in order to inhibit COXs or if it can blockit as a whole molecule. NCX-4016 in vivo undergoesmetabolization into different metabolites that maycontain aspirin or salicylic acid. The aim of our studywas to evaluate the effects of NCX-4016 and itsmetabolites on purified ovine COX-1 and COX-2 andto compare them with those of aspirin, using a spectrophotometricassay. We found that NCX-4016directly inhibits the activity of both oCOX-1 andoCOX-2 with IC50 values of 65 µM and 45 µM,respectively, similar to aspirin (IC50: 35 µM and 87microM, respectively). Among NCX-4016 metabolites,those containing acetylsalicylic acid (NCX-4017, NCX-4215), inhibited oCOX-1 and oCOX-2activity. On the contrary, the metabolites containingsalicylic acid (NCX-4023, NCX-4019), the nitricoxide-donating moiety (NCX-4015) and the spacer(3-hydroxybenzyl alcohol) were ineffective on bothenzymes. Dialysis experiments showed that oCOX-1inhibition, by both aspirin and NCX-4016, was irreversible.Finally, competition experiments withreversible COX inhibitors (indomethacin, ibuprofen)showed that both aspirin and NCX-4016 are displacedfrom COX-1 by a previous addition of areversible COX inhibitor. In conclusion, in vitro in apurified system, NCX-4016 interacts with COXenzymes in the same way as aspirin, excluding thatsteric hindrance, due to the presence of a spacer anda NO-molocule, prevents nitroaspirin from reachingits target within the enzymatic channel of COX.PO-092PLATELET ANTIAGGREGATING ACTIVITY OF QUINOLIZIDINYL-AND OF QUINOLIZIDINYLMETHYL- BENZOAZOLESMina L,* Terranova E,* Boido V,* Sparatore F,*Piana A,° Armani U°*Dipartimento di Scienze Farmaceutiche, Universitàdi Genova; °Centro di ricerca sulla trombosi edell'aterosclerosi, DIMI, Università di Genova, Italy2-Phenyl-3-(quinolizidin-1-yl)-5-susbstitutedindoles have recently shown a significant antiaggregatingactivity on human platelets activated byadenosine diphosphate, collagen and adrenaline. 1Very interestingly, this activity was in some casesassociated with antihypercholesterolemic action. Inorder to broaden our SAR studies, other analogueshave been considered, bearing a quinolizidinylmethylmoiety in the position 3 or 2 of the indole nucleus,and it was observed that the platelet antiaggregatingactivity was still present. 2 The antiaggregatingactivity was observed in the past in many otherquinolizidine derivatives, synthesized for differentpharmacological aims [1: ref. 4-12]. We describe nownew sets of compounds derived from benzoazolicnuclei which are isosters or bioisosters of indole andcorrespond to the general structures I, II and III(Z=O,S):RNRNRNR'R'CH 2NHNZ HCH 2HNNNI II IIIData so far available, and referring to the plateletantiaggregatingaction stimulated by ADP 2 µM,show in some of the compounds tested a moderateactivity.References1. Ercoli M, Mina L, Canu Boido C, Boido V, Sparatore F, Armani U, etal. Farmaco 2004:59:101.2. Mina L, Bennicelli A, Boido V, Sparatore D. XVIth Int Symp on MedChem. Bologna; 18-22 September 2000. [abstracts 219].haematologica vol. 89(suppl. n. 8):september 2004

142PostersPO-093MONITORING PLATELET GPIIB/IIIA ANTAGONIST THERAPYUSING A FLOW CYTOMETRY TECHNIQUEGallo L,* Seregni R,° Del Maschio A,* Ferrandi P,*Ieri M,* Rossi E**S.I.M.T. and °Lab. Emodinamica, AziendaOspedaliera L. Sacco, Milan, ItalyInhibition of soluble fibrinogen binding to activatedplatelet is the target of tirofiban, a GPIIb/IIIa (alfaIIb β 3) complex antagonist. GPIIb/IIIa antagonistsdrugs are used to prevent thrombotic complicationsof percutaneous coronary interventions. In this studywe used a whole blood flow cytometric assay to evaluatethe effect of tirofiban therapy in patients withcoronary artery disease undergoing to pèercutaneoustransluminal coronary angioplasty (PTCA). We usedPAC-1, a monoclonal antibody (Mo Ab) that recognizeactivated GPIIb/IIIa, to assess the amount ofGPIIb/IIIa complex inhibited by tirofiban. We studied20 patients before treatment, 15 minutes and 3hours after bolus infusion. The patients where treatedwith aspirin, heparin and tirofiban with the followingscheme: - bolus of 25 µg/kg in 3 minutes; -continuous infusion with 0.15 µg/Kg/minute for 12hours. We have also evaluated platelets function inpresence of tirofiban, measuring P-selectin (Mo AbCD 62 P) exposition after in vitro stimulation withagonist (U46619-Sigma). As controls we studied 20healthy volunteers (basal conditions). The percentageof GPIIb/IIIa receptors blocked by tirofiban had rangeof 50-94% at 15 minutes and range of 70-97% at 3hours after treatment. Some patients showed anincrease of receptors blocked after 15 minute, otherpatients showed the same effect at 15 minute andat 3 hours. Two patients had 83-94% of effect at 15minute and < 70% at 3 hours after bolus infusion.All the patients platelets showed normal exposure ofP-selectin in basal conditions ad during tirofibantreatment. These preliminary data confirm theinterindividual variation in response to GPIIb/IIIaantagonist therapy and indicate that the describedflow cytomety technique could be a suitable methodfor assessing the effects of GPIIb/IIIa antagonist andfor monitoring these therapy.PO-094ANTIAGGREGANTING AGENTS DO NOT INFLUENCE THESONOCLOT PROFILEPaniccia R,* Tellini I,* Costanzo M,* Innocenti D,*Bolli P,* Valente S,° Giglioli C,° Lombardi A,*Evangelisti L,* Gensini GF,* Prisco D*, Abbate R**Dipartimento Area Critica Medico-Chirurgica,Università degli Studi di Firenze; °DipartimentoCardiologico e dei Vasi, Azienda Ospedaliero,Universitaria Careggi, Florence, ItalyTwo new point-of-care devices, which identifyplatelet function (PF) have recently become available.The PFA–100 system (DADE, USA) records theclosure time (CT) taken by platelets to occlude amembrane coated with collagen and either epinephrine(CT/EPI) or ADP (CT/ADP). The Sonoclot analyzer(SIENCO Inc, USA) records changes in blood viscoelasticproperties in the form of a graph. Sonoclotsignatures show a lag period (SonACT), correspondingto ACT, and a primary wave whose slope reflectsthe rate of fibrin formation (Clot rate – CR). Then, aninflection is produced as platelets are incorporatedinto the fibrin mesh and a secondary upslope leadsto peak, which occurs at completion of fibrin formation.Subsequent downslope is produced as plateletsinduce further clot retraction The time to peak (minutes)(TP) reflects clot retraction, and is consideredan indicator of PF. The purpose of this study was toinvestigate the changes of Sonoclot analysis and PFA100 CT in 42 patients suffering from idiopathic suddensensoneurinal hearing loss (ISSHL), treated withASA (100 mg/die) for at least 1 month and in 54patients undergoing PCI treated with ASA (325mg/die) and clopidogrel (bolus: 300 mg followed by75 mg/die) for a period of 2-5 days. A control groupof 67 subjects, who had not taken any antiaggregatingagent for 15 days before blood sampling, wasalso studied. In both patient groups the CT/EPI wasprolonged compared to control group, so indicatingASA response. CT/ADP was prolonged in 22/42 ISSHLpatients (52%) and in 18/56 PCI patients (33%).Instead, Sonoclot signature did not show any effectof ASA administration. In ISSHL patients TPs wassimilar compared to control group (113.6 vs 112.7min) and only 1 patient showed a Sonoclot signaturewithout clot retraction curve. In PCI patients a slightnon significant prolongation of TP was observed incomparison with control group (117;5.8 vs 1117;2.7min) and only 2 patients showed a Sonoclot signaturewithout clot retraction curve. The SonACTs weresignificantly (p

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 2004143that antiaggregating agents in ASA responderpatients - as evaluated by PFA 100 analysis - do notaffect the Sonoclot profiles.PO-095EFFECT OF PARNAPARIN, A LOW MOLECULAR WEIGHTHEPARIN, ON PLATELET-PMN LEUKOCYTE INTERACTIONMaugeri N, De Gaetano G, Donati MB, Cerletti CLaboratory of Cell Biology and Pharmacology ofThrombosis. Center for High Technology Researchand Education in Biomedical Sciences, CatholicUniversity, Campobasso, ItalyThe antithrombotic effect of heparin may be partlydue to some anti-inflammatory properties. Aim: totest the effects of parnaparin (PNP, Alfa Wassermann,Bologna), a low-molecular weight heparin, onadhesive molecule-mediated platelet-PMN interactionand on leukocyte function. Methods: Washedplatelets and PMN (from healthy volunteers) wereincubated with PNP or unfractionated heparin (UFH),before cell activation. Dual color FACs was used fordetection of platelet-PMN mixed aggregates (MCA),L-selectin(CD62L), CD142 (TF) expression, andmyeloperoxidase (MPO) content and expressed aspercent of controls (mean±SEM; n=4-6). Results:MCA, formed by stirring PMN at 1000 rpm for 5 minin the presence of thrombin-activated, fixedplatelets, were 54±13% of total PMN population.Between 0.3 and 3 IUaXa/mL PNP inhibited MCA formationin a concentration-dependent way up to70%, while UFH was inhibitory up to 40%. CD62Lexpressed on PMN (% of CD62L+), stirred for 5 minin the absence of platelets, was 28±12%. PNP (0.3-0.8IUaXa/mL) completely prevented the shedding ofCD62L induced by either P-selectin (5µg/mL) or fMLP(0.5 µM), corresponding to about 60% of unstimulatedPMN, an effect independent of the incubationtime (5-20 min). PNP also effectively preventedCD62L shedding occurring during PMN stirring, inthe absence of any added chemical agonist. Increasein TF expression on PMN (% CD142 + ) induced by P-selectin or fMLP was 185±10 and 241±80% of basalvalue, respectively, and was completely prevented(>95% inhibition) by 0.8 IUaXa/mL PNP. PNP completelyprotected PMN degranulation, after challengewith P-selectin or fMLP (>95% inhibition). All theeffects of PNP were similarly observed with UFH.Conclusions: The process of fractionation to obtainlow molecular weight PNP did not remove the anticoagulant-independenteffects on PMN function.Thus, if the overall anti-thrombotic properties ofheparin are partly due to a leukocyte-mediated antiinflammatoryeffect, PNP would share a similarmechanism at concentrations in the same range asthose of UFH.PO-096NITRIC OXIDE(NO)-RELEASING FLUVASTATIN IMPROVESFLUVASTATIN ACTIVITY WITHOUT REVERTING STATINS’NEGATIVE EFFECT ON ATHERO-ENDOTHELIAL LEUKOCYTEADHESION MOLECULES (ELAM) EXPRESSION IN THEVASCULAR ENDOTHELIUMZampolli A,* Massaro M,* Basta G,* Del Turco S,*De Caterina R*°*Institute of Clinical Physiology, CNR Pisa and Lecce,°G. d'Annunzio University, Chieti, ItalyAlthough statins improve many pathogeneticaspects of atherothrombosis, even independently oftheir cholesterol-lowering action, some detrimentaleffects have been observed. In the vascular endothelium,statins increase nitric oxide (NO) bioavailabilityimproving endothelial function, while inmacrophages, they decrease matrix-metalloproteinases(MMP) secretion, contributing to plaquestabilization. However, statins do not decrease circulatinglevels of soluble cell adhesion molecules.Moreover, statins increase athero-ELAM expression(VCAM-1 and E-selectin) in human umbilical veinendothelial cells (HUVEC). In the same system, theinduced athero-ELAM expression is inhibited byexogenous NO. We therefore hypothesized that thenew NO-releasing fluvastatin-NO could reverse thenegative effect of fluvastatin alone on ELAM expression.We also assayed the effect of fluvastatin-NO onMMP-9 secretion by U937 monocytoid cells. HUVECwere incubated with fluvastatin and fluvastatin-NO(0.01-1 µmol/L) for 16 h followed by IL-1α (1 ng/mL)stimulation for 12 h (VCAM-1) or 4 h (E-selectin). Ascontrol, 200 µmol/L of the NO donor S-nitroso-glutathione(GSNO), was incubated with fluvastatin.VCAM-1 and E-selectin expression were determinedby EIA. U937 cells were incubated with fluvastatin orfluvastatin–NO (0.1- 50 µmol/L) for 24 h, followedby stimulation with phorbol myristate acetate (PMA)for 20 h, after which culture medium was collectedand MMP-9 activity assessed by zymography. Fluvastatinpotentiated the IL-1α-mediated athero-ELAM expression in HUVEC (+ 15%, p

144PostersPostersACQUIRED COAGULATION DISORDERSPO-097ACQUIRED FACTOR VIII HEMOPHILIA IN A GERIATRIC PATIENTBoddi W, Machetti S, Sodi N, Sammicheli L, Cati G,Marotta G, Cappelli R*U.O. Medicina Interna Ospedale CampostaggiaA.S.L. 7 - Siena; * Dipartimento di Medicina Interna,Cardiovascolare e Geriatrica, Università di SienaAcquired hemophilia is a rare coagulopathy in adultcaused by the development of autoantibodies againstto factor VIII and is associated with bleeding complicationthat can be life threatening. Although the aetiologyof this disorder remains obscure, about half ofcases are associated with other conditions, mainly thepost partum, underlying cancer, autoimmune diseaseor drug administration. An 81-year old male wasadmitted at hospital with extensive haematomas complaintsappearing one week before. There was no familyor personal history of congenital bleeding diathesis.He had chronic bronchitis and smoke; any drugwas used in the last month. On examination, thepatient was in a reduced condition with largehaematomas (neck, chest, arms and lower limbs). Theother systems were intact. Laboratory parametersrevealed: haemoglobin 7.9 g dL-1, haematocrit 25%,mean cell volume 94, normal number of platelets andwhite blood cells. Coagulation studies revealed an activatedpartial thromboplastin time (aPTT) prolonged (98sec.), with normal prothrombin time and fibrinogenconcentration. An aPTT mixing study did no show correction,suggesting a coagulation inhibitor. Biochemical,immunological tests and tumour markers werenormal. Lupus anticoagulant and anticardiolipin antibodieswere negative. Thoracic and abdominal computedtomographic scan did not reveal pathologicalimages or haematomas. Analysis of clotting factorsrevealed decreased factor VIII (< 2%) and elevated factorVIII inhibitor (55 Bethesda units). From clinical andlaboratory findings, idiopathic acquired hemophiliadiagnosis was made. Fresh plasma, red blood cells andHuman Factor VIII ( 2000 u/day for 7 days) infusionwas initiated, associated with 80 mg/day of metilprenisoloneintravenously. A progressive improvementof clinical conditions and laboratory parameters wasobserved. After 18 days the patient was dischargedand treated with predinosone 50 mg /day. After onemonth at follow-up control the clinical conditions andlaboratory parameters were normal.PO-098ACQUIRED HEMOPHILIA TYPE A IN A VERY OLD WOMAN:A CASE REPORTMasotti L, 1 Vecce R, 1 Gianchecchi D, 1 Cantini R, 1Cannistraro D, 1 Matteucci A, 2 Cecconi N 31U.O. Medicina Interna, Ospedale di Cecina, AziendaUSL 6 Livorno, 2 U.O. Patologia Clinica, Ospedale diLivorno, Azienda USL 6 Livorno, 3 Dipartimenti diOncologia, divisione di Ematologia, Ospedale SantaChiara, Università degli Studi di Pisa, ItalyAcquired hemophilia (AH) is a severe, potentiallylife-threatening condition usually involving elderlypatients, caused by antibody against factor VIII ofcoagulation. Case report: A 92-years-old womancame to our observation for severe acute anemia (6,5g/dl) associated to many diffuse skin bleedings. Thehistory didn’t reveal diseases, pharmacological treatmentor trauma. The physical examination didn’treveal significant signs. The first performed laboratoryassays demonstrated as abnormal: aPTT 110 sec;leucocyte count (37800×10 9 /L, 32 neutrophils, 64%lymphocytes); platelets count of 94000×10 6 /L withantibodies against platelets; ESR of 71 mm/h; D-Dimer 0,61 ng/mL. The dosage of the coagulationfactors demonstrated a value of factor VIII < 1%with a titer of Factor VIII inhibitor of 5,5 BethesdaUnits. The following biochemical examinationsresulted in normal range: tumor markers, autoimmunityprofile, thyroid functionality, markers ofhepatitis. The instrumental examinations of chestand abdomen only revealed moderate spleenmegaly.Lymphocytes typization showed an increasing of Blymphocytes CD5 + /CD19 + with expression of surfaceimmunoglobulins and K clonality, diagnostic forchronic lymphocytic leukemia. We treated thepatients with blood transfusion and intravenousprednisolone with prompt reduction of aPTT andinhibitor Factor VIII titer. At hospital discharge theclinical condition of the patient were good: Hb 11,5g/dL, aPTT 51 sec, Factor VIII 18%, Factor VIIIinhibitor 2,1 Bethesda Units, leucocytes were15600×10 9 /L. At home the patients was treated withprednisone. Actually, after six months any bleedingsoccurred; biochemical values are aPTT 34 sec, FactorVIII 101%, 6400×10 9 /L lymphocytes, Hb 13,7 g/dl,platelets count 60000×10 6 /L. AH is a rare but seriouscoagulopathy usually affecting elderly people,both in absence of pathologies than in many diseases,especially in tumors. The present case reportinvolving a very old patients could represent anexample of AH as first manifestation of a lymphoproliferativesyndrome.haematologica vol. 89(suppl. n. 8):september 2004

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 2004145PO-099ACQUIRED HEMOPHILIA: AN EARLY MARKER OF RELAPSE INA PATIENT WITH A HISTORY OF MULTIPLE MYELOMADe Simone C, Catalano L,* Coppola A, Cimino E,Pollio G,* Buonanno MT,* Tufano A, Cerbone AM,Rotoli B,* Di Minno GRegional Reference Centre for Coagulation Disease,and *Chair of Hematology, Dep. of Clinical andExperimental Medicine, “Federico II” University,Naples, ItalyAcquired hemophilia is a rare, life-threateningbleeding disorder, due to anti-Factor (F) VIII autoantibodies,often related to immunological or lymphoproliferativedisease. A 59-yr old man wasreferred to our Institution in April 2003 because ofa recently-onset bleeding tendency (post-traumatichematomas, persistent hematuria) and prolongedAPTT (ratio 2.03). A moderate Factor (F) VIII deficiency(4.2%) and a low-titre (3.4 BU) FVIII inhibitorwas diagnosed. He had a history of multiple myeloma(IgGk, stage IIIA according to Durie and Salomon)and had achieved a complete remission since 2001,after double bone marrow transplantation. After 3mo-follow-up, in which no clinical or laboratorysigns of relapse were detected, a severe hematomaof the right arm was treated with desmopressin andrecombinant FVIII concentrate, and a 5-d 400 mg/Kgi.v. Ig course and a 15 mg/Kg i.v cyclophosphamidebolus (preferred because of uncompensated diabetesmellitus) were started as eradication strategy. Twoweeks later, the onset of unstable angina (treatedonly by anti-ischemic drugs, as coagulation abnormalitieshampered to perform angiographic proceduresand to start antithrombotic treatment) led tointroduce oral prednisone (0.5 mg/Kg) andcyclophosphamide (1.5 mg/Kg). APTT and inhibitortitre progressively lowered over the following 6weeks of treatment; however, when doses of prednisonewere reduced, a tendency to a new increaseof inhibitor was shown. Therefore, after reducing andwithdrawing cyclophosphamide, a very slow reductionof prednisone was carried out. FVIII inhibitorwas undetectable since November 2003 and prednisonewas cessated on January 2004. Two monthslater a significant increase of a monoclonal IgGkparaprotein was detected, in the absence of coagulationabnormalities. In this patient with a history ofmultiple myeloma, the onset of acquired hemophiliawas likely to be an early marker of derangementof the immunologic network due to the malignantrelapse.PO-100DISLIPIDEMIA AND REDUCED FIBRINOLYSIS IN A CHILDWITH UREMIC HEMOLYTIC SYNDROMECerneca F, Bruno GIstituto per l'Infanzia IRCCS Burlo Garofolo, Trieste,ItalyMany pathogenetic factors may enhance coagulationprocess and induce thrombosis. The Authorsreport a case of uremic hemolytic syndrome, withmarked evidence of macroscopic kidney thromboticinvolvement, in which an important dislipidemia(triglyceridemia 384 mg/dl, colesterolemia 428 mg/dL)was detected during the phase of clinical improvement.These findings, and the contemporary markedreduction of fibrinolytic activity (t-PA before stimulation2.6 ng/mL, t-PA after stimulation 2.5 ng/mL;PAI-1 before stimulation 25.7 AU/mL, PAI-1 afterstimulation 40.4 AU/mL), seem to be relevant pathogeneticfactors in this case. The treatment with polynsaturatedfatty acids Omega 3 may have been helpfulin modifying these serum abnormalities (triglyceridemia101 mg/dL; colesterolemia 295 mg/dl; t-PAbefore stimulation 3.6 ng/mL, t-PA after stimulation8.3 ng/mL; PAI-1 before stimulation 7.8 AU/mL, PAI-1 after stimulation 2.2 AU/mL) and maybe could havebrought to the clinical improvement.PO-101CD40LIGAND ENHANCES MONOCYTE TISSUE FACTOREXPRESSION AND THROMBIN GENERATION VIA OXIDATIVESTRESS IN PATIENTS WITH HYPERCHOLESTEROLEMIAFerro D,* Sanguigni V, Pignatelli P, Del Ben M,Tini N, Saliola M,* Sorge R, Violi FDepartment of Experimental Medicine andPathology; University of Rome "La Sapienza:*Department of Internal Medicine, Universityof Rome "Tor Vergata”, Rome, ItalyPatients with hypercholesterolemia show an ongoingprothrombotic state, but the underlying mechanismis still unclear. We tested the hypothesis thatCD40 ligand induces a prothrombotic state byenhancing oxidative stress. Circulating levels of thesoluble form of CD40 ligand (sCD40L) (QuantikineCD40 ligand, R&D Systems, Minneapolis, USA), ofprothrombin fragment 1+2 (F1+2)(Enzygnost F1+2,Behringwerke, Marburg, Germany), a marker ofthrombin generation, and of 8-hydroxy-2-deoxyguanosine(8OHdG) (Bioxytech 8-OHdG-EIA, OXISHealth products, Portland, USA), a marker of oxidativestress, were measured in 40 hypercholesterolemicpatients and in 20 age and sex matchedhealthy subjects. Patients with hypercholesterolemiahaematologica vol. 89(suppl. n. 8):september 2004

146Postersshowed significantly higher levels of sCD40L(p

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 2004147On day 22, Fondaparinux was administered at thedose of 7.5 mg/day for 27 days before re-insitutionof warfarin treatment, with platelet counts remainingconsistently above 250,000. The patient wasreadmitted for additional surgery after 6 and 8months and always treated with Fondaparinux withoutany drop in platelet counts. This is the first reportof successful LMW-HIT type II treatment with Fondaparinux.In view of its safety, this pentasaccharideshould be preferred to hirudin in the treatment ofheparin-induced thrombocytopenia.PostersANTIPHOSPHOLIPID ANTIBODIESPO-104TRANSIENT LUPUS ANTICOAGULANT SECONDARY TOMENINGOCOCCAL MENINGITISSantoro R, Iannaccaro P, Sottilotta G, Elia L,Zappalà D, Bulotta GCentro Emofilia - Centro di Riferimento Regionaleper la Patologia Emorragica e Trombotica Ereditaria,Azienda Ospedaliera Pugliese-Ciaccio, Catanzaro,ItalyInfectious agents have been implicated in theinduction of antiphospholipid (aPL) antibodies, inthe positivity for lupus anticoagulant (LA) and in thedevelopment of the antiphospholipid syndrome(APS). Many viral infections including hepatitis Cvirus, human immunodeficiency virus, cytomegalovirus,varicella zoster virus, Epstein-Barr virus, adenovirus,parvovirus B, but also bacterial infectionslike relapsing streptococcal tonsillitis, have beendescribed. We report a case of a previously well threemonths old infant (female), come to our observationbecause she developed elevated fever and neck stiffness.She was diagnosed to have meningococcalmeningitis. Hematologic testing revealed derangedclotting time with an aPTT of 63.4 sec (ratio 2.19),aPTT after mixing with normal plasma ratio 1.68 andINR of 1.29. Platelets were within the normal range.Diluted Russell’s viper venom test was consistentwith the presence of a lupus anticoagulant. However,anticardiolipin antibodies were negative.Although meningococcal meningitis sometimes presentswith haemorrhagic disorders and disseminatedintravascular coagulation (DIC), in this case theinfection was the triggering factor for the appearanceof lupus anticoagulant. The infant had an excellentoutcome with complete recovery, gradual normalizationof the aPTT and disappearance of thelupus anticoagulant two months after. The clinicalsignificance of finding aPL antibodies and lupus anticoagulantpositivity in patients with viral and bacterialinfections remains unknown. In some patients,these antibodies may be transient, often disappearwithin 2 or 3 months and are not usually associatedwith thrombotic complication. In other susceptibleindividuals, they may persist and raise the questionof whether infections may trigger the developmentof aPL antibodies and/or lupus anticoagulant inautoimmune diseases.haematologica vol. 89(suppl. n. 8):september 2004

148PostersPO-105PROGNOSTIC SIGNIFICANCE OF ENCEPHALIC NMR INPATIENTS WITH ANTIPHOSPHOLIPID SINDROME (APS)La Rosa L, Rosana A, Cefis M, Restifo D, Uccellini MAmbulatorio di Emostasi e Trombosi del Servizio diMedicina Trasfusionale, Azienda Ospedaliera Civiledi Vimercate, ItalyIntroduction: One of the major concerns of theantiphospholipid syndrome (APS) is the propensityof antiphospholipid antibodies to cause thrombosisin the vessels of the brain. Although encephalic NMRalterations have been described in APS, their clinicalcorrelates are still undefined. Patients and Methods:Twelve patients diagnosed as having APS were studied.At the diagnosis of APS, three patients wereadmitted in the Neurology Division for a transientischemic attack, one was referred for an hypoacusia,one was followed-up by the rheumathologist for aLupus Anti-Coagulant (LAC) sindrome, two werereferred to the hematologist for the occurrence ofanemia and thrombocitopenia. Five cases were occasionallydiagnosed as having APS during admissionin the Internal Medicine Department. All the patientswere administered long term oral anticoagulanttherapy with warfarin. LAC was detected in 7 out of12 patients (58.3%). In all the cases, NMR of thebrain was performed at the diagnosis of the APS andduring follow-up. Results: In 6 out of 12 patients(50%), encephalic NMR imaging was suggestive forlocal brain microembolism. Two out of 6 patients(33%) with stable brain NMR alterations developeda progressive loss of memory during follow-up. Conclusions:Although the significance of brain NMRalterations in patients with APS remains uncertain,a significant proportion of patients with the radiologicalevidence of brain microembolism developsneurological symptoms during follow-up despitewarfarin treatment. To better define the prognosticsignificance of NMR imaging for neurological complicationsin patients with APS, a prospective observationalstudy has been undertaken in our Center.PO-106STUDY OF AUTOANTIBODIES AND THE RELATIONSHIPBETWEEN ANTICARDIOLIPIN ANTIBODIES AND LUPUSANTICOAGULANT IN FEMALE PATIENTS WITHPREGNANCY-INDUCED ARTERIAL HYPERTENSIONCerneca F, Bruno G, Bogatti PIstituto per l'Infanzia IRCCS Burlo Garofolo, Trieste,ItalyThe aim of this study was to evaluate the relationshipbetween anticardiolipin antibodies andlupus anticoagulant, and the prevalence of autoantibodiesin a group of 48 hypertensive third trimesterpregnant women. There was no correlation betweenlevels of anticardiolipin and tests of lupus anticoagulantactivity. Prevalence of autoantibodies was low,and the presence of anticardiolipin antibodies wasnot a marker for severe hypertension, trisymptomaticgestosis, or intrauterine growth retardation. In conclusion,our study did not reveal the presence ofantiphospholipid antibodies in hypertensive thirdtrimester pregnant women, but the results failed toshow any concordance between anticardiolipin antibodiesand lupus anticoagulant activity.PO-107CASE REPORT: MIDDLE CEREBRAL ARTERY THROMBOSIS INA YOUNG WOMAN WITH ANTIPHOSPHOLIPID SYNDROMELo Pinto G, Schenone A, Bianchini D*Thrombosis Unit: Medical and *NeurologicalDepartment Galliera Hospital, Genoa, ItalyWe describe a case of an unmarried woman aged32, with a family history of coronary artery disease(father) and moderate low platelet count (100.000 cumm) known since many months. She has developeda sudden transitory aphasia with asthenia of thebody right side with progression to aphasia and ipsilateralhemiplegia. The patient was submitted tobrain TC both without and with contrast medium,MR and angio MR with contrast medium as well asto angiographic evaluation that showed a middlecerebral artery occlusion near its origin. The ultrasoundexamination of the neck vessels and the heartultrasonography were normal. We performed a coagulationscreening and found Lupus AntiCoagulantpositivity and a high title of anticardiolipin antibody:IgG 94 U/mL and IgM 36 U/mL (N.V. 1 - 10 U/mL) andpositivity of antinuclear antibody (title 1/320 withhomogeneous pattern). At the beginning we performedan anticoagulant therapy with heparin andsoon after with warfarin. At present the patient istreated with warfarin and physiotherapy. A pharmacologicalimmunosoppression may be useful. Wethink that an early attention to the low plateletcount would have been helpful for a preventivetreatment of such severe ischemic clinical event.PO-108ADAMTS13 IN PATIENTS WITH ANTIPHOSPHOLIPID SYNDROME(APS)Birolini A, Del Maschio A, Lamagna R, Carraro MC,Arrigoni L, Rossi ES.I.M.T. Azienda Ospedaliera Luigi Sacco, Milan, ItalyAntiphospholipid antibody syndrome (APS) definesa clinical picture derived by association of antibodieshaematologica vol. 89(suppl. n. 8):september 2004

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 2004149against phospholipid-binding proteins and thromboticdiathesis. The most severe form of APS is CatastrophicAntiphospholipid Syndrome; (CAPS), a thromboticmicroangiopathy, clinically and histologicallysimilar to Thrombotic Thrombocytopenic Purpura(TTP). Most frequent form of TTP has an autoimmunepathogenesis, as it is due to the inhibition ofADAMTS13 by an IgG antibody. We studied 26patients suffering from APS in order to verify if alsoin these patients, like in TTP, there was a deficiency ofADAMTS13, due to the presence of autoantibodiesable to block the activity of ADAMTS13, possiblyexplaining their thrombosis. Patients’ plasma wastested for laboratory confirmation of antiphospholipidantibodies, activity of ADAMTS13, functionalVWF level (VWF:RCo) and VWF antigen (VWF:Ag).Plasma from 20 blood donors, matched for sex andage with patients, was used as control group. Resultsindicated that ADAMTS13, VWF:RCo, VWF:Ag were:98.6±14.6, 104.2±35.5 and 111.7±34.2, versus153.5±37.7, 187.7±72.4 and 194.3±64.2, respectivelyfor control and patient group (p

150Postersscreen and SCT confirm were obtained by replicativeanalysis of normal pool (NP) and an LA sample usedas a positive control (LA+), 6 times within the samerun (NP = 1.0 and LA+ = 3.1 for SCT screen and NP= 2.0 and LA+ = 2.6 for SCT confirm), and six timesin different runs (NP = 3.9 and LA+ = 5.2 for SCTscreen and NP = 4.8 and LA+ = 5.7 for SCT confirm).Specificity of the SCT for the LA was investigated bystudying patients with different coagulation abnormalities:patients on low molecular weight heparintherapy [LMWH, blood sample collected 12 hoursafter the injection (n = 12)], patients on oral anticoagulanttherapy [OAT (n = 23)], patients with factordeficiencies of intrinsic coagulation system [n=3: 1factor VIII:C = 37%, 1 factor IX:C = 39% and 1 factorXI:C = 41%], patients with type 1 von Willebranddisease [vWD (n = 3)]. 6/12 patients on LMHW, allthe subjects with defects of intrinsic coagulationfactors and 1/3 patients with vWD had prolongedSCT screen; [cut off (95 th percentile) of SCT screenratio in healthy subjects = 1.21] assay but all of themwere identified as LA negative by SCT confirm assay[cut off (95 th percentile) of SCT confirm ratio inhealthy subjects) = 1.22]. 19/23 patients on OAT hadprolonged SCT screen ratio: 7/19 previously identifiedas having a LA were confirmed to be LA positiveby SCT confirm assay, 12/19 were identified to be LAnegative. In order to evaluate the screening performanceof this new test, SCT was carried out on 136plasma samples from anti-coagulant free patients inwhich a LA determination was requested. All 136plasma samples were further analysed for the presenceof LA by means of our laboratory routine LAassays [aPTT (APTT-SP, IL), DRVVT (LAC, IL) and SCT (inhouse method) screening and confirmation tests]. 46patients were identified as having a LA by our routineLA tests. Of these samples, a prolonged SCTscreentest was found in 40 plasma samples. Inhibitoractivity observed in these patients was confirmed tobe of the LA type by the SCT confirm assay. 6 out of46 samples identified as having a LA were found tobe SCT negative, in these patients the only test foundto be positive was DRVVT. 6 out of 90 LA negativepatients were found to be SCT positive. Among thesepatients, 2 were found to be positive for anti cardiolipinIgM (18 and 12 MPL/mL), 1 for anti prothrombinIgG (12,5 U/mL), 1 for anti protein S IgM(21 U/mL) and 1 for anti protein C IgM (28,4 U/mL)autoantibodies. These data allow us to calculate theperformance characteristics of the SCT assay for theLA. Sensitivity, specificity, PPV, NPV and diagnosticaccuracy of the SCT were: 87%, 93%, 87%, 95% and90% respectively. The easy and rapid performanceon photooptical coagulometers and the good screeningperformances make this new commercial SCTuseful, in combination with DRVVT, for large scalescreening test for LA.PostersCEREBROVASCULAR AND CORONARY DISEASESPO-111INFLAMMATION AND BRAIN ABNORMALITIES INSTROKE-PRONE RATS: EFFECTS OF ROSUVASTATINSironi L, Gelosa P, Gianazza E, Guerrini U, Nobili E,Gianella A, Paoletti R, Tremoli EDipartimento di Scienze Farmacologiche, Universitàdegli Studi di Milano; Centro Cardiologio Monzino,Milan, ItalyInflammatory processes play a critical role in thepathogenesis of stroke. Spontaneously hypertensivestroke-prone rats (SHRSP) develop, before theappearance of brain damage, an inflammatory conditioncharacterized by the accumulation in the bodyfluids of several acute-phase proteins. Thus, thisexperimental model represents an useful tool toevaluate the contribution of inflammation to braininjury. Statins have beneficial effects in brainischemia independent of their lipid-lowering properties,including anti-inflammatory actions. Weinvestigated whether rosuvastatin influences thedevelopment of inflammation associated with brainabnormalities in salt-loaded SHRSP. Methods andResults Male SHRSP fed a high salt-diet were treatedlong-term with vehicle or rosuvastatin (1 and 10mg/kg/day). Brain abnormalities developed respectivelyafter 40±5 days and after 60±5 days (p

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 2004151PO-112VALUE OF ACTIVATED FACTOR TWELVE AND PLASMAIMMUNOLOGIC DERANGEMENTS IN THE PATHOGENESIS OFCEREBROVASCULAR ISCHEMIC DISORDERSde Lucia D, 1 Sessa M, 1 Misso M, 1 Adamo S, 1Tagliaferri A, 1 De Francesco F, 1 Del Prete SM, 2Pignalosa O, 1 Russo T, 1 Galante M, 1 Bernacchi M, 1Caruso S, 2 Maisto G, 2 Perricone F, 2 Lupone MR 21Clinical Pathology, Laboratory of Haemostasis andThrombosis; II University of Naples: 2 RegionalCenter for Haemocoagulopathies, PausilliponHospital, Naples, ItalyCerebral thrombosis is a multicausal disease. Inmost cases two or more risk factors both genetic andacquired are necessary before thrombotic eventoccurs. Such a combination of risk factors are hyperfibrinogenemia(> 340 mg/dL), high FVIIa and FVIIIlevels (> 150 IU/dL, >155 IU/dL respectively), highlevels of antiphospholipid antibodies/aPL Abs (> 20.0U GPL/mL), F1+2 (>1.1 nM) and D-Dimer(s) (> 450ng/mL). For these observations, it is now mandatoryto have a screening assay which is able to recognizethis combination of defects. We aimed to evaluateactivation of FXII (FXIIa), FVIIa, and peptides releasedduring thrombin generation (prothrombin fragment1+2/F1+2, thrombin-antithrombin complex/T-AT,and fibrinolysis (D-dimer/DD) in ischemic strokepatients (cardioembolic and atherothrombotic subtypes)with (n=40) and without (n=30) aPL Abs. Seventyhealthy subjects acted as a control group. FXI-Ia (Shield Diagnostics Ltd), T-AT and F1+2 (Enzygnost,Dade Behring), and DD (Dimertest, Roche) wereassessed by ELISA method. FVIIa was measured usinga recombinant mutant form of human tissue factor(TF 219) as a cofactor for FVIIa catalysed activationof FX. FXIIa levels in aPL (+) stroke patients were significantlyhigher (10.86±2.48 ng/mL) than in aPL (-)stroke patients (2.49±1.75 ng/mL) and normals(1.15±0.37 ng/mL), which was further associatedwith a history of cerebral thrombosis (p= 0.021).Patients with high aPL titers had significantly elevatedT-AT (6.5±3.5 mg 3 /mL vs 2.85±2.25 mg 3 /mLand 2.45±2.05 mg 3 /mL), F1+2 (2.55±2.05 nM vs0.95±1.05 nM and 0.45±0.35 nM), DD (635±245ng/mL vs 375±225 ng/mL and 355±175 ng/mL) andFVIIa (2.80±1.95 ng/mL vs 1.40±1.55 ng/mL and1.20±1.35 ng/mL) levels compared to aPL (-) patientsand controls, thus reflecting a generalised prothromboticstate. The authors feel that the mechanismof FXIIa generation in cerebrovascular ischemicpatients is unknown, but may reflect activation ofthe plasma kallikrein/kinin system on damagedendothelial cells activated by high titers of aPL(s).PO-113PREVALENCE OF PROTHROMBIN G20210A MUTATION AMONGPATIENTS WITH CEREBRAL ISCHAEMIAStefanin C, Garghetti E, Cozzi MG, Duca MI,Rampinini F, Morelli BLaboratorio di Ematologia, Ospedale Civile diLegnano, ItalyThe prothrombin G20210A mutation is an inhereditedthrombofilic factor that is associated with anincreased risk for venous and arterial thromboembolism.The prevalence of G20210A mutation isapproximately 2,3% in general population (more frequentin southern Europe as compared to northernEurope and very rare in Asia and Africa), approximately18% in young patients with venous thrombosis andapproximately 6,2% in non selected patients. In arterialthrombosis the role of G20210A mutation is stillunknown. Evaluate the role of G20210A mutation inarterial thrombotic events (TIA, RIND, minor stroke,ictus). The study was performed on 200 patients (53men and 147 women), median age 38 years (range16-50), in addition to 350 healthy controls. All selectedpatients had cerebral ischaemia without anythrombotic risk factors, such as hypertension, diabetes,elevated levels of cholesterol and triglycerides, obesity,etc. The patients were hospitalized in the Neurology’sDepartment of Legnano hospital. Cerebralischemia was confirmed on all patients by CT, MRI,angiography and EEG. Blood samples were collected insodium citrate. Mutation has been tested usinggenomic DNA on whole blood, by PCR and sequencyanalysis. Results. In the group of patients we found 14heterozigous individuals (7%; 95% CI, 3,5% - 10,5%):4 men and 10 women, whereas in the group of controlswe found 8 heterozigous individuals (2,2%; 95%CI, 0,6% - 3,7%): 2 men and 6 women. None of themwas homozigous. The prevalence of factor II G20210Amutation is 7% among patients showing a significantdifference versus group control (2,2%; p=0,006). Theodd ratio shows an increase of relative risk duringcerebral ischaemia of 3.2 fold associated with prothrombinmutation (95% CI, 1,3 - 7,7). Conclusion.Prevalence of prothrombin mutation (7%) in patientswith cerebral ischaemia is comparable to the prevalencefound in non selected patients having venousthrombotic events. Presence of this mutation is associatedwith an increased risk of arterial cerebralthrombosis.ReferencesLongstreth WT Jr, Rosendaal FR, Siscovick DS, Vos HL, Schwartz SM,Psaty BM, et al. Risk of stroke in young women and two prothromboticmutations: factor V Leiden and prothrombin gene variant(G20210A). Stroke 1998;29:577-80haematologica vol. 89(suppl. n. 8):september 2004

152PostersPO-114ECAPS STUDY: MULTICENTRIC EVALUATION OF CEREBRALHEMORRHAGE IN ANTICOAGULATED/ANTIAGGREGATEDPATIENTS IN THE ITALIAN EMERGENCY ROOMSAltomonte F, Altomonte M, Iorio A, Ghirarduzzi A,Ricciardi A, Remollino V, Spisni L, Saporito A,Caiazza A, Musso G, Cervellin G, Lamberti S,Buzzalino M, de Giorgi F, Del Prato C, Golinelli MP,Gai V, Monsù R, Gioffrè M, Giovanardi D, CattaneoS, Frumento F, Caporrella A, Re G, de Iaco F,Bologna G, Nocenti F, Lorenzi C, Zoratti R,Sciolla A, Tiscione V, Pastorello M, Vandelli A,Villa A, Zanna M, De Palma AAz. Ospedale Università San Martino (GE) on behalfof other 32 First-care Departments, ItalyBackground. Cerebral hemorrhage still constitute amajor drawback of antithrombotic treatment. Riskfactors and natural history of the disease are far frombeing completely clarified Objective. To evaluate theincidence rate, natural history and risk factors ofintracerebral hemorrhage (ICH) in the italian emergencyrooms (ER) setting. Design and methods. Multicentricobservational investigator driven study. Consecutivepatients admitted for CT proven ICH andtreated with antithrombotics were enrolled in thestudy. A consecutive cohort of ICH non antithrombotictreated patients was enrolled in a single centre.History, clinical and therapeutic data were collectedthrough a computer-based form. Univariate and multivariatelogistic regression was used to evaluate theeffect on mortality (Rankin score = 6) at ER dischargeand three months later. Kaplan-Mayer analysis andlog rank test were used to compare survival versusthe control group in the coordinating centre. Results.From 01/02/03 to 31/01/04 695 patients wereenrolled in 33 ER out of a population of 6.672.830patients (1/10,000/year) and 1.711.348 admission tothe ER (4/10,000). 199 controls were enrolled. At univariateanalysis the variables significantly associatedwith early and late mortality were the spontaneouscause of the event, low GCS, neurosurgical treatment,ICH below the tentorium or associated with ventricularbleeding. Anticoagulant treated patient showeda significantly higher mortality than those antiaggregated.The multivariate model was highly significant,accounting for 76% of the variance, and indicatedage, spontaneous events and low GCS as the majorpredictors of mortality. The control group was statisticallydifferent for an higher incidence of ventilationassisted, cerebral vascular malformation and youngerpatients. The log rank test showed a higher mortalityin the anticoagulated/antiaggregated patients(p=0.014) than in controls. Conclusions. Our studyconfirms that ICH constitutes a common complicationof antithrombotic treatment, statistically more severein orally anticoagulated patients. If a rapid reversal ofthe iatrogenic coagulation defect could reduce mortalityand morbidity is worth to be tested in a clinicaltrial.PO-115HEMORHEOLOGY AND ATRIAL FIBRILLATION: A NEW MARKERFOR EMBOLIC COMPLICATIONS?Mannini L, Cecchi E, Bruschettini A, Costanzo M,Poli D, Gensini GF, Abbate R, Prisco DDipartimento di Area Critica Medico-Chirurgica,Centro Trombosi, Azienda Ospedaliero-UniversitariaCareggi, Florence, ItalyAtrial fibrillation (AF) is a frequent tachyarrhythmiacharacterized by an increased risk of stroke. Oralanticoagulant therapy (OAT) can reduce the incidenceof stroke by about two thirds. Recent studieshave suggested that hyperviscosity is frequent in AFpatients. Aim of this study was to evaluate if somehaemorheological alterations can play a role in theoccurrence of ischemic cerebrovascular events in AFpatients. We studied 23 AF patients [13 M, 10F;median age 75 (54-85) years] with history of at leastone embolic event and 63 AF patients without [35 M,28F; median age 74 (59-84) years]; the two groupswere matched for age and sex. Hemorheologicalstudies were performed by assessing whole bloodviscosity (WBV) at 0.512 and 94.5 s-1, plasma viscosity(PLV) at 94.5 s-1 and erythrocyte filtration test(EFT). WBV and PLV were studied by rotational viscosimeterContraves LS 30 and EFT was performed byerythrocyte filtrometer Myrenne MF4. Blood sampleswere collected at least one month after theembolic event and during a period of optimal anticoagulationlevel. No differences were foundbetween the two groups in WBV at 0.512 s-1, PLV,EFT, hematocrit and fibrinogen levels. As WBV at 94.5s-1 is concerned, results were considered normal orabnormal according to a nomogram taking intoaccount different ranges of hematocrit values. Analtered WBV at 94.5 s-1 was found more frequentlyin patients with a previous embolic event at bothunivariate (OR: 4.60, CI 95: 1.41-15.06; p=0.012) andmultivariate analysis (OR:5.72, CI 95: 1.39-23.50;p=0.016) adjusted for age, sex, hypertension, diabetes,coronary artery disease, left ventricular dysfunction,smoking habitus and dyslipidemia. Theseresults stimulate to further investigate the mechanismsthat could determine a hyperviscosity state inAF patients, and in particular an increased WBV at94.5 s-1. Moreover, the possible mechanisms throughwhich hyperviscosity may lead to an increased risk ofthromboembolic complications in AF patients mustbe explored.haematologica vol. 89(suppl. n. 8):september 2004

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 2004153This work was supported by Grants from the Ministerodell’Istruzione, dell’Università e della Ricerca,Rome, Italy.PO-116MICROCIRCULATORY ALTERATIONS AND ENDOTHELIALDYSFUNCTION IN HEART FAILURETurchetti V, Boschi L, Mastronuzzi VMA, Coppola D,Dragoni S, Donati G, Trabalzini L, Forconi SDepartment of Vascular and Geriatric InternalMedicine, University of Siena, ItalyThe aim of our study was to evaluate endotheliumdependentdilatation induced by an ACE-inhibitor,Ca++ antagonist and β-blocker in patients sufferingfrom heart failure (NYHA class II and III). We studied34 patients (19M, 15F, mean age 76,96±8,82) inpharmacological wash-out for at least one week,divided into 3 groups: Group A (15 patients, 9M and6F) taking ramipril (5 mg/die); Group B (10 patients,6M and 4F) taking amlodipine (10 mg/die), Group C:(9 patients, 4M and 5F) taking carvedilole (25mg/die). The groups were homologous for NYHA classand instrumental echographic parameters (mean EF= 22,5±6,7 and mean sAPP 38,4±8,7). At the beginningand after 3 weeks of therapy, we performed aclinical and instrumental assessment; we studiedendothelial function by determination of L-Arginineand L-Citrulline (amino acids of the Nitric Oxidemetabolic pathway), the L-Citrulline/L-Arginine ratio(an index of NOS activity) and VCAM-1 (endothelialdysfunction index); hemorheological parameters(blood viscosity, plasma fibrinogen and erythrocytemorphology); coagulative/fibrinolytic parameters (PT,aPTT, fibrinogen and PAI-1). The results show that L-Citrulline and L-Arginine increase, while VCAM-1decreases. The L-Citrulline/L-Arginine ratio increasesin a statistically significant way. This trend ismaintained in each group. These results demonstratethat the drugs used induce an improvement ofendothelium-dependent dilatation. In addition, thereis progressive haemorheological and fibrinolyticimprovement, with a reduction of PAI-1 and bloodviscosity.PO-117INFLAMMATORY MARKERS AND C-IMT IN PATIENTS WITHPREVIOUS MYOCARDIAL INFARCTION. THE ATORVASTATINEFFECT. MIAMI STUDYArquati M,* Porta B, Camera M,° Tremoli E,°Cortellaro M,* On Behalf Of The Miami StudyGroup*Dipartimento di Medicina Interna, IstitutoPoliclinico San Donato, Milan University, Italy; °Dipartimento di Scienze Farmacologiche, MilanUniversity, ItalyIntroduction: Plasma levels of Hs-CRP may be usefulto select subjects at high risk of cardiovascularevents. This inflammatory marker also helps identifypatients most likely to respond to statins. Aim: Aprospective clinical study was designed to investigate:(i)the regression of CRP levels and other solubleinflammatory markers; (ii)the correlationsbetween levels of Carotid Intima Media Thickness(C-IMT) and soluble markers at different times;(iii)the influence of Hs-CRP levels on C-IMT regression;(iv)the utility of C-IMT as an additional markerto identify patients likely to respond to statins.Materials and Methods: MIAMI is an open, multicenter,independent study. In 100 patients with previousMI (> two months), in stable clinical conditions,with normal cholesterol and blood glucose,receiving atorvastatin (20 mg/daily) for 24 months,the following variables were measured at 0, 12 and24 month: VCAM, ICAM, E-selectins, IL-6, -8, -10, -18, TNF-α, hs-CRP, MMP-9, TF, TFPI, Fg, TC, HDL, LDL,TG, urinary isoprostanes. C-IMT was measured blindlyby computerised determination of images recordedin three examinations. The study started in January2003 and is scheduled to end in December 2005.PO-118EMERGING PROGNOSTIC FACTORS IN ISCHEMIC HEARTDISEASEBrogi D,* Sofi F,* Marcucci R,* Gori AM,*Bandinelli B,* Valente S,° Giglioli C,° Comeglio M,°Margheri M,° Prisco D,* Abbate R,* Gensini GF**Dipartimento di Area Critica Medico-Chirugica,Università degli Studi di Firenze; °DipartimentoCardiologico e dei VasiDifferent studies investigated the role of emerginghaemostasis-related factors on the pathogenesis ofcoronary artery disease but their prognostic valuehave not yet been defined. Aim of our study was toevaluate the prognostic value of emerging geneticand metabolic haemostasis-related risk factors in agroup of consecutive patients referred to the Coro-haematologica vol. 89(suppl. n. 8):september 2004

154Postersnary Intensive Therapy Unit of University of Florencewith diagnosis of acute coronary syndrome (ACS).We investigated, at the time of admission, the presenceof Factor V Leiden and prothrombin G20210Amutations, homocysteine (Hcy), lipoprotein (a) [Lp(a)]and plasminogen activator inhibitor 1 (PAI-1) plasmalevels in 416 patients and in 416 healthy subjects.Hyperhomocysteinemia, defined as a concentrationof Hcy above the 95th percentile of controls, wasdiagnosed in 160/416 patients (38.5%) and in20/416 controls (5%); Lp (a) levels above 300 mg/dLwere found in 146/416 patients (35.1%) and in41/416 (9.8%) controls and PAI-1 >15 IU/mL in204/416 patients (49%) and in 74/416 controls(17.7%). There was no statistically significant differenceconcerning the presence of the 2 mutations inpatients compared to controls. Patients were followedfor a mean period of 15 months: 80/416 hada major adverse cardiac event (MACE): 41/416 diedfor cardiovascular causes and target vessel revascularizationwas recorded in 34 patients. Patients withMACE had elevated PAI-1 levels (63.4%) and hyperhomocysteinemia(31.3%). At the multivariate analysis,adjusted for traditional and haemostasis-relatedrisk factors, hyperhomocysteinemia and high PAI-1levels resulted independent predictive factors forMACE (OR= 2.1, 95%CI 1.2-3.8, p=0.001; OR= 1.795%CI 1.03-2.9, p=0.004). This study reveals thathyperhomocysteinemia and high levels of PAI-1could be predictors for MACE in patient with ACS.PO-119DISTINCT ROLES FOR PAR1- AND PAR2-MEDIATEDVASOMOTOR MODULATION IN HUMAN ARTERIAL AND VENOUSCONDUITS USED IN CORONARY ARTERY BYPASS SURGERYBallerio R,^ Brambilla M, # Banas A, # Parolari A,^Agrifoglio M,^ Alamanni F,^ Cannata A,^Camera M,^# Tremoli E,^# Mussoni L ##Dipartimento di Scienze Farmacologiche,Università degli Studi di Milano;^Centro CardiologicoMonzino, IRCCS, Milan, ItalyInternal mammary artery (IMA) is the preferredconduit for coronary artery bypass grafting (CABG),because of improved patency rates in comparisonwith greater saphenous vein (GSV); this may be relatedto endothelial release of vasodilators that antagonisevascular contraction. Recently, a family of seven-transmembraneG protein-coupled receptors,protease-activated receptors (PARs), have beenshown to modulate endothelium-dependent vasodilatation.In the vascular system, PAR1 and PAR2 areexpressed in smooth muscle and in endothelial cells,and PAR2 only can be up-regulated in pathologicalconditions such as inflammation. In the presentstudy we compared the presence and functionalactivity of PAR1 and PAR2 in IMAs and GSVsobtained from patients undergoing CABG. PAR1 andPAR2 mRNAs were detected by RT-PCR both inendothelium intact IMAs and in GSVs. In order tostudy the role of these receptors in mediating vasculartone, IMAs and GSVs rings were suspended forisometric tension recording and challenged withselective PAR-activating peptides (PAR-AP). Theselective PAR1-AP, TFLLR-NH2 (0.001 to 10 µmol/L),caused marked endothelium-dependent relaxationin pre-contracted IMAs and GSVs rings (Emax 55%and 54%, respectively p=n.s), whereas PAR2-AP,SLIGKV-NH2 (0.01 to 100 µmol/L), elicited only aslight change in vascular tension in both arterial andvenous vessels. The pre-incubation (14h) of vesselswith the inflammatory stimulus, TNF-α (3 nanomol/L),followed by exposure to PAR2-AP, increasedthe relaxation in both vessels, with a more prominenteffect on IMAs (48±5% and 24±4%, for IMAs andGSVs respectively, p

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 2004155domized to undergo OPCAB (n=17) or CABG (n=18).Blood samples were collected before the interventionand up to one month after surgery. Prothrombinfragment F1.2 (F1.2), thrombin-antithrombin complex(TAT) and D-dimer (XDP) increased after surgeryand were persistently higher than preoperative valuesup to 30 postoperative days both in OPCAB andCABG; higher levels of these variables were detectedin CABG with respect to OPCAB only at the timepoint after termination of CPB (F1.2 and TAT) or fromCPB end up to 8 postoperative days (XDP). Fibrinogenlevels decreased after surgery, then increased inparallel in both groups up to 8 days after surgery. vonWillebrand factor (vWF) increased postoperatively inboth groups and returned to baseline 30 days aftersurgery, being higher in CABG from CPB end up to 8postoperative days; soluble Vascular Cell AdhesionMolecule-1 (sVCAM) increased significantly overbaseline in both groups 30 days after surgery with nodifference between groups. In conclusion OPCABpatients show a protection against activation ofcoagulation and fibrinolysis and of endothelium onlyduring the intraoperative period, followed by thedevelopment of a prothrombotic pattern comparableto that of CABG patients lasting at least up to 30days after surgery.PostersHEREDITARY THROMBOPHILIAPO-121FACTOR V LEIDEN AND G20210A PROTHROMBIN MUTATIONSIN LATE PREGNANCY LOSSSantoro R, Iannaccaro P, Sottilotta G, Papaleo GCentro Emofilia, Centro di Riferimento Regionaleper la Patologia Emorragica e Trombotica Ereditaria,Azienda Ospedaliera “Pugliese-Ciaccio”, CatanzaroIntroduction: The role of inherited trombophilias inthe occurrence of unexplained recurrent spontaneousabortions, fetal loss, abruptio placentae, intrauterinegrowth restriction (IUGR) and pre-eclampsia has beenstudied and established. Although fetal loss in the firsttrimester is a common complication of pregnancy andhas many possible causes, fetal loss in the second andthird trimesters is often associated with placentalinsufficiency. In the recent years a growing number ofreports have suggested that prothrombotic moleculardefects are associated with vascular pathologies,resulting in poor gestational outcome. The aim of thisstudy was to investigate the prevalence of factor VLeiden (FVL) and factor II prothrombin mutation (PTM)in women with at least one previous intrauterine fetaldeath, comparated with the prevalence of the samemutations in our control group. Methods: We have retrospectivelyevaluated 27 women with a history ofunexplained late pregnancy loss (fetal death at 20weeks or more of gestation), with a median age of 31years, and we have comparated the prevalence of theFVL mutation and of the FIIG20210A mutation in thesewomen and in a control group of 115 healthy womenwithout history of pregnancy loss. Markers for thethrombophilias were assessed by PCR analysis. Results:The FVL mutation and the FII G20210A mutation wereshown in 6/27 (22,2%) and in 5/27 (18,5%) womenwith history of late pregnancy loss, respectively, incomparison with a prevalence of 2,6% of both FVL andFII G20210A mutations in the control group. The differencewas statistically significant (p=0,0001 for theFVL and p=0,001 for the FII G20210A ). Conclusions: Inour experience the prevalence of FVL and the FIIG20210A mutations was significantly higher in womenwith unexplained late pregnancy loss, suggesting thatthe screening for these mutations might be indicatedin this setting.haematologica vol. 89(suppl. n. 8):september 2004

156PostersPO-122GENOTYPE/PHENOTYPE CORRELATION IN ACTIVATED PROTEINC RESISTANCE DUE TO FACTOR V LEIDENRolla R, Suno A, Cau C, Albertario M, Bellomo G,Pergolini PLaboratorio di Ricerche Chimico-Cliniche,Università del Piemonte Orientale, ASO Maggiore dellaCarità, Novara, ItalyResistance to activated protein C (APC-R) is a consequenceof the resistance of activated coagulationfactor V (FV) to the degradation promoted by activatedprotein C. APC-R remains the most important causeof thrombophilia. Deficiencies of protein C (PC) or itscofactor protein S (PS) are less frequent. Resistanceusually results from nucleotide substitution (1691 GA)in the factor V gene (Leiden mutation). We studied thecorrelation between a functional phenotypic approachand a molecular genotypic approach in the diagnosisof APC-R in 220 consecutive patients simultaneouslyinvestigated for PC, PS, APC-R and for the occurrenceof Leiden mutation in FV. Pro C Global is a functionalcoagulation test (Dade Behring) for the determinationof the anticoagulant capacity of Protein C system.When used in combination with FV-deficient plasma(Pro C Global/FV), ProC Global/FV (Dade Behring) hasbeen suggested as suitable test for the determinationof FV Leiden (0.74-1.35 normalized ratio, NR). The Leidenmutation was investigated using polymerase chainreaction (PCR) and reverse-hybridization assay(Nuclear Laser Medicine srl). The mean value for APC-R was 1.04±0.17 NR, while PC and PS activities were124±30% and 83±18% respectively. 30 patients wereetherozigotes and 190 wild-type for FV Leiden. Furtheranalyses showed that etherozigotes for Leidenmutation displayed lower APC-R (0.61 NR; 95% CI0.58-0.63), below the threshold value, than wild-typepatients (1.08 NR; 95% CI 1.06-1.10). No statisticallysignificant difference was instead observed for PC orPS. The Pro C Global/FV test is then able to detect allpatients with Factor V Leiden mutation and no falsepositives has been found in the wild-type subgroup.Therefore in the thrombophilia screening, we recommendthe use of the functional test Pro C Global/FV asa preliminar approach, before the subsequent geneticdiagnosis to identify homo- or ethero-zigosity for FVLeiden or other thrombophilic mutations.PO-123ASSESSMENT OF PERFORMANCE OF CLINICAL LABORATORIESFOR DNA ANALYSES TO DETECT THREE THROMBOPHILICMUTATIONSChantarangkul V, Menegatti M, Tagliabue L,Peyvandi F, Tripodi AAngelo Bianchi Bonomi Hemophilia and ThrombosisCenter, Department of Internal Medicine, Universityand IRCCS Maggiore Hospital, Milano, ItalyThis is the second report of the Italian External QualityAssurance program for DNA analyses. The exercisewas carried out as part of the activity of the InterlaboratoryCoagulation Survey (ICS) of the CISMEL (ItalianCommittee for Standardization), which enrolls 250participants and aims at assessing the laboratory performanceof basic coagulation and thrombophilia testing.All ICS participants were asked to join this specialexercise and 52 accepted. Upon informed consent, DNAsamples were prepared at the organizing center from4 patients whose genotype was previously identified onthe occasion of thrombophilia screening. Genotypeswere as follows: 3 were homozygous mutations ofeither FVLeiden or prothrombin or MTHFR and 1 withtriple heterozygotes for FVLeiden, prothrombin andMTHFR. DNA samples were coded and stored at 4 °Cuntil shipment. The participants were provided with10 µl of DNA aliquots (400ng/L) and asked to detectthe mutations with their methods. Forty of 52 participantsreturned results. Thirty-three of 40 performedanalyses for all three mutations. Fourteen used inhouseand 26 used commercial methods. Eleven didnot complete their assessment on all samples. FVLeiden.All participants identified correctly the wild-type,2 misclassified the heterozygote as either homozygoteor wild-type and 1 misclassified the homozygote asheterozygote. Prothrombin. Two participants misclassifiedthe wild-type as heterozygote, 1 misclassifiedthe homozygote as heterozygote and 2 misclassifiedthe heterozygote as either homozygote or wild-type.MTHFR. Two participants misclassified the wild-type aseither heterozygote or homozygote, 1 misclassified theheterozygote as wild-type and 2 misclassified thehomozygote as heterozygote. Only 72.5% identifiedcorrectly all of the genotypes of 4 samples and 5-6%identified false-positive genotypes for prothrombin andMTHFR. In conclusions, this exercise shows that DNAanalyses meant to detect 3 thrombophilic mutationsare not devoid of problems. Standardization and qualitycontrol programs aimed at identifying causes offailure are warranted.haematologica vol. 89(suppl. n. 8):september 2004

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 2004157PO-124THE ROLE OF INHERITED THROMBOPHILIA IN TEN PATIENTSAFFECTED BY UPPER EXTREMITIES DEEP VENOUS THROMBOSISDi Micco P, 1 Niglio A, 1 Lucania A, 2De Renzo A, 2 Scudiero O, 3 Di Fiore R, 3 Izzo T, 1Viggiano G, 1 Castaldo G, 1 Castaldo G 3-41IV Divisione di Medicina Interna e PatologieEpato-bilio-Metaboliche Avanzate, SecondaUniversità di Napoli, Napoli; 2 Divisione diEmatologia, Università di Napoli “Federico II”, Napoli;3Dipartimento di Biochimica e Biotecnologie Medichee CEINGE-Bioteconologie Avanzate,Università di Napoli "Federico II", Napoli; 4 Facoltàdi Scienze, Università del Molise, Isernia, ItalyBackground. Congenital thrombotic risk factors hasbeen often investigated in patients affected by upperextremities deep venous thrombosis (UEDVT). Recentlywe described on a small population of oncohaematologicalpatients an increased incidence of inheritedthrombophilia. We report below the incidence of inheritedthrombophilia (MTHFR C677T, PTHRA20210G andFactor V Leiden) in ten consecutive patients referred toour division for an UEDVT. Patients and Methods. Westudied ten patients with UEDVT, eight patients bearinglymphoma (one Hodgkin’s and seven non-Hodgkin’s,two at diagnosis and six during chemotherapy;two of these six cases had implantation of a centralvenous catheter and four received Growth ColonyStimulating Factors in addition to chemotherapy) whodeveloped UEDVT, one elderly woman was affected bynon valvular atrial fibrillation (ongoing oral anticoagulationwith INR 2.91) and peritoneal metastasis andone by twice pregnancy and throacic oulet syndrome.Patients were screened for: factor V G1691A (Leiden),prothrombin G20210A, methylene tetrahydrofolatereductase (MTHFR) C677T mutations and antithrombinIII, proteins C and S plasma activity. Results. Allpatients were wild-type homozygotes for G20210A.One was heterozygote for factor V G1691A, the other6 were wild-type homozygotes. Three of the 7 patientswere homozygotes and 2 heterozygotes for the MTH-FR mutation; the remaining 2 were wild-type homozygotes.Clotting inhibitor levels were normal in allpatients. Conclusions. 50% of reported patientsshowed an inherited thrombophilia, while all patientsshowed also one or more acquired risk factors for deepvenous thrombosis. In particular, UEDVT in patientsbearing hematologic malignancies can occur irrespectiveof congenital thrombophilic alterations confirmingone more time the relevant role of malignancy andits care as acquired risk factor for UEDVT. However, ina subgroup of patients UEDVT could also depend oncongenital thrombophilic alterations and a screeningfor inherited thrombophilia can identify high riskpatients that could be specifically treated to preventthrombotic complications before to undergo to oncologicalcare.PO-125CAUSES OF THROMBOPHILIA IN PATIENTS WITH THROMBOSISAND IN INDIVIDUALS WITH A POSITIVE HEREDITARY RECORDOF THROMBOSIS WHO WERE CHECKED IN GENERAL HOSPITALOF CHANIA/CRETE/GREECESkordilaki A, 1 Samiotaki-Bisceglia F, 1 Kidona C, 1Sfiridaki A, 2 Livadiotaki A, 2 Tsagarakis N 11Transfusion Service-General Hospital of Chania,Crete, Greece; 2 Transfusion Centre-VenizelioHospital of Heraklion, Crete, GreeceThe aim of our study was to evaluate the results ofa thrombofilia control in patients with a thromboticepisode, that had no apparent predisposing factor orobstetric complication (recrudescent recurrent fetalloses, preeclampsia, eclampsia) and had been examinedin our department. 236 patients were checked: 134women and 102 men from 1/2003 until 12/2003. Meanage of women 43,97 years, of men 43,50. 29 women(21,7%) had a history of venous thrombosis (DVT, PE)34 (25,3%) an arterial thrombosis ( cerebral ischemicstroke, myocardial infraction, arterial thrombosis) 35(26,1%) obstetric complication and 36 (26,8%) had apositive hereditary record. The control included for allpatients: PT, APTT, FIB, DD, FDP, PrC, PrS, ATIII, FV, FVI-II, FX, FXII, APCR, APA, ACA, PTT-LA, PLG, PAI-1, t PA, tHCy, B12, FOLIC ACID and genetic control for FVLeiden,FIIG20210A, MTHFR C677T. DVT 28 (11,86%), PE 19(8,7%), cerebral ischemic stroke 44 (18,64%), arterialthrombosis 38 (16,1%), obstetric complication 35(14,83%) Positive Hereditary Record 72 (30,5%), NOR-MALS 11 (39,2%), 8 (42,2%), 27 (61,4%), 21 (55,26%)18 (51,42%), 34 (47,7%); Het. FVLeiden 9 (32,2%) 5(26,3%) 6 (13,73%) 5 (13,18%) 3 (8,57%) 9 (12,5%);Het. FIIG20210A 1 (3,6%) 1 (5,26%) 1 (2,27%) 2(5,26%) - 2 (2,8%) Het. FVLeiden Het.;HFR 2 (7,2%) -2 (4,54%) 2 (5,26%) 2 (5,7%) 6 (8,3%); Hom.; HFR - -2 (4,54%) 2 (5,26%) 2 (5,7%) 11 (15,3%): Het. ;HFR 5(17,8%) 2 (10,5%) 2 (4,54%) 4 (10,52%) 8 (22,9%) 9(12,5%); APA, ACA - 3 (15,8%) 3 (6,81%) 2 (5,26%) 2(5,7%) 1 (1,4%); III - - 1 (2,27%). - - - Conclusions. (i).We must suspect thrombofilia in all women with a historyof recrudescent miscarriage. The risk of obstetriccomplications in those women is even higher than inhealthy ones. (ii). The research for thrombofilia is advisablefor all those who develop a thrombotic episodewithout a predisposing factor and in those who havea positive hereditary or personal record, so that bothprecaution and treatment of thrombotic episodes canbe determined.haematologica vol. 89(suppl. n. 8):september 2004

158PostersPO-126MATERNAL-FETAL THROMBOPHILIC MUTANTS OBSERVATIONET RATIOLarciprete G,*° Angelucci PA, # Di Pierro G, §Stroppolo A, § Ameglio F, # Barbati G,* Arduini D,° §Cirese E #*AFaR, Associazione Fatebenefratelli per la Ricerca,Isola Tiberina, Rome; °Dottorato di ricerca in MedicinaPrenatale, § Università Tor Vergata, Ginecologia edOstetricia, Rome; # Ospedale Fatebenefratelli IsolaTiberina, Rome, ItalyObjectives. Aim of this study was to verify if anythrombophilic mutants could correlate with adversepregnancy outcomes. Materials and methods. 65 pregnants,matched for age (figure 1), were enrolled in 5pathology groups. The 1st helded 15 patients withthrombotic-hemorragic disorders of term pregnancyand puerperium. The 2 nd group of 16 patients compliedof hypertensive disorders as like preclampsia/eclampsia,PIH syndrome, DIC and HELLP syndrome. The 3 rdgroup had 3 pregnants with abruptio placentae. The 4 thgroup enrolled 8 patients with the US diagnosis ofIUGR. The last group was the control group, made of23 normal pregnants. MTHFR mutants C677T and 1298were evaluated along with Factor V and II mutants, bymeans of gene amplification techniques. The resistanceto the activated C protein was evaluated too. Results.The analysis of the carachteristics of the study populationsshowed a normal distribution of maternal ageand of the birthweights, (Figure 1).Figure 1. Characteristics of studied population.The categorial regression analysis underlined thatthe APCR occurrance was linked with the fetal IUGRwherever the occurrance of eterozygous mutantsMTHFR 1298 is deeply linked with a reduction in DICand abruptio placentae (Table 1).Standardized coefficientBeta Standard Error DF F Sig.APCR 0.478 0.200 1 5.585 0.022Factor V Leiden +/- -0.386 0.201 1 3.690 0.061MTHFR 1298 +/- -0.490 0.119 1 17.042 0.000Even the eterozygous factor V mutants showed thesame behaviour of the APCR, but it might be due toa strong multicollinearity effect. Discussion. We donot find any correlation between MTHFR mutantsand the IUGR, and we do find it when looking to theAPCR. It looks as not only one specific trombophilictract but a set of genes could be avaraged in determininga microvascular damage at the level of placentalvessels. The new finding is the interestingsafeting effect of the 1298 MTHFR gene. It seems tobe correlated to a decreasing rate of abruptio placentae,may be trought a faster homocysteine degradation.PO-127PREDISPOSING FACTORS IN PATIENTS WITH EARLY-ONSETCEREBRAL VEIN THROMBOSISTufano A, De Simone C, Coppola A,Macarone Palmieri N, Varricchione N,Lombardini D, Cirillo F, Cerbone AMReference Regional Centre for Coagulation Disease,Dep. of Clinical and Experimental Medicine,"Federico II" University of Naples, ItalyWe have screened the pro-thrombotic mutationsG20210A of the prothrombin (FII) gene, the Factor VLeiden mutation, the 677TT of the methylenetetrahydrofolatereductase (MTHFR) gene, and some transientfactors predisposing to thrombophilia (fastingtotal plasma homocysteine levels, oral contraceptiveuse, pregnancy/puerperium) in 20 consecutivepatients (8 men and 12 women; 34.45±12.44 yrs old)referred to our Centre because of a recently documented(CT and/or MRI) cerebral vein thrombosis(CVT). As many as 328 age- and sex-matched apparentlyhealthy subjects (133 men and 195 women;mean age 36.57±9.76 years), from the same ethnicbackground served as controls. Four out of the 20 CVThaematologica vol. 89(suppl. n. 8):september 2004

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 2004159patients (4/20; 20%) had the G20210A FII allele variant.This frequency was 4-fold higher (p=0.025 Fisher'stest; OR 4.57, CI 1.4-15.2) than that of healthycontrols (17/328; 5.2%). The frequency of the FactorV Leiden allele was 10% (2/20) in patients and almosthalf (5.8%, 19/328) in controls (p=0.34, Fisher's test).In contrast, the frequency of the omozygosity forMTHFR was 20% (4/20) in patients and 18.3%(60/328) in controls (p=0.75), and hyperhomocysteinemia(HHcy) was 19% in patients and 28% incontrols (p=0.56 Fisher's test). Within female patients,5 out of 12 developed CVT while using oral contraceptives(41.7%), and 2 out of 12 during pregnancy/puerperium(16.7%). None of the mutationsreported above was present in these 7 cases. Despitethe limitations of the sample size, these data confirmthe role of the G20210A FII mutation as well as ofFactor V Leiden in CVT; they also document that in>1/3 of cases acquired rather than inherited predisposingfactors are present in such patients.PO-128THROMBOPHILIA IN YOUNG ADULTS WITH ISCHEMIC STROKENOT RELATED TO CARDIOVASCULAR RISK FACTORSAnastasio R, Lupo E,* Grimaudo S, Bonifacio G,Malato A, Siragusa S, Mariani G**Cattedra di Ematologia, Azienda OspedalieraUniversitaria Policlinico di Palermo; *Cattedra di Neurologia,Azienda Ospedaliera UniversitariaPoliclinico di Palermo; **Dipartimento di MedicinaInterna e Sanità Pubblica, Università dell'Aquila, ItalyThe role of thrombophilia for causing ischemicstroke in young adults is still debated. During the lastyears several trials investigated the role of prothromboticconditions in this setting reporting discordantresults. During a period of 2 years (2001-2003), weevaluated 73 consecutive patients referred to ourInstitution for an episode of adult ischemic stroke inwhom common cardiovascular risk factors (cigarettesmoking, hypertension, hyperlipidemia and diabete)were not considered as responsible for the disease. Inthis setting of patients, the prevalence of coagulationabnormalities (lupus antiCoagulant –LAC-, deficiencyof anti-thrombin, protein C and protein S, elevatedlevels of FVIII and Fibrinogen, activated protein C resistance–APCR-), genetic prothrombotic risk factors (FVLeiden, FII mutation -G20210A-, C677T and A1298C5,10-methylenetethrahydropholate reductase –MTH-FR-) and fasting and post-methionine loading (PML)serum homocysteine levels were determined. One hundredthirty-eight healthy subject served as controls.The results are shown in the table: the prevalence ofcommon cardiovascular risk factors did not differbetween case and controls.Patients Controls Odds Ratio p value(95% ConfidenceIntervals)No. of patients (M/F) 73 (38/35) 138 (66/72) n.s.Mean age, y. 37.6±10.9 38.8±11.3 n.s.Age at the first event 33.7 ±10.2Risk factorsCigarette smoking 31.2% 28.7% 1.1 (0.6-1.6) n.s.Hypertension 11.4% 8.6% 1.3 (0.5-2.1) n.s.Hyperlipidemia 7.3% 6.5% 1.1 (0.2-2.0) n.s.Diabetes 1.9% 1.4% 1.4 (0.5-2.1) n.s.ThrombophiliaLAC 6.8% 1.4% 4.9 (1.7-8.19)

160PostersPO-129RISK FACTOR FOR EXTRA-HEPATIC PORTAL VEINOBSTRUCTIONBattaglioli T,* Martinelli I,* Bucciarelli P,* Reati R,°Fabris F,° Primignani M,° Mannucci PM**Angelo Bianchi Bonomi Hemophilia andThrombosis Center and °Gastroenterology Unit,Department of Internal Medicine and Dermatology,IRCCS Ospedale Maggiore Policlinico, Universityof Milano, ItalyHereditary thrombophilia is associated with anincreased risk of deep vein thrombosis (DVT), but fewinformation is avaliable on its role in patients withextra-hepatic portal vein obstruction (EHPVO). Wecarried out a case-control study on 65 patientsreferred to the Thrombosis Center (January 1994 toMay 2003) for a thrombophilia screening after a firstepisode of EHPVO and 700 healthy controls. Fivehundred patients with a first episode of lower limbDVT (LL-DVT) referred in the same study period werealso investigated. Screening for hereditary thrombophiliaand hyperhomocysteinemia was performed.Transient risk factors for thrombosis at the time ofthrombosis (for patients) or the visit (for controls)were recorded. The prevalence of thrombophilicabnormalities in EHPVO patients, LL-DVT patientsand healthy controls was respectively: factor V Leiden:3%, 16% and 2%; prothrombin mutation: 22%,11%, 3%; antithrombin, protein C and protein S deficienciestaken together: 5%, 4%, 1%; hyperhomocysteinemia:12%, 16%, 7%. Compared to controls,the odds ratio (OR) for EHPVO was 0.8 (95%CI 0.1-6.6) for factor V Leiden, 7.4 (95%CI 3.4-16.2) for theprothrombin mutation, 4.5 (95% CI 1.1-18.2) for thedeficiencies, 2.2 (95%CI 0.9-5.0) for hyperhomocysteinemia.The OR for LL-DVT was 7.5 (95%CI 4.4-13.0) for factor V Leiden and 4.8 (95%CI 1.9-12.3) forprothrombin mutation. The use of oral contraceptiveswas associated with an OR of 0.8 (95%CI 0.3-2.0)for EHPVO and 3.3 (95%CI 2.2-4.8) for LL-DVT.Forty patients with EHPVO were investigated for thepresence of myeloproliferative disorders, which werediagnosed in 22 (55%). In 8 patients (20%) a myeloproliferativedisorder and thrombophilia coexisted.In conclusion, the prothrombin mutation is associatedwith an increased risk of EHPVO whereas, atvariance with LL-DVT, factor V Leiden and oral contraceptivesuse are not. Myeloproliferative disordersare a strong risk factor for EHPVO.PostersVENOUS THROMBOEMOLISM:EPIDEMIOLOGYPO-130VENOUS THROMBOSIS OF UNUSUAL SITES: ANALYSIS OF 150PROSPECTIVE CASES FROM TWELVE ITALIAN THROMBOSISCENTRES (ITALIAN REGISTRY)Ghirarduzzi A, Ageno W, Agnelli G, Antonelli MI,Barcellona D, Dentali F, Imberti D, Iotti M,Marongiu F, Palareti G, Prandoni P, Quintavalla R,Ria L, Sabatini M, Scannapieco G, Scoditti U,Taliani MR, Tosetto ACentri Trombosi di Reggio Emilia, Albano Laziale,Bologna, Lecce, Piacenza, Padova, Parma, Perugia,Piacenza, Treviso, Varese, Vicenza; ItalyBackground. Venous Thrombosis (VT) at unusualsites (visceral and cerebral) are less frequentlyencountered events than DVT-PE but account forhigh morbidity and mortality (mostly due to intestinalinfarcts and sequaele of portal hypertension).Modern imaging techniques improved clinical understandingand allow prompt and aggressive antithrombotictherapy. Nevertheless, the natural historyand optimal duration of anticoagulant therapy inidiopathic cases are uncertain. Objective. To evaluateclinical presentation, prevalence of inherited andacquired thrombophila and current diagnostic andtherapeutic workup in both visceral (VVT) and cerebral(CVT) VT. Methods. Patients referred for VVT orCVT were considered, except patients with cancer,myeloproliferative or chronic liver disease. Allpatients were screened for ATIII, protein C, protein S,factor V Leiden, prothrombin 20210 G/A mutation,fasting homocysteine, antiphospholipid antibodies.Results. 150 patients, 75 visceral VT (mean age 46(range 18-75) years, male/female 37/38), and 75cerebral VT (mean age 37 (range 17-69) male/female17/58)were included in seven years (1997-2003).Major risk factors were: previous DVT/PE (16% inVVT), oral contraceptives use (46% in CVT), previoussplenectomy (8% in VVT). No risk factor was foundin 49% of VVT and 33% of CVT patients. Commonclinical presentations for VVT were moderate abdominalpain (58%) and surgical acute abdomen (24%),while headaches were experienced in the majority ofthe CVT cases. VVT diagnosis was carried out with CT(57%) or echocolorDoppler (31)%, while CVT diagnosiswas best established by MRI/MRA. VVT treat-haematologica vol. 89(suppl. n. 8):september 2004

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 2004161ment: surgery was performed in 20 cases (27%); 2patients underwent unsuccessful thrombolysis withrt-PA. 39% of pts were on oral anticoagulant therapyfor 6-12 months and showed 34% of recurrencesafter stopping warfarin. 28% of patients wereassigned to lifelong warfarin therapy, of which 2showed thrombosis progression into inferior cavalvein. CVT treatment: 92% of patients were on oralanticoagulant therapy for 3-12 months and showed2% of recurrences after stopping warfarin. 8% ofpatients were assigned to lifelong warfarin. Sevendeath were recorded in VVT group (death rate 5.5%per year). Acquired hypercoagulable states werefound during the follow-up: myeloproliferative disorders(15% of VVT), antiphospholipid syndrome(12% of VVT). Inherited thrombophilia was found in37% of VVT and in 33% of CVT pts: ATIII deficiency(2), protein C deficiency (3), protein S deficiency (5),factor V Leiden (9), prothrombin 20210 G/A mutation(7), hyperhomocysteinemia (9). Conclusions. In ourcase series, VVT and CVT were found to be commonlyrelated to inherited and acquired thrombophilicdisorders. VVT showed a high recurrency rate afterwithdrawal of antithrombotic therapy.PO-131GENETIC AND ACQUIRED THROMBOPHYLIC FACTORS INPATIENTS WITH UNUSUAL VENOUS THROMBOSISRupoli S, Pulini S, Barulli S, Da Lio L,° Logullo F,*Scortechini AR, Tassetti A, Ravaglia F,° Leoni PClinica Ematologica, *Clinica Neurologica, °LaboratorioAnalisi; Università Politecnica delle MarcheAzienda Ospedaliero Universitaria Ospedali RiunitiUmberto I-G.M. Lancisi-G. Salesi, Ancona, ItalyUnusual venous thrombosis are frequently associatedwith genetic and acquired risk factors. We evaluatedthe frequency of such factors in 6 patients(median age 38 years, range 26-56) with cerebralvenous thrombosis (CVT) and in 9 patients (medianage of 41 years, range 18-56) with splancnic veinthrombosis (SVT). Among the first group a completethrombophylic screening was performed in 5/6. Onlyone patient had no risk factors. The others presented,respectively: 1)APC resistance and high plasmalevels of coagulation factor VIII together with oralcontraceptive therapy (OCT); 2) LAC/ACA antibodies,factor V Leiden with OCT; 3)OCT and breast cancer;4)essential thrombocytemia (ET). Among the patientswith SVT only one patient had no risk factors. Threepatients presented a combination of genetic andacquired risk factors: factor V Leiden and OCT;G20210A prothrombin gene mutation and OCT;hyperhomocysteinemia and ET. Another woman, intherapy with OC, was diagnosed has having ET at thetime of SVT. With regards to the remaining patients,one had various genetic polymorphism of fibrinogen,PAI and coagulation factor XIII genes; one wasaffected by idiopathic myelofibrosis; one was affectedby ET and one by myelo-monocitic chronicleukemia. The majority of these 15 patients wasquickly treated after diagnosis with LMWH and warfarin.The outcome of thrombosis was: complete resolutionin 10/13 valuable patients; an unfavourableresult in one; 2 patients are still in treatment; 2patients were lost at follow-up. At present no recurrenceshave been observed. Patients with unusualvenous thrombosis need an accurate research of anunderlying myeloproliferative disorder or othermalignancy in order to optimise treatment for boththe thrombosis and the baseline disorder. Despite thesmall sample size, our data suggest that geneticthrombophilias are often associated with predisposingfactors. xAmong them, OCT seems to be animportant determinant in the global thrombotic riskof CVT and SVT and chronic myeloproliferative disordersin the pathogenesis of SVT.PO-132VENOUS THROMBOEMBOLIC DISEASE: CLINICAL FEATURES OF1282 PROSPECTIVE CONSECUTIVE CASES AT ANGIOLOGY UNITOF REGGIO EMILIAGhirarduzzi A, Iotti M, Cattabiani L, Ieran M,Iorio A,* Silingardi M, Iori ISSD Angiologia and Thrombosis Centre, Dpt areaMedica 1^, Arcispedale Santa Maria Nuova,Reggio Emilia.*Dept Internal Medicine, Universitàdi Perugia, ItalyBackground. In the last decade the validation ofcompressive ultrasonography, D-dimer testing andpre-clinical probability assessment for the diagnosisand the availability of low molecular weight heparinfor the prophylaxis and initial treatment of venousthromboembolism (VTE), have both induced significantchanges in the management of proximal deepvenous thrombosis (DVT) and pulmonary embolism(PE). Aim of the study. We referred to the registriesof the Angiology Unit of Reggio Emilia, built up in1997, to trace the effect of the availability of preferreddiagnostic pathways, adoption and maintenanceof prophylaxis-therapeutic guidelines, progressiveincrease of home treatment and reductionlenght of hospital stay on the epidemiology and naturalhistory of VTE. Patients and methods. Wereviewed all the patients admitted for VTE to ourhospital between January 1997 and December 2003.Data were collected from Angiology Unit data baseand cross-matched with those of hospital data base(DRGs codes 451 and 411). All the patients with ahaematologica vol. 89(suppl. n. 8):september 2004

162Postersfirst VTE episode were enrolled and prospectively followed-upto estimate mortality, recurrences and longterm sequelae (pulmonary hypertension and postthromboticsyndrome). Results. 1282 patients wereenrolled (729 male / 553 female, mean age 70 years,range 18 – 96) with estimated incidence in the generalpopulation of 1.07 cases/1,000/year. The maincharacteristics of study population are shown in thetable.DVT DVT+PE Isolated Massive Unusual Idiopathic Surg/Orthp.PE PE Site VT VTE VTE(or RVD)558 210 448 97 66 610 358Cancer Intern./CO Recurrent Cr.Pulm. Fatal Total MajorVTE VTE VTE Hypert. PE Deaths Bleedings279 148/30 163 17 29 129 32Conclusions. During the seven years of follow-upwe observed a steadiness in VTE incidence rate, withsignificant reduction both in incidence and in deathrate massive-PE related. Cancer-VTE increased withconsistent effect on late recurrence, mortality andhemorrhagic events.PO-133INCIDENCE OF POSTOPERATIVE PULMONARY EMBOLISMDETECTED BY MULTI-SLICE COMPUTERIZED TOMOGRAPHYIN LUNG SURGERY FOR CANCERMilillo G, Daddi G, lupattelli L, Ragusa M, Lemmi A,Puma F, lomonaco A, Agnelli G for The PulmonaryEmbolism In Thoracic Surgery (PETS) Study GroupDivisions of Internal and CardiovascularMedicine,Thoracic Surgery and Radiology,University of Perugia, ItalyThe incidence of pulmonary embolism (PE) inpatients undergoing lung surgery for cancer remainsundefined. In these patients the risk for venous thromboembolismis potentially high due to the combinationof prothrombotic effects of cancer, extensivesurgery, prolonged immobilization and parietal andendothelial local alteration consequent to surgery. Theaim of this study was to assess the incidence of PEafter lung surgery for cancer using multi-slice computerizedtomography (MSCT). Methods: Consecutivepatients undergoing surgery for lung cancer wereincluded in this study. PE was detected by MSCT (GELight-Speed 4×1,25) scan performed 7-15 days aftersurgery. Diagnostic criteria for PE were complete orpartial intraluminal filling defect. Patients werescheduled to receive pharmacological prophylaxis forvenous thromboembolism with low-molecular-weightheparin (LMWH) starting the first post-operative dayand continued until discharge. Results: Fifty patientswere included in this study. The average age was 66,5(range 26 to 90). Diagnosis of cancer was confirmedin all except one patient found to be affected bytuberculosis. Thirty-six patients underwent lobectomy,11 pneumonectomy, and the remaining 3 patientswedge resection. Histology showed epidermoid carcinomain 20 cases, adenocarcinoma in 15, anaplasticcarcinoma in 7, and other types in the remaining 8cases. All patients but two received prophylaxis forvenous thromboembolism. Seven patients (14%)showed PE at MSCT scan. Of the PE, 5 involved centralarteries (principal, lobar and segmentary) and 2subsegmentary arteries. Two of the patients withMSCT scan detected PE were symptomatic. PE is acommon complication in patients undergoing lungsurgery for cancer, despite antithrombotic prophylaxis.In such high-risk population prophylactic regimensshould be optimized.PO-134CARDIOVASCULAR RISK FACTORS AND THE RISK OF VENOUSTHROMBOEMBOLISMTufano A, Varricchione N, Coppola A, Cirillo F,De Simone C, Meola M, Lombardini D,Sidiropulos M, Albisinni R, Cerbone AMRegional Reference Centre for Coagulation Disease,Dep. of Clinical and Experimental Medicine;Federico II University, Naples, ItalyThe association between established cardiovascularrisk factors and the risk of venous thromboembolism(VTE) is not entirely understood. We havescreened for cigarette smoking, hypertension, hypercholesterolemia,diabetes mellitus, obesity, andhyperhomocysteinemia 397 consecutive patients(172 men and 225 women; 42.69±14.89 yrs) referredto our Centre because of recently documented venousthromboembolic events (deep venous thrombosisand/or pulmonary embolism, and venous thrombosisin abdominal veins, and cerebral vein thrombosis). Asmany as 407 age- and sex-matched apparentlyhealthy subjects (164 men and 243 women;39.53±13.57 yrs), from the same ethnic backgroung,served as controls. Arterial hypertension was presentin 18.5% of VTE patients and in 12.8% of controls(p=0.026; OR 1.5, 95% CI 1.1-2.3). Hypercholesterolemia(total cholesterol levels >5.2 mmol/L inrepeated evaluations over a 1-yr period) was morecommon in VTE patients than in controls (53.6% vs.39.1%, p=0.001, OR 1.8, 95% CI 1.3-2.5), as was BMI>25 (59.5% vs 40.4%; p

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 2004163plasma levels >15 µmol/L; 32.5% vs. 33.0%) and ofdiabetes mellitus (6.6% vs. 3.6%) did not differ fromcontrols. In conclusion some established cardiovascularrisk factor (hypertension, hypercholesterolemiaand overweight/obesity) may have a role in VTE, suggestingthat treatment and/or prophylaxis of theseconditions may contribute to primary and secondaryprevention of VTE.PO-135TISSUE FACTOR PATHWAY INHIBITOR (TFPI) AND THROMBINACTIVABLE FIBRINOLYSIS INHIBITOR (TAFI) IN JUVENILE DEEPVEIN THROMBOSISRogolino A,* Cellai AP,* Blagojevich J, Marcucci R,Gori AM, Cesari F, Lenti M, Sestini I, Abbate R,Gensini GF, Prisco DDepartment of Medical and Surgical Critical Area,University of Florence, Florence, Italy; *Dipartimentodel Cuore e dei Vasi, Azienda OspedalieraUniversitaria CareggiLow levels of tissue factor pathway inhibitor (TFPI)and elevated levels of thrombin activatable fibrinolysisinhibitor (TAFI) have been recently reported to beassociated with an increased risk of deep venousthrombosis (DVT). Scarce data are available on therole of these parameters in juvenile DVT. We investigated116 patients with a first episode of DVT before50 years (median age 38, 17-50 yrs; 51 males/65females) and 60 apparently health subjects comparablefor age and sex. TFPI and TAFI plasma levels weremeasured by ELISAs (Asserachrom total- and free-TFPI, Diagnostica Stago; Asniere sur Seine, France;COALIZA TAFI, Chromogenix, IL, Milan, Italy). Venousblood samples were collected at least 6 months afterthe acute event. No patient was under anticoagulanttreatment for at least three months. Free-TFPI plasmalevels were not significantly different betweenpatients and controls (10.7, 7-28.2 ng/mL vs 11.5, 5.6-48.7 ng/mL). Ten percent of healthy subjects had TFPIfree antigen levels below 7.1 ng/mL (10 th percentile).A similar percentage (10.3%) of patients had free-TFPI below this cut-off. Total-TFPI plasma levels werenot significantly different between patients and controlsalthough a trend to higher levels was observedin patients (78.6, 42.7-190.9 ng/mL vs 69.8, 42.1-198.1 ng/mL). Ten percent of healthy subjects had TFPItotal antigen levels below 51.2 ng/mL (10th percentile).A similar percentage (7.5%) of patients hadtotal-TFPI below this cut-off. As TAFI plasma levels areconcerned, no significant differences were foundbetween patients and controls (103, 47.6-262.9% vs102, 57-233%). Ten out of 116 DVT patients (8.6%)had TAFI levels above 90 th percentile of control distribution(150.4%). These preliminary results do not supporta relevant role for TFPI defect and TAFI excess injuvenile DVT. Further studies are needed on a largernumber of patients and healthy controls to betterassess the utility of their determination in the clinicalmanagement of patients with DVT.PO-136USE OF BLOOD SALVAGE AND RE-INFUSION IN PATIENTSUNDERGOING TOTAL HIP ARTHROPLASTY AND TOTALKNEE ARTHROPLASTY AND POSSIBLE CORRELATION WITHVTE COMPLICATIONSFilippucci E,* Agnelli G,* Gallus AS,° Kuznetsova P, §Anderson FA Jr § for The Glory Investigators*Dipartimento di Medicina Interna, Università diPerugia, Perugia, Italia; °Flinders Medical Centre,Bedford Park, South Australia, Australia; § Centerfor outcomes research, University of MassachusettsMedical School, Worcester, MA, USAIntroduction: In patients undergoing major orthopedicsurgery, the risk of viral transmission associatedwith blood transfusion promoted the developmentof perioperative blood salvage and other alternativesto donor blood transfusion. Recent data suggestthat perioperative salvage and re-infusion ofunwashed whole blood is associated with an activationof blood coagulation in patients undergoingtotal knee arthroplasty (TKA). We analysed the use oftransfusion and blood re-infusion in patients undergoingTKA and total hip arthroplasty (THA). Methods:Patients undergoing THA or TKA between January2002 and December 2003 in 68 hospitals located in11 countries were enrolled in the GLORY registry. TheGLORY registry is a large prospective study on theoutcome of patients undergoing major orthopedicsurgery. Data on VTE events were collected aftersurgery and at 3 months following the hospital discharge.Results: Of 4995 patients, 2364 (47.3%)underwent THA and 2631 (52.7%) underwent TKA.Blood salvage was used in about one fourth ofpatients (26%), and its diffusion is higher in USA incomparison to non-USA countries (37% vs 16%,respectively). Among blood salvage procedures, postoperativere-infusion is the most used (postoperative14.8%, intraoperative 1.0%, both 9.5%). Unwashedblood is the re-infusion procedure most largerly usedin USA, in comparison to washed blood (93.9% vs5.8%), while in the non-USA countries the use ofthe unwashed and washed devices is quite similar(43.3% vs 39.8% respectively). 64% of THA and 59%of TKA patients received LMWH as VTE prophylaxis.Conclusion: Re-infusion procedures are frequentlyused in patients undergoing THA and TKA. No dataare currently available about the incidence of venousthromboembolism in patients undergoing perioper-haematologica vol. 89(suppl. n. 8):september 2004

164Postersative salvage and re-infusion of shed blood. The followup of GLORY patients will provide informationsabout the correlation between the use of re-infusionprocedure and occurrence of VTE complications.PO-137THE FIRST AMBULATORY SCREENING ON THROMBOEMBOLISM(FAST) STUDY: A MULTICENTRE, CROSS-SECTIONAL,OBSERVATIONAL STUDY ON THROMBOEMBOLIC RISK FACTORSDi Minno G,* Tufano A,* Palareti G,° Moia M,^Giudici GA,** Baccaglini U,^^ Mannucci PM^ onBehalf Of The FAST Study*Centro di Riferimento Regionale per leemocoagulopatie. II Policlinico Di Napoli. UniversitàFederico II, Napoli.°Dipartimento di Angiologia, UniversityHospital S. Orsola Malpighi, Bologna. ^AngeloBianchi Bonomi Hemophilia and thrombosis Center,Dip. Medicina Interna IRCCS Osp. MaggioreMilano and University of Milan. **Sanofi-Synthelabo.^^OspedaleBusonera, Padova, ItalyDesign: multicentre, cross-sectional, observationalstudy. Objectives: to assess the prevalence of riskfactors for venous thromboembolism (VTE) and theprevalence of recent VTE (within 1 year) in subjectswho consulted their General Practitioner (GP) for ahealth disorder. Setting: 1,536 GP surgeries. Participants:15,180 adult, co-operative subjects, who consultedtheir GP for a health disorder and signed theinformed consent form. Main outcome measures:prevalence of known thromboembolic risk factors,graded according to importance (strong, moderate,weak risk factors), and of recent VTE events (within1 year), based on interviews. Results: about 1:5patients had at least one strong risk factor for VTEand about 1:20 had at least two, with no differencebetween sexes; their prevalence increased with age.Most were related to medical conditions: history ofsuperficial venous thrombosis (VT) and/or deepvenous thrombosis (DVT)/pulmonary embolism, heartfailure and malignancy. About 3:4 women and 2:3men had at least one moderate to weak risk factor;nearly 1:2 women and 1:3 men had at least two. Themost common were history of VTE, smoking, historyof miscarriage, oestrogen therapy, obesity and varicoseveins. Overall, 80% women and 67% men hadat least one risk factor and 50% women and 35%men had at least two risk factors. The prevalence ofrecent VTE was 3.4% in women and 2.4% in men,and increased with age. The majority of cases weresuperficial VT, in both sexes (2.5% in women, and1.5% in men). Conclusions: the prevalence of riskfactors for VTE in patients attending GP surgeries ishigh. GPs should bear this in mind during their dailypractice.PO-138LOW PLASMA LEVELS OF PYRIDOXAL-5’-PHOSPHATE ARE ARISK FACTOR FOR RECURRENT VENOUS THROMBOEMBOLISMCattaneo M, 1,2 Kyrle PA, 3 Lombardi R, 2Weltermann A, 3 Lecchi A, 2 Eichinger S 31Hematology and Thrombosis Unit, Ospedale SanPaolo, Dept. of Medicine, Surgery and Dentistry,and, 2 A.Bianchi Bonomi Hemophilia and ThrombosisCenter, IRCCS Ospedale Maggiore, University ofMilano, Italy; 3 Dept. of Internal Medicine I,Division of Hematology/Hemostasis, University ofVienna, AustriaIn a case-control study, we showed that low plasmalevels of pyridoxal-5’-phosphate (PLP), the coenzymeform of vitamin B6, are associated with aheightened risk for venous thromboembolism (VTE).This association was independent of known risk factorsfor VTE, including high plasma levels of homocysteine,which is under the metabolic control of Bvitamins, including vitamin B6. In a prospective,cohort study, we investigated whether or not lowplasma levels of PLP are associated with a heightenedrisk for recurrent VTE. 766 patients with a singleepisode of VTE were prospectively followed for37±31 (mean±SD) months after discontinuation oforal anticoagulants (OA). The outcome events studiedwere deep-vein thrombosis (DVT) and/or pulmonaryembolism (PE). Testing for known thromboticrisk factors was performed 3 weeks after discontinuationof OA. PLP was measured by the tyrosinedecarboxylase method in the same EDTA-anticoagulatedplasma samples that were used for totalhomocysteine measurement 6+9 months after discontinuationof OA. Recurrent VTE occurred in 98patients. The mean plasma levels of PLP were56.3±59 nmol/L in patients with recurrences and64.4±72.3 in patients without recurrences (P=0.1).VTE recurrences were observed in 22/117 patientswith plasma levels of PLP that were lower than the15th percentile and in 76/649 patients with plasmaPLP levels higher than the 15th percentile. The cumulativeprobability of VTE recurrence was 26% (95%CI: 16-37%) in patients with low PLP levels and 17%(13-21%; p =0.05) in those with normal PLP levels.The crude relative risk of recurrent VTE in patientswith low PLP levels was 1.60 (0.99-2.57) andremained practically unmodified after adjustmentfor VTE risk factors, including hyperhomocysteinemia:1.5 (0.92-2.5). Therefore, low plasma levels ofPLP are associated with a heightened risk for recurrentVTE, independently of other VTE risk factors,including hyperhomocysteinemia.haematologica vol. 89(suppl. n. 8):september 2004

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 2004165PO-139THROMBOPHILIA IS FREQUENT IN SUPERFICIAL VEINTHROMBOSIS AND IS ASSOCIATED WITH RECURRENCE:A RETROSPECTIVE STUDYMarcucci R, Ciuti G, Blagojevic J, Lenti M, Sofi F,Fatini C, Sticchi E, Gazzini A, Evangelisti L,Gensini GF, Abbate R, Prisco DDipartimento Area Critica Medico Chirurgica,Azienda Ospedaliero-Universitaria Careggi,FirenzeSuperficial vein thrombosis (SVT) is a common diseaseand the recent recognition of its associationwith deep venous thrombosis (DVT) and pulmonaryembolism has revived the interest in this disease. Aimof our study was to determine the thrombophilic riskprofile of patients with SVT by evaluating: Factor VLeiden, prothrombin polymorphism, physiologicalclotting inhibitors, antiphospholipid antibodies,homocysteine, plasminogen activator inhibitor-1(PAI-1) and lipoprotein (a). The study population consistedof 183 patients (138 F/ 45 M; age 42 (15-74)yrs) and 183 age and sex matched (138 F/ 45 M; age42 (15-74) yrs) controls. At the multivariate analysisadjusted for age, sex, circumstantial and thrombophilicrisk factors, independent risk factors for SVTwere: factor V Leiden (OR 12.3 (95% CI 5.0-30.2),p

166PostersPO-141CYTOMEGALOVIRUS INFECTION AND ACUTE THROMBOSISSquizzato A, Gerdes VEA, Büller HRDepartment of Vascular Medicine, AcademicMedical Center, University of Amsterdam,Amsterdam, The NetherlandsDifferent infectious pathogens have been advocatedas responsible agents for thrombotic disorders.Cytomegalovirus (CMV) is one of the infectiousagents supposed to play a role in apparently unprovokedacute thrombosis. In vitro studies show a clearprocoagulant influence of CMV, either directly or byinfection of endothelial cells and monocytes. However,the frequency of thrombotic complications inCMV-infected patients is unknown, since welldesignedstudies are lacking. METHODS. We performedan extensive search in the all-languages literaturethroughout the MEDLINE and EMBASE databases,utilizing the terms of cytomegalovirus, CMV,and thrombosis to identify all published case reportsof acute thrombosis occurring during an acute CMVinfection. A detailed review of the references completedthe search. We identified 36 cases of acutethrombosis in patients with acute CMV infection, 15in immunocompromised and 21 in immunocompetentpatients. Venous thrombosis was present in 29patients, arterial thrombosis in 9, and both in 2.Mean age was 39.3 years (range: neonatal to 74),and 66% of the patients was male. HIV infection waspresent in 50% of the immunocompromised patients.A positive association between positive acute CMVinfection serology and early and late hepatic arterialthrombosis after liver transplantation is alsoreported. Our systematic review of case reports indicatesthat thrombosis is a possible and not rare complicationof acute CMV infection. A possible role ofsubclinical CMV infection in apparently unprovokedarterial and venous acute thrombosis is unclear. Adequateprospective studies evaluating the role of CMVas risk factor for thrombosis are warranted.PO-142INCIDENCE OF DEEP VEIN THROMBOSIS IN PATIENTS WITHCOPD AT I AND II STAGE OF GOLD CLASSIFICATIONLessiani G,° Shamasbloo M, Santozzi M, Laglia G,Febo F, Grazziosetto R, Franzone G, Gentili M,D'Amico M, Di Bernardo C,° Feliciani MDepts. of Internal Medicine and Angiology Unit°,Pescara, ItalyThere are increasing data in literature on the roleof the deep vein thrombosis (DVT) as a complicationof numerous medical conditions. Patients group withinternistical deseases presents an intermediate risk(15%). Nevertheless in this group of patients lots ofheterogeneous pathologies are represented. In theCronic Obstructive pulmonary desease (COPD)patients there aren’t till now certain datas. The DVTrisk in patients with COPD fluctuates between valuesof 10%-20%,untill 28% in decompensatedCOPD. Target of our study is to verify with venousultrasonography the presence of DVT, in the affectedpatients from mild-moderate COPD curbedaccording to the GOLD classification. We tested 100consecutive patients, arrived at our institute forexacebation of COPD between Jannuary and May2003 (average : 68.02 years; 61 male and 39 female).All were checked with spirometry, Rx Thorax, bloodgas test to diagnose COPD I-II (A ;B) stadium (GOLDI:1) VEMS/CVF< 70%, 2) VEMS>80% of the abovementioned,3) with or without clinical symptoms.GOLD II:1) VEMS/CVF

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 2004167PO-143THROMBOPHILIA IN PATIENTS WITH SPINAL CORD INJURYMarcucci R, Ricci L,* Fedi S, Alessandrello Liotta A, #Cesari F, Evangelisti L, Falciani M, Ferrini S,Gazzini A, Ilari I, Aito S,* Abbate RDipartimento Area Critica Medico Chirurgica;#Dipartimento Cardiologico e dei Vasi; *UnitàSpinale, Azienda Ospedaliero-Universitaria Careggi,Florence, ItalyDeep venous thrombosis (DVT) and pulmonaryembolism are major causes of morbidity and mortalityin patients with spinal injuries. A wide rangeof thromboprophylactic measures have been proposedbut the optimum treatment and duration forthromboprophylaxis is unknown. No data are availableon the role of thrombophilic risk factors. Aim ofour study was to determine the thrombophilic riskprofile of patients with spinal cord injury (SCI) withor without a previous deep venous thrombosis byevaluating: antithrombin, protein C ans S, factor VLeiden, prothrombin polymorphism, homocysteine(Hcy), plasminogen activator inhibitor-1 (PAI.1) andlipoprotein(a) (Lp(a)). We studied 46 patients withSCI without DVT (group A) (35 M/11 F; age: 50.5 (21-82) and 43 patients with SCI and a previous episodeof DVT (group B) (34 M/9 F; age: 50 (13-78). Nopatient had a deficiency of clotting inhibitors, and inonly 1 patient of group A we found the presence ofprothrombin polymorphism. Lp(a) levels were similarin the two groups (124 (1-487) mg/L vs 84.5 (1-1079)mg/L; p=ns). Hcy and PAI-1 levels were significantlyhigher in group B with respect to A (Hcy: 12.3(6.2-30.0) µmole/L vs 18.7 (5.4-35) µmole/L;p=0.003/ PAI-1: 15.4 (1-40) IU/mL vs 10 (1-36.8)IU/mol; p=0.008). At the multivariate analysisadjusted for age, sex, smoking, hypertension, dyslipidemiaand BMI, independent risk factors for DVTin patients with SCI were hyperHcy (OR=3.6 (95%CI1.4-9.5), p=0.007) and high levels of PAI-1 (OR= 4.9(1.7-14); p=0.002). Our data demonstrate thathyperHcy and impaired fibrinolysis documented byhigh levels of PAI-1 are independent risk factors forthe occurrence of DVT in patients with SCI, suggestingthe possible benefit of a vitamin supplementation.PostersVENOUS THROMBOEMOLISM:DIAGNOSIS & THERAPYPO-144ACUTE ILIO-CAVAL THROMBOSIS ASSOCIATED WITH HIGHFEVER: A DIFFICULT CLINICAL PRESENTATIONFlorioi A,* Tripodi MF,° Andreana L°*Vascular Surgery, °Internal Medicine 2 nd Universityof Naples, Medical School, Naples, ItalyIntroduction: Behçet disease is a systemic vasculitisin which few diagnostic features, i.e. oral andgenital ulcers, are often associated with differentclinical manifestations, the variability of which maymislead the diagnosis. We report on a case of Behçetdisease which was difficult to diagnose and severe inits clinical presentation. Case report: A male,19 years,presented with an history of high fever,up to 40°Csince the last 2 months, generalized arthralgia,fatigue, anorexia.He was referred to our unit formassive ilio-caval thrombosis after he had beenexhaustively screened for infectious, neoplastic andautoimmune disease Echoduplex showed bilateralobstrucion of iliac veins and complete obstruction ofinferior cava up to the renal vein origin.Moreovercavography showed involvement of left renal vein,partial obstruction of retrohepatic cava (lumen size< 2 cm) up to the right atrium, the blood flow beingshunted to the right renal vein. Laboratory data:VES,117; fibrinogen,800mg/dl; PCR, 22mg/dl; d-Dimers,16mg/mL ; serum iron,4mg/dl; C-protein, S-protein, Antithrombin III, Fattore V Leiden, antiphospholipids,no abnormalities. A caval filter was notjudged technically suitable and clinically appropriate.The patient was treated with warfarin, elastic compressionbandage and steroids with resolution ofedema, pain and fever. One year later, the patientstopped maintenance steroids and again experiencedhigh fever, arthralgia, oral and genital ulcers; laboratorydata refelected acute inflammatory response;MR-angiography showed an unchanged venousobstruction with development of collaterals. The historyrevealed that oral and genital ulcers hadappared in the past as well. Behçet disease was diagnosedsince Duffy criteria were fullfilled (oral andgenital ulcers, arthralgias, deep venous thrombosis).Conclusions: the clinical presentation of deep venousthrombosis, fever and acute inflammation shouldarise the diagnostic suspicion of Behçet disease.haematologica vol. 89(suppl. n. 8):september 2004

168PostersPO-145COMPLETE COMPRESSION ULTRASONOGRAPHY OF THE LEGVEINS AS A SINGLE TEST FOR THE DIAGNOSIS OF DEEP VEINTHROMBOSIS: A RETROSPECTIVE STUDYCaponi C, Agnelli G, Felici ADivision of Internal and Cardiovascular Medicine,Department of Internal Medicine, University ofPerugia, ItalyBackground. Noninvasive diagnosis of deep veinthrombosis (DVT) is based on ultrasound examinationof the leg veins, usually restricted to compression ofthe proximal veins (CUS). However, due to the limitedaccuracy of CUS for the diagnosis of calf DVT, serialtesting was mandatory to rule out the extension ofcalf vein thrombosis to proximal veins (serial CUS, D-dimer). Material and methods. This retrospective clinicaloutcome study included consecutive patients whoreceived a single complete compression ultrasound ofall proximal and distal veins (CCUS) as the only diagnostictest. Patients with a clinical suspicion of DVTwere seen at our Thrombosis Centre from August 2003to December 2003. Patients in whom CCUS findingswere negative were follow up for the occurrence ofsymptomatic and objectively confirmed venousthromboembolism in the 3 months after the indexCCUS evaluation. Results. Overall, 234 patients wereincluded in the study. CCUS revealed a DVT in 90patients (38%). Among the patients in whom CCUSwas negative, three (2%) experienced a symptomaticobjectively confirmed venous thromboembolic eventwithin the 3 months after the index CCUS evaluation.VTE was DVT in 1 patients and PE in 1 patients. Weconclude that the CCUS protocol is relative safe withrespect to excluding DVT, thereby reducing the diagnosticworkup of patients with suspected DVT to asingle ultrasound examination.PO-146A SURVEY OF THROMBOPROPHYLAXIS MANAGEMENTIN PATIENTS WITH MAJOR TRAUMAImberti D, Dentali F,* Ageno W*Department of Internal Medicine, Piacenza Hospitaland *Department of Internal Medicine, University ofInsubria, Varese, ItalyVenous thromboembolism (VTE) is a common lifethreateningcomplication of major trauma and optimalthromboprophylaxis management is still challengingin this specific group of patients, in spite ofboth very high thrombotic and bleeding risk. Clinicalguidelines clearly recommend routine thrombosisprevention with low molecular weight heparin(LMWH) in patients with major trauma in absence ofcontraindications due to concomitant haemorrage;however, there is still a lack of strong recommendationsabout the timing and the modality of LMWHadministration and the role of mechanical prophylaxisin case of bleeding events. We tested thehypothesis that there is significant practice variationin thromboprophylaxis management of patientswith major trauma among Intensive Care Unit (ICU)specialists. Two hundred Italian ICU specialists, representing200 ICU throughout the country, were contactedby telephone and were asked a) if they routinelyprescribe antithrombotic prophylaxis inpatients with major trauma, if prophylaxis is prescribedto all patients or to selected patients, thetype of prophylaxis and the timing of administrationb) if they recommend physical prevention and if it isprescribed to all patients or to selected patients, thetype of physical prophylaxis. In patients with majortrauma, 85% of the interviewed ICU specialistsanswered they prescribe pharmacologic prophylaxisfor VTE. Of them, 37.6% prescribes prophylaxis onlyto selected patients based on the level of risk, 87.7%prescribes LMWH, 42.4% starts propylaxis immediatelyafter hospitalization. Only 61% of the interviewedspecialists prescribes physical prophylaxis ; ofthem 82.8% uses elastic stokings (ES), 9.8% InferiorVena Cava (IVC) filter, 7.4 other mechanicaldevices. 41% of them prescribes physical prophylaxisin all the patients, 59% only in case of contraindicationto pharmacological prevention. 47%would prescribe IVC filter insertion if anticoagulationin contraindicated; of them 91.4% recommends IVCfilter only if deep vein thrombosis (DVT) is demonstrated.Even in the presence of clinical guidelines,prescription of VTE prevention with LMWH inpatients with major trauma is underused and timingand modality of prophylaxis are rather heterogenous;if anticoagulation is contraindicate, IVC filter arecommonly recommended only in the presence of DVT.PO-147A SURVEY OF THROMBOPROPHYLAXIS MANAGEMENTIN MAJOR ORTHOPAEDIC SURGERYImberti D, Dentali F,* Ageno W*Department of Internal Medicine, Piacenza Hospitaland *Department of Internal Medicine, University ofInsubria, Varese, ItalyVenous thromboembolism (VTE) is a common lifethreateningcomplication of major orthopedic surgeryof the lower limbs. Low-molecular-weight heparins(LMWHs) offer in this group of patients the greatestefficacy with an acceptable risk of bleeding and clinicalguidelines clearly recommend their use in routinethromboprophylaxis; however, optimal timing ofhaematologica vol. 89(suppl. n. 8):september 2004

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 2004169treatment initiation and the modality of LMWHadministration in case of regional anesthesia are stillmatter of debate. We tested the hypothesis that thereis significant practice variation in thromboprophylaxismanagement of patients undergoing majororthopaedic surgery among anesthesiologists. Twohundred Italian anesthesiologists, representing 200anesthesiology departments throughout the country,were contacted by telephone and were asked a) incase of general anesthesia, if they routinely prescribeantithrombotic prophylaxis in major orthopaedic surgicalpatients, the type of prophylaxis and the timingof initiation b) in case of regional antesthesia, ifthey use thomboprophylaxis, the type and the timingof initiation of treatment and the most importantcriteria they evaluate to reduce the risk of spinalhematoma. Results: 95.5% of the interviewed anesthesiologistsanswered they prescribe pharmacologicprophylaxis in case of general anesthesia in majororthopaedic surgery. Of them 97.7% prescribesLMWH, 89.5% starts prophylaxis preoperatively, 8%postoperatively, only 2.5% perioperatively. 69% ofthe interviewed specialists routinary uses regionalanesthestesia; 86.5% of them prescribes pharmacologicprophylaxis in all the patients, 97.7% usesLMWH and 67% starts prophylaxis preoperatively.The interviewed specialists answered that the mostimportant criteria they evaluated to reduce the riskof spinal hematoma are the diameter of the needle/catheters(54.3%), the timing of prophylaxisadministration related to the spinal/epidural procedure(53.8%), the presence of hemostatic balanceimpairment (50.3%). Conclusions: LMWHs are thecommonest drugs used in thromboprophylaxis inpatients undergoing major orthopaedic surgery;according to European guidelines, treatment usuallystarts preoperatively, regardeless of the use of generalor regional anesthesia.PO-148THROMBOPROPHYLAXIS FOLLOWING MINOR ORTHOPEDICSURGERY: RESULTS OF A SURVEY ON CLINICAL PRACTICEPATTERNSAgeno W,* Dentali F,* Romualdi E,* Imberti D°*Department of Clinical Medicine, Universityof Insubria, Varese, and °Department ofMedicine, Piacenza Hospital, Piacenza, ItalyThe risk of venous thromboembolism (VTE) followingminimally invasive joint surgery or in patientswith lower limb plaster cast immobilization is poorlydefined. Clinical guidelines usually omit recommendationson thromboprophylaxis in this setting.We hypothesized that there is significant practicevariation amongst orthopedic surgeons in the use ofantithrombotic prophylaxis in such patients. Twohundred Italian orthopedic surgeons, representing200 orthopedic departments throughout the country,were contacted by telephone and were asked ifthey routinely prescribe antithrombotic prophylaxisin patients undergoing knee arthroscopy, in patientswith lower limb fractures, and in patients with lowerlimb plaster cast immobilization, if prophylaxis isprescribed to all patients or to selected patients, thetype of prophylaxis and the timing of administration.Following knee arthroscopy, 94% of the interviewedorthopedic surgeons prescribed pharmacologicprophylaxis for the prevention of VTE. Of them,only 17% prescribed prophylaxis based on thepatients risk stratification, 97.3% prescribed LMWH,59% started prophylaxis 12 hours before the procedure,and 59% prescribed prophylaxis for more than10 days. In patients with lower limb fractures, 94.5%prescribed some form of VTE prophylaxis. Of them,93.1% prescribed prophylaxis to all patients, regardlessof individual risk stratification, 96.8% usedLMWH, 74.6% started prophylaxis at the time oftheir diagnosis, and 55% prolonged prophylaxis untilthe patient returned to normal weight bearing. Inpatients with plaster cast immobilization, 91% prescribedsome form of VTE prophylaxis. Of them,25.8% prescribed prophylaxis based on individualrisk stratification, 96.2% used LMWH, which is startedbefore the cast is applied in 41.8% of cases, and65.4% prescribed prophylaxis until the patientreturned to normal weight bearing. Even in theabsence of supporting clinical guidelines, mostpatients who have had minor orthopedic proceduresreceive some form of thromboprophylaxis in Italy,regardless of their level of risk. The timing and durationof prophylaxis is heterogeneous.PO-149FINDINGS OF THE INTERNATIONAL MEDICAL PREVENTIONREGISTRY ON VENOUS THROMBOEMBOLISM (IMPROVE):A MULTINATIONAL OBSERVATIONAL COHORT STUDY INACUTELY ILL MEDICAL PATIENTS OF PRACTICES INPREVENTION OF VENOUS THROMBOEMBOLISMPiovella F,* Pini M,° Tapson VF,^ Decousus H,**Zotz RB,°° Allegrone J,^^ Anderson FA,^^ for theIMPROVE Investigators*IRCCS Policlinico San Matteo Servizio MalattieTromboemboliche, Pavia, Italy; °Ospedale di FidenzaMedicina Interna, Fidenza, Italy; ^Duke UniversityMedical Center, Durham, USA; **Centre HospitalierUniversitaire de Bellevue, Saint-Etienne, France;°°Universitätsklinikum Düsseldorf, Düsseldorf, Germany;^^Center for Outcomes Research, Universityof Massachusetts Medical School, Worcester, USAhaematologica vol. 89(suppl. n. 8):september 2004

170PostersBackground: Acutely ill medical patients are at significantrisk from venous thromboembolism (VTE).IMPROVE is a prospective cohort study designed to(i.) Assess routine VTE prophylaxis provided to acutelyill medical patients, and 2.) Test predictive modelsof the relationship between patient characteristics,prophylaxis use and key clinical endpoints. Methods:Patient recruitment began in July 2002. Patients 18years old and hospitalized for 3 days with an acutemedical illness are enrolled consecutively. Exclusioncriteria are: therapeutic antithrombotic agents orthrombolytics at admission, major surgery or traumaduring 3 months prior to admission, and VTE treatmentwithin 24 hours of admission. Data are recordedat discharge and 3 months after discharge. A centraladjudication committee reviews key clinical endpoints.Results: As of March 31, 2004, 3727 patientshave been enrolled at 33 hospitals in 11 countries.Three-month follow-up data have been received for2796 (75%) of patients. In-hospital VTE prophylaxis (1type) was received by 40% of patients. The most commontypes of prophylaxis were low-molecular-weightheparin 23%, unfractionated heparin 13%, elasticstockings and intermittent pneumatic compression(6% each). In total, 13% of IMPROVE patients met theMEDENOX study eligibility criteria, and of these, only51% received prophylaxis in hospital. Prophylaxis wasgiven to 5% of patients during the 3-month followupperiod, and continued after discharge in 11% ofpatients who received prophylaxis in hospital. 5% ofpatients died in hospital and an additional 8% diedwithin 3 months after discharge. Pulmonary embolismwas the suspected cause of death for six patients,though this was not confirmed by autopsy. Key clinicalendpoints are shown in the Table.Endpoint In-hospital events 3-month follow-upN=3727 N=2796Received VTE prophylaxis, n (%) 1490 (40) 1415 (5)Suspected VTE, n (%) 394 (11) 129 (5)Treated VTE, n (%) 62 (2) 53 (2)Bleeding, n (%) 115 (3) 70 (3)Death, n (%) 177 (5) 219 (8)Conclusion. Despite consensus groups recommendingthat acutely ill medical patients should receive VTEprophylaxis, this practice appears to be under-utilized.Even among patients enrolled in the IMPROVE registrywho would have qualified for inclusion in the MEDE-NOX study, the rates of VTE prophylaxis were surprisinglylow.Disclosures: IMPROVE is supported by an unrestrictededucational grant from Aventis Pharma.PO-150VENOUS THROMBOEMBOLIC EVENTS DURING THERAPY WITHORAL LOW FORMULATION CONTRACEPTIVES IN HEREDITARYDYSTROPHIC EPIDERMOLYSIS BULLOSAMusso R, Cultrera D,* Stella M,** Cipolla N,Burgio N, Giustolisi R*Haemophilia and Thrombosis Reference RegionalCenter; *Institute of Hematology, University of Catania;**Plastic Surgeon Consultant, ItalyEpidermolysis bullosa (EB) recognizes a variety ofgenetically rare devastating diseases with characteristicblisters and erosions in the skin and mucous membranesincluding nail dystrophy, scars and epidermalcists (milia). Although no cure exists for any form ofEB, several medical and surgical treatments have beenaddressed. Among them the administration of oralcontraceptives (OC) reduces dramatically blister formationand skin lesions (Schachner L. et al Br J Dermatol1977). While the effect of OC on the haemostasishas studied in normal women using OC currently,no data published on their effect on the possible associationwith thrombosis in EB. We here report theoccurrence of repeated deep vein thromboses (n=5)and thrombophlebitis (n=8) in 3 female EB patients,age ranging 19-27 yrs, treated with low formulation(estrogen 35g) OCs. Eleven healthy women not on OCserved as controls. PT, aPTT, thrombin time and fibrinogen,Factor VII:C and Factor VIII:C (one stagemethod) were performed by ACL 3000 Plus coagulometer(IL, Milan). Antithrombin III (ATIII) activity(chromogenis substrate, Stago) was evaluated. ProteinC activity and Protein S activity were measured (DadeBehring, Milan). Plasminogen activator inhibitor-1(PAI-1) activity and tissue plasminogen activator (t-PA)antigen (total fibrinolytic capacity) were also determined(Biopool AB-Menarini). Thrombin/antithrombinIII (TAT) complexes, prothrombin fragment (F1+2) andD-dimer (DD) were assayed by ELISA (Dade Behring,Milan). Circulating antiphospholipid antibodies (APA)were measured (APA IgG-M, Stago).Controls (n=11)no OC useEpidermolysis Bullosapatients (n=3) OC useATIII activity (%) 88.6±12 45-57Protein C (%) 92.9±15 55-68Free Protein S (%) 44±12.5 18-25PAI-1 act. (U/mL) 2.15±1.1 3.8-4.5TAT (µg/L) 3.0±0.9 4.68-6.1F1+2 (nM/L) 0.87±0.43 2.1-3.72D-dimer (ng/mL) 221±102 710-835The table summarizes main results. In the past, fewstudies reported a shortened whole blood clotting timeand low levels of hageman factor in certain EBhaematologica vol. 89(suppl. n. 8):september 2004

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 2004171patients (Homby MA et al. Am J Dis Child 1973) nevertreated with OC formulations. In this regard, our EBpatients no OC user showed an activation of bloodcoagulation and a slight decrease of total fibrinolyticcapacity. These findings therefore would suggest thatthe generalization of OC use in EB implies a permanentassessment of its safety toward the risk of thromboembolicevents as well as genetic screening forinherited thrombophilia.PO-151ELASTIC STOCKINGS, HYDROXYETHYLRUTOSIDES OR BOTHFOR THE TREATMENT OF THE POST-THROMBOTIC SYNDROME.A PILOT RANDOMIZED STUDYFrulla M, Marchiori A, Sartor D, Mosena L,Concolato A, Hartman L, Tormene D, Prandoni PDipartimento di Scienze Mediche e Chirurgiche,Università di Padova, ItalyIntroduction. In contrast with the extensive documentationon epidemiology, diagnosis, and preventionof the post-thrombotic syndrome (PTS), little isknown about the treatment of this common andsometimes frustrating complication of deep veinthrombosis (DVT) of the lower extremities. Aims. Toevaluate the efficacy of elastic compression stockings(ECS), hydroxyethylrutosides (HR), or both forthe treatment of PTS. Materials and Methods. 120consecutive patients with post-thrombotic manifestations,arising after one or more episodes of objectivelydocumented DVT, were randomized to receivebelow-knee ECS (30-40 mm Hg at the ankle), theoral administration of HR (1000 mg b.i.d.), or bothregimens for one year. Each group consisted of 40patients. In all patients follow-up visits were plannedafter 3 and 6 months, and then after the completionof the scheduled 12 months. The presence and severityof PTS was scored using a standardized score (theVillalta scale). The efficacy of ECS and HR, alone andcombined, was calculated using Cox regression models,and expressed as hazard ratio and its 95% confidenceintervals (CI). Results. After adjustment forage, sex, modality of clinical presentation, locationof the thrombotic episode, history of multiple ipsilateralthrombotic episodes, time elapsed betweenDVT episode and patient’s recruitment, and PTSseverity, hazard ratios for PTS recovery (defined as aPTS score lower than 5) for ECS versus HR, ECS aloneversus the combined regimens, and HR alone versusthe combined regimens were 1.48 (0.55 to 3.94), 1.46(0.59 to 3.60), and 0.94 (0.37 to 2.40), respectively.The mean PTS score registered at each follow-up visitdid not differ between the study groups. Conclusions.The combination of ECS with HR does notimprove the results obtained by each strategy alonefor the treatment of established PTS. ECS and HR areequally effective, and, therefore, interchangeable inpatients with PTS. Larger studies are needed to confirmthese findings.haematologica vol. 89(suppl. n. 8):september 2004

172PostersPostersORAL ANTICOAGULANT THERAPYPO-152HOME TERAPY OF DEEP VENOUS THROMBOSIS:WHAT CAN BE DONE?Landini G,° Panigada G,* Tatini S,° Alessandrì A,*Laureano R*°Ospedale S.M.Annunziata, Firenze;*OspedalePescia, PT, ItalyWith the introduction of low-molecular-weightHeparin(LMWH), DTV can be treated safely at homewith a reduction in cost and improvement in thequality of live. To make possible this type of assistancewe have activated in our local health area adiagnostic-therapeutic pathway connecting the differentmedical systems involved. We have organizedmeetings with general pratictioners to put in usediagnostic clinical pre-test. The diagnosis is carriedout with a fast track detailed ultrasonography in theAngiological Departiment (direct access by telephonewithin 24-48 hours). In a case of confirmation acourse of anticoagulation with LMWH followed byoral anticoagulation is started and patient is sent tohome with the exception of clinically discovered pulmonaryembolism, actually bleeding, actual pepticulcers, disorders of coagulation, thrombosis of cava,lack of compliant assistance. Elastic stockings(IIclass) are prescribed and given allowed to walk.Where indicated, diagnostic protocols are introducedto find hidden tumors, and coagulation disorders.The duration of anticoagulant treatment is decidedand follow-up is implemented. During 2003 GeneralPratictioners sent in our outpatient’s Departmentof Angiology 280 patients of avarage age of 55 years(160 males and 120 famales) with suspected DVT, and102 were positive with ultrasound ( 32.3%). Patientswith prossimal DVT were 68(66.7%), with distal DVTwere 34(33.3%). Oncology patients were 32 (31.4%).We treated at home 92 patients with DTV (90.2%)without thromboembolic recurrences and with 2major bleedings(1.9%) and 4 minor bleedings. INR inthe range in 60% of patients.PO-153ORAL ANTICOAGULANT THERAPY MANAGEMENT IN PATIENTSELF-TESTINGAlatri A, Morstabilini G, Testa SCentro Emostasi e Trombosi, Azienda IstitutiOspitalieri, Cremona, ItalyRecent studies demonstrated that INR portablemonitors have good accuracy and are user friendly.Patients on PST have shown the major time spent inthe therapeutic range, as compared with PSM, anticoagulationclinic (AC) and routine medical care(RMC). Since few data are available on major complicationsin patients on PSM, we connected ourAnticoagulation Clinic with patients at home, usinga new net supported program TAOnet®, already online with general physicians, health territorial careunits of the Cremona area. To ensure effectivenessand security of oral anticoagulant treatment inpatients using portable monitors; - to obtain goodmanagement of PST and PSM, objectively definedthrough the availability of laboratory and clinicalparameters; - to demonstrate the efficiency of a bidirectionalnet supported system. All patientsattended courses on anticoagulation therapy,portable monitors self-testing and/or self managementand on computer utilization procedures. Twentypatients, mean age 54 years (29-81y), are connectedwith our AC from November 2002, afterinformed consent was obtained. The main reason forpatient acceptance was to save time and generallyto improve quality of life. In summary, they referdirectly to the hospital sending:1) their own clinicaldata, 2) the quality control, and 3) the INR value,through the compilation of an electronic data-sheet.They can also send to the AC further informationsregarding their clinical status, co-medication,etc. Inthe standard model the patient receive the adjusteddosage through the net system by e-mail. All patientsexpressed their satisfaction, as evaluated through anopen questionnaire. We observed an increase of thetime spent in the therapeutic range as compared tothe previous six months (AC= 58%, home patients=72%; p

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 2004173PO-154LOW DOSE ORAL VITAMIN K TO RESTORE NORMAL LEVELS OFINR AND COAGULATION FACTORS IN PATIENTS ELECTIVELYDISCOUNTINUING WARFARIN: A RANDOMIZED CONTROLLEDSTUDYAgeno W,* Manfredi E,* Micieli E,* Crowther M°*Department of Clinical Medicine, University ofInsubria, Varese, Italy and °Department of Medicine,McMaster University, Hamilton, CanadaPatients on warfarin therapy must temporarilyinterrupt their treatment when undergoing an electivesurgical procedure. Low-dose oral vitamin K canshorten the time period during which patients havesubtherapeutic anticoagulation, thus obviating theneed for bridging therapy. We assessed whether oralvitamin K produces an INR fall into a range that isconsidered safe for surgery, and whether the levelsof the vitamin K dependent coagulation factorsincrease to restore the integrity of the coagulationcascade. Patients receiving warfarin for more than 30days with a target INR between 2.0 and 3.0 whowere electively discontinuing warfarin receivedeither 2.5 mg of oral vitamin K or placebo. Plasmasamples were analyzed for INR and for functionallevels of factors II, VII and V on days 0 (immediatelyprior to study drug), 1, 2, 3 and 5. Thirty patientswere enrolled from 2 clinical centers. The INR in theplacebo group declined by 0.26 INR units (from 1.94to 1.68), compared with 1.31 INR units in the vitaminK group (from 2.58 to 1.27, p 0.007). On the dayafter study drug 6 of 15 patients allocated to placebo,compared with 10 of 15 allocated to vitamin K,had an INR less than 1.4. On the second day after vitaminK administration, these proportions were 6 of15 and 14 of 15 (p < 0.01). The levels of factor VIIincreased more quickly over the first 24 hours in thevitamin K group than in the placebo group (meandifference 0.47 U [95% confidence interval (CI)0.28,0.66] compared with 0.10 U [0.03,0.17], p =0.002). The average rise in the factor II level was 0.27U [0.17,0.37] in patients allocated to vitamin K, comparedwith 0.06 U [0.01,0.11] in patients allocated toplacebo (p = 0.001). Our results support the potentialuse of low doses of oral vitamin K in patients whorequire correction of their INR, for elective surgery orother interventions, between 24 to 48 hours after itsadministration.PO-155IQC AND EQC FOR COAGULATION DIAGNOSTICS PARAMETERSMalatesti V, Di Maira G, Mori MDivision of Laboratory Medicine E.O. Galliera Genoa,italyWe report our present experience with a web-basedexternal quality control system for coagulation diagnosticsparameters. We are using prothrombin time,activated partial thromboplastin time, fibrinogenassay as screening coagulation tests. Oral anticoagulanttherapy is monitored by noting changes in a tissuefactor-induced coagulation time (PT) test on plasmaand expressed as INR. We made a summary reportof internal and external QC of some coagulation testsin Division of Laboratory Medicine, Galliera Hospital.Quality Control schemes are valuable to investigateour performance in daily practice. Our approach todata management is Biorad QC On-Call, an integrated,web-based, quality control system that allows toplain our quality control strategy and put operatingrules into practise. The system incorporates currentlyavailable reference standards, medical relevance andbiological variability, which allow our laboratory todefine the maximum error for each analyte and set anoperative rule to reach the required quality standard.This tool includes a polling function, which automaticallyimport an LIS QC data file without user intervention.The system includes an extensive graphmenu, including performance reports and graphics;we have a review of daily quality control data and theinterlaboratory reports, which provides a fast evaluationof quality control trends across peer and methodgroups. The control graphs, multilevel and evolutiongraphs such as Levey-Jennings charts support our laboratoryto optimise the statistical rules for qualitycontrol evaluation. This strategy provides to eliminatenonessential reset and calibrations saving time andmoney. Analytical goals help improvement of laboratorystatus performance by providing valuable informationabout the appropriateness of the process controlsystem, using tools based on biological variation,medical relevance, and state of the art performancemetrics.haematologica vol. 89(suppl. n. 8):september 2004

174PostersPO-156DIAGNOSTIC EFFICACY OF LUPUS ANTICOAGULANT TESTAFTER THE ONSET OF ORAL ANTICOAGULANT TREATMENT:COMPARISION OF RESULTS FROM TWO LABORATORIESDel Bono R, Martini G, Volpi R, Testa S,*Morstabilini P,* Caimi LII Laboratorio Analisi (Centro FCSA n°28), AOSpedali Civili - Brescia Italy; *Istituto di PatologiaClinica, Centro di Emostasi e Trombosi, IstitutiOspedalieri di Cremona, ItalyBackground. Oral anticoagulant therapy (OAT) is atool to prevent thrombosis in patients affected byantiphospholipid syndrome (APS). Laboratory testingfor Lupus Anticoagulant (LA) is often problematicin anticoagulated patients because of their prolongedclotting times. Purpose. The aim of the studyis to evaluate the diagnostic efficacy of LA testingbefore and after the onset of OAT, comparing resultsfrom two different settings to improve accuracy.Patients. We enrolled 96 patients in OAT with (27)and without (69) APS. We tested 41 patients so far.All of them but one had been investigated for LAbefore the onset of OAT. Methods. Filtered plasmaswere tested for dilute Russell viper venom screen andconfirm test (IL dRVVT) at the II Laboratory, Brescia(BS) and with a four steps LA analysis at theHaemostasis and Thrombosis Centre, Cremona (CR):Stago PTT-LA sensitive, STA aPTT Kaolin, STA silicaaPTT, and the Cheshire modified dRVVT test (plateletderived reagent as the phospholipid trigger). Apooled plasma from pre-OAT LA negative anticoagulatedpatients was also prepared and run within theanalytical IL dRVVT session. Results. Results of testsrun in BS and in CR are shown in Table 1 and Table2, their comparision is shown in Table 3. Conclusions.We found a good agreement between LA testedbefore and after the onset of OAT and between thelaboratories. We had just three controversies: 1) Oneof the patients tested in BS showed a negative LAcompared to her previous result and to CR’s, likelydue to an excessive anticoagulation (INR=7.5). Infact, when the patient’s INR dropped to 2.0, her LAtested positive.Table 1.Pre-OATLA Test run i BS + − TOTTable 2.Pre-OATLA Test run in CR + − TOT+ 5 1 6Post-OAT − 1< 33 346 34 40K value= 0.80, p

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 2004175PO-157THROMBOTIC AND HAEMORRHAGIC RISK SCORE: A TOOLTO IMPROVE ORAL ANTICOAGULANT THERAPY MANAGEMENTManotti C,* Tassoni MI,* Lombardi MR,*Pattacini C,* Quintavalla R,* Tagliaferri A**Dip. Medicina 3, Centro per le Malattiedell’Emostasi e Cura dell’Emofilia, AziendaOspedaliera di Parma, ItalyIn recent years several studies demonstrated thattogether with INR values other factors could increasebleeding or thromboembolic risk: age, sex, cancer,diabetes, previous thromboembolism, the first 90days of anticoagulant treatment, the gender andarterial associated diseases. On this basis we havethought that it was possible to implement a riskscore in the Parma program (PARMAvers5-InstrumentationLaboratory-Milano)to influence the algorithmsuggestions on the basis of the clinical conditionof each patient. The bleeding and thrombosisscore of Parma 5 have been built into the programto be calculated directly by the software at each controlin real time during the daily prescription sessionand to be able to influence the therapeutic proposalalgorithm. PARMA5 calculates the bleeding/thromboticscore, giving risk points to patientconditions. Different pathology will be assigned to aspecific degree of point score, derived from publisheddata. Some points are automatically assigned at eachpatient visit by the software on the basis of age, INRtarget or actual or length of therapy. The performanceof the new algorithm implemented in PAR-MA5 in comparison with that used in the Parma4,the version used and validated in APROAT study, wasinvestigated. We examined all 2890 patients (in stabilisationor in stable phase) attending our clinic 6months after implementation of the new algorithm(700 p/y,21820 visits) in comparison with all patients2402 (670 p/y,18847visits) attending the clinic lastyear during the same period and managed with theold algorithm. The computer algorithm of Parma5suggested a therapeutic proposal in 91% of total visitsin comparison with 77% obtained with Parma 4.Patients managed with Parma5 spent more timewithin therapeutic range (69%) than patients managedwith the old algorithm (67%), the improvementwas obtained reducing the time spent under thetherapeutic range.PO-158AN ALTERNATIVE MODEL FOR MANAGEMENT OF ORALANTICOAGULANT THERAPY: THE ROLE OF GENERALPRACTITIONERSManotti C,* Lombardi MR,* Pattacini C,*Quintavalla R,* Tagliaferri A,* Tassoni MI,*Zurlini C #*Dip. Medicina 3, Centro per le Malattiedell’ Emostasi e Cura dell’Emofilia, AziendaOspedaliera di Parma, # Dip.Patologia ClinicaAzienda USL di Parma, ItalyThe clinical indications for anticoagulation areincreasing. The pressure on anticoagulation centers(AC) intensifies and alternative models of servicedelivery had to be developed. The aim of the projectis to maintain the treatment efficacy, to involvedirectly General Practitioners (GP) in AO surveillance,to reduce access of stabilised patients to AC and toimprove the patient’s life quality. The proposed modelprovides a decentralisation of delivery servicethrough (GP) who are directly involved in completemanagement of their own patients. Two importanttools allowed this great degree of decentralization:a) near-patients test devices;b) computerised decision support systems;The project started on 01/01/2002: twelve GP ofParma area were recruited on voluntary basis tomanage their own patients (about 10-15 per GP).GP work as an AC:a) patients attend to the GP office;b) capillary blood is taken and analyzed with anear-patient testing device.c) dosing is provided directly by GP using a computeriseddecision support system (P.A.R.M.A. systemvers 4.2).d) all patient data are stored in the central databaseof our AC and GP can see only data of his ownpatients. All GP participant were trained andinstructed to use of near-patient testing device andcomputer program. Dedicated educational trainingand regular audit have been carried out and planned.To assess the efficacy of this model oral anticoagulanttreatment analyses will be carried out beforeand after its implementation. Also clinical end point(hemorrhagic and thromboembolic complications)will be checked.haematologica vol. 89(suppl. n. 8):september 2004

176PostersPO-159ORAL ANTICOAGULATION WITH WARFARIN ANDPOLYMORPHISMS IN FACTOR II AND FACTOR VII GENESD’Ambrosio RL,* Cappucci F,° D’Andrea G,°Brancaccio V, § Ciampa A,^ Grandone E,°Margaglione M*Istituto di Genetica Medica, Dipartimento diScienze Biomediche, Università di Foggia,* Unita'di Aterosclerosi e Trombosi, I.R.C.C.S. "Casa Sollievodella Sofferenza", S. Giovanni Rotondo,° andDivisione di Ematologia, Ospedale "G. Moscati",Avellino,^ ItalySince a large inter-individual variability, patientsrequire different warfarin dosages to achieve the targettherapeutic anticoagulation. The variability islargely genetically determined and it can be onlypartly explained by genetic variability in thecytochrome CYP2C9 locus. In a cohort of 147patients followed up at one specialized clinic fromthe start of the anticoagulation with warfarin, wehave investigated whether factor II and factor VIIpolymorphisms have affected doses of drug prescribedto acquire the target anticoagulation intensity.No significant effect was found for factor IIA20210G and factor VII G-402A polymorphisms.Regardless of the presence of confounding variables,the mean adjusted dose required of warfarin washigher among patients with the factor II Thr/Thr 165genotype (4.2 mg) than those of patients carryingthe Met165 allele (2.9 mg; p=0.041) and in carriersof the factor VII GG-401 genotype (4.1 mg) as comparedwith those presenting with the T-401 allele(3.1 mg; p

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 2004177PO-161SURGERY IN PATIENTS ON LONG TERM ORAL ANTICOAGULANTTREATMENT (OAT): A PROSPECTIVE OBSERVATIONAL STUDYBaudo F, Ghirarduzzi A,^ Mostarda G, Molinatti M, +Pengo V,^^ Poli D, Tiraferri E,° Tosetto A,* Morra Eon behalf of FCSA (Federazione Centri SorveglianzaAnticoagulati)Niguarda Hospital, Milan; ^Santa Maria NuovaHospital, Reggio Emilia; + Maria Vittoria Hospital,Torino; ^^Ex Busonera Hospital, Padua; Universityof Florence; °Ospedale degli Infermi, Rimini; SanBortolo Hospital, Vicenza, ItalyThe perioperative management of patients (pts) onlong term OAT is an increasing issue in clinical practice.When surgery is carried out the problem arisesof balancing the thromboembolic (TE) with thebleeding risk. The issue is unsettled. The therapeuticoptions are: discontinuation of OAT to obtain an INRvalue

178PostersPO-163BLEEDING RISK IN PATIENTS RECEIVING BOTH ORALANTICOAGULANT AND ANTIPLATELET THERAPYPoli D, Antonucci E, Cecchi E, Mannini L, Ilari I,Ferrini S, Gazzini A, Coppo M, Falciani M,Gensini GF, Abbate R, Prisco DCentro di Riferimento Regionale per la Trombosi,Dipartimento dell’Area Critica Medico Chirurgica,Azienda Ospedaliero-Universitaria Careggi,Florence, ItalyIn selected patients oral anticoagulant therapy(OAT) cannot sufficiently prevent thrombotic eventsand the association of antiplatelet therapy (APT) isrequired. This practice is debatable because ofincreased risk of bleeding. Aim of our study was toevaluate the rate of adverse events in patientsreceiving OAT+APT treatments. We studied 1596consecutive unselected patients (942 males and 654females; mean age 64±13.1 yrs; follow-up 3364patient-years), referred to our Anticoagulation Clinicfor OAT monitoring. Among these patients, 44 (33males and 11 females; mean age 64.6±11.7 yrs)received also APT, for a total period of 115 patientyears.Patients received both treatments for differentindications: mechanical prosthetic heart valves (13),arterial vascular disease (12), coronary artery disease(13), atrial fibrillation with embolic complication (4),other indications (2). Looking at the quality of OAT,time spent within, above and below the intendedtherapeutic range was similar in treated or not withAPT (67%, 15% and 18% in the whole populationand 70%, 15% and 15% in patients with APT). 2/44APT patients had major bleeding events (1.7/100patient-years), none was fatal. In all the otherpatients we recorded 36/1552 major bleeding eventswith a similar rate of incidence (1.1/100 patientyears),3 were fatal (p=0.5). 9/44 APT patients hadmajor thrombotic events (rate 7.8/100 patientyears),none was fatal. In all the other patients, werecorded 95/1552 major thrombotic events (rate2.9/100 patient-years), 3 were fatal. This differencewas statistically significant [RR 2.6 (1.1-5.3 95%CI) p=0.01], and may reflect the higher thromboticrisk that lead to the choice of adding APT to OAT. Inconclusion, patients at high thrombotic risk whoreceived OAT associated with APT seems not to be atsignificantly higher risk of serious bleeding.FCSA PrizePO-164ORAL ANTICOAGULANT TREATMENT FOR STROKE PREVENTIONIN ATRIAL FIBRILLATION PATIENTS: FEMALES ARE STILL ATRISKPoli D, Cecchi E, Antonucci E, Mannini L, Gensini F,Lucarini L, Saracini C, Attanasio M, Fatini C,Gensini GF, Abbate R, Prisco DCentro di Riferimento Regionale per la Trombosi,Dipartimento dell’ Area Critica Medico Chirurgica,Azienda Ospedaliero-Universitaria Careggi,Florence, ItalyOver the last years, oral anticoagulant treatment(OAT), has become the cornerstone for stroke preventionin patients with atrial fibrillation (AF). Thisstudy was aimed at evaluating the efficacy and safetyof OAT managing in a real-practice situation. Westudied 1596 consecutive unselected patients (follow-up3364 patient-years), referred for the controlof OAT to our Anticoagulation Clinic. Among thesepatients, AF was the indication for OAT in 558 (35%)(follow-up 958 patient-years). Looking at the qualityof OAT, time spent within, above and below theintended therapeutic range was 66%, 16% and 18%respectively in the whole population and 71%, 13%and 16% in AF patients. 12/558 AF patients hadmajor bleeding events (1.2/100 patient-years), 2were fatal. In all the other patients, we recorded26/1038 major bleeding events with a similar rate ofincidence (1.1/100 patient-years), one was fatal. Inboth groups females showed a trend to higher risk ofbleeding (p=0.4). 26/558 AF patients had thromboticevents, 17 of which were major (2.7 and1.7/100 patient-years, respectively), one was fatal.The other patients had 78/1038 thrombotic events,46 of which were major (3.2 and 2.2/100 patientyears,respectively). In all patients no significant differencewas found in the rate of total thromboticcomplications in relation to sex. Instead, the rate ofstroke in patients with AF was significantly higher infemales with respect to males (1.87 and 0.5/100patient-years respectively; [RR 3.7 (1.0-22.2 95%CI;p=0.05)]. This difference was not found in patientson OAT for others reasons (RR 1.4 (0.5-3.6 95% CI;p=0.3). On the whole AF patients on OAT show a lowrate of complications however, females show a trendto high risk of bleeding and are at higher risk ofstroke in spite of OAT.haematologica vol. 89(suppl. n. 8):september 2004

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 2004179PO-165THROMBOTIC COMPLICATIONS IN NONVALVULAR ATRIALFIBRILLATION PATIENTS DESPITE OATPoli D, Antonucci E, Cecchi E, Mannini L,Bruschettini A, Paniccia R, Poggi F, Rossi L,Sestini I, Abbate R, Gensini GF, Prisco DCentro di Riferimento Regionale per la Trombosi,Dipartimento dell’Area Critica Medico Chirurgica,Azienda Ospedaliero-Universitaria Careggi,Florence, ItalyOral anticoagulant treatment (OAT) is the cornerstonefor stroke prevention in patients with atrialfibrillation (AF). The aims of our study were: 1)toevaluate the efficacy of OAT in the prevention of cardiovascularcomplications in AF patients and 2)toevaluate if comorbidity and cardiovascular risk factorsmay play a role in unsuccessful OAT. We studied303 AF patients on OAT (186M, 117F, mean age72.9±8.2, follow-up 571 patient-years). During follow-up,20 patients (12M, 8F) had thrombotic complications(rate 3.5/100 patient-years): 7 TIA, 8stroke, 4 peripheral embolisms, 1 myocardial infarction.Time spent within, above and below the intendedtherapeutic range was 71%, 13% and 16% in AFpatients without thrombotic events and 64%, 14%and 22% in AF patients with thrombotic events. Themean INR related to the thrombotic event was1.9±0.5. Univariate analysis showed no correlationbetween the occurrence of thrombotic event despiteOAT and sex, the presence of either hypertension,coronary artery disease, diabetes, hypercholesterolemia,peripheral artery disease, or hearth failurerespectively. Hyperhomocysteinemia was more frequent,in patients with thrombotic events than inthose without, but this difference was not statisticallysignificant (p=0.07). On the contrary, a significantcorrelation was found between a positive historyof a previous thrombotic event and the recurrenceof thrombotic event and [OR 8 (2.3-28.0),p=0.001]. A multivariate analysis adjusted for theother above-mentioned variables, confirmed the historyfor a previous thrombotic event as a strong riskfactor for unsuccessful OAT [OR 6.8(1.8-24.6),p=0.003]. Moreover, these patients showed a worseOAT control, with longer time spent below theintended INR. In conclusion, OAT is highly efficaciousfor the prevention of cardiovascular events. Specialefforts are needed to maintain INR within the therapeuticrange, specially in patients with previousthrombotic complications for whom closer INR controlsmay be indicated.PostersCONTROL OF ANTICOAGULATIONPO-166IS IT POSSIBLE TO ADD A STATISTICAL SIGNIFICANCE TO INRTHERAPEUTIC RANGES? IMPROVEMENTS IN LABORATORYMONITORING FOR INTERPRETING BORDERLINEMEASUREMENTSIntrocaso GServizio di Medicina di Laboratorio, Settore diEmocoagulazione, Centro Cardiologico ''Monzino''Milan, ItalyRetrospective data of anticoagulated patients wereused to calculate a mean of observations and controllimits X±1s and X±2s (mean±standard deviation).During 18 months of coagulation monitoring,patients without major oral anticoagulant therapycomplications, with more INR determinations intherapeutic range and with normal distribution ofINR values according to Kurtosis coefficient, wereselected. Considering 1338 citrated plasma thecumulative distribution functions allowed us toobtain critical limits of international normalized ratio(INR) with a cumulative probability (p). Control limits,calculated for a therapeutic control chart,showed through different control rules 1S2 or 2S1 analarm signal to analyse the cause of INR outsidetherapeutic range. Our investigation suggested thatfor results at level of the therapeutic control limitsneeded at least 2 consecutive INR results to detecta significative over or under anticoagulation. Therapeuticcontrol chart method should be a useful meanin clinical practice evaluating the statistical significanceof thearapeutic ranges then of consecutiveand borderline INR outcomes. Analytical improvementsand control rules applied to clinical monitoringmay optimise the drug dose administration.PO-167THE QUALITY CONTROL OF THE ORAL ANTICOAGULANTTHERAPY (OAT): ROLE OF ANTICOAGULATION CLINICS INTHE APPROACH TO MAJOR BLEEDING EVENTSErba N,* De Micheli V,* Brambilla P,* Mochetti F,°Bassi P,^ Frigerio L, § Restifo DGruppo Di Studio Coagulazione Lombardia,*Presidio Ospedaliero di Merate (Lc), °Ospedaledi Legnano (Mi), ^Ospedale San Carlo Milano,§Ospedale Valduce Como, Ospedale di Vimercate(MI), Italyhaematologica vol. 89(suppl. n. 8):september 2004

180PostersHemorrhages represent the main side effects ofthe oral anticoagulant treatment (OAT). Amongthese, the major ones have high mortality rate. Themajor bleeding events can lead the patient to hospital’semergency room, in these cases INR valuemust be normalized as soon as possible. The AnticoagulationClinics are hospital’s structures appointedto clinical and laboratory monitoring of anticoagulatedsubjects and operate with medical specialistsin all disciplines in defining the therapeutic protocolsfor the management of OAT patients. Five anticoagulationClinics of the Lombardia Coagulation Groupanalyzed the management of the major bleedingevents occurred in 2003.84 major bleeding eventswere analysed (1.41% patient-year), 9 of them werefatal (0.15 patient-year). Only 53 patients (62%)were admitted to the same Hospital of the AnticoagulationClinic. 17 were admitted to other hospitals,whereas 14 were not admitted at all. Among thepatients of the first group, only in 16 cases (30%) thehaemorrhage was treated according to the advice orto the guidelines of the Anticoagulation Clinic. Inthis group, only 1 patient out of 16 had INR value >2 at 48 hours from the admission, versus 10 out of25 patients in the group which did not receive theadvice from Anticoagulation Clinic (p< 0.05). Themortality rate was 6.3% vs 22% (n.s.). The approachto major bleeding event directly or indirectly managedby Anticoagulation Clinic might produce betteroutcome. The practice of consulting the AnticoagulationClinic or use of clinical guidelines have stillnow restricted application. In OAT clinical qualitypolicy, the management of patients with majorbleeding can be improved by coordinating the activitybetween clinical care departements and AnticoagulationClinic.PO-168A PROJECT FOR ORAL ANTICOAGULANT MONITORING BY AWEB-BASED PATIENT MANAGEMENT SYSTEM (ANTHEMA)Erba N,* Brambilla P,* Vercelloni S,° Villa I, §Pasquini M^*Presidio Ospedaliero di Merate, AziendaOspedaliera di Lecco, ° Sevizio di Medicina diBase GCP Olgiale (LC), § R.S.A. I.R.A.M. Lecco, Italy.^e-MedicalMonitorThe oral anticoagulant treatment (OAT) is recommendedin thromboembolic conditions and involvesmore than 1% of the general population. The needof clinical and laboratory monitoring stimulated thegrowth of Anticoagulation Clinics. Moreover, theextended use of information technology improvedtreatment’s quality. Difficulties in the OAT surveillanceare related to clinic crowding, to the olderpatients and those with chronic or multiple pathologies,to disabled patients nursed at home. The developmentof a network of peripheral specialists underAnticoagulation Clinic’s co-ordination might becomean appropriate instrument for the solution of theseproblems. The Merate’s Anticoagulation Clinic isactually the only referral point for almost all patientsliving in the district. Anthema, a web based system(ASP) dedicated to the oral anticoagulant management,was recently introduced by e-MedicalMonitor(an Instrumentation Laboratory company). This webapplication links the Anticoagulation Clinic that hasthe responsibility of the treatment to peripheralstructures such as nursing homes or general practitioners.The main features are: patient reception andquestionnaire, manual option for result entry or onlineimport of the INR test results from the analyzers,weekly or daily dosage therapy proposal and settingthe date of the next visit. Anthema incorporatesall therapeutic decision support systems used in“PARMA software. e-MedicalMonitor and the Merate’sAnticoagulation Clinic started a collaborativeproject with two main aims: anthema evaluation bya team of operators involved in the OAT management;Optimization of network strategy in thedecentralized patient’s management, involving 4general practitioners, 3 nursing homes, and Merate’sAnticoagulation Clinic as referral point. Trainingplanes, evaluation questionnaires for users andpatients were provided.PO-169OAT WITH POC DEVICES AND REMOTE COMPUTERMONITORING IN THE SETTING OF THE ITALIAN NHS:A CONTROLLED CLINICAL TRIALPaccamiccio E, Ferrante F, Trottini S, Rosafalco M, §Duca E,* Vecchioli M,* Nenci GG, Iorio ASezione di Medicina Interna e Cardiovascolare,Università di Perugia and § Instrumentation §Laboratory, IL, Milan, Italy and *Distretto delPerugino ASL2, Perugia, ItalyBackground. The number of patients chronicallytaking oral anticoagulant therapy (OAT) is progressivelygrowing due to aging of the population andwidening of the indications to treatment. Moreover,it has been shown that the best clinical results aregained by specialised centres, that are not frequentlyable to cope with the needs of far living patients.A comprehensive solution could be to decentralizeOAT testing maintaining the advantage of a specialisedmanagement. Objective. To test the effectivenessand feasibility of specialised OAT monitoringwith a remote computer modality in the setting ofthe Italian National Health System (INHS). Design andhaematologica vol. 89(suppl. n. 8):september 2004

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 2004181methods. Seven outpatient peripheral units from theINHS were identified. Three hundred unselectedpatients referring to the Thrombosis Centre (TC) ofPerugia were enrolled on a first come basis afterobtaining informed consent. As a control group, westudied a series of matched patients of the TC. ThePoint-of-care (POC) device for the capillary determinationof INR was the Protime Microcoagulation System(IL, Milan), while the software used to manageOAT was P.A.R.M.A. 5 (IL, Milan, Italy). The project wasplanned and conducted with a research grant given byRegion Umbria. Results. Patients recruitment startedon October 2002 and fifty patients were assigned toeach unit. Five patients asked to withdraw. The meannumber of monthly accesses was 1.5 per patient. Theanalysis of the INR values showed that 62.1% of controlswere in the therapeutic range and that 72.0% oftime was spent within the therapeutic range. Neithermajor bleedings nor thromboembolic complicationswere documented. Interpretation and conclusions.These figures were not different from those measuredin the control group and in the historical analysis ofthe study group confirming the feasibility of this typeof management of OAT.PO-170QUALITY CONTROL PROGRAMS FOR POC DEVICES TOMEASURE INR. RESULTS OF A PILOT EXERCISEFerrante F, Paccamiccio E, Servettini I, Iorio ASezione di Medicina Interna e Cardiovascolare –Università di Perugia, ItalyBackground. INR testing is usually performed onvenous plasma with automated coagulometers, andmany external and internal quality controls programare available. To date, several studies have demonstratedthat Point-Of-Care (POC) coagulation monitorsrepresent an attractive and efficient option forOAT monitoring, but specific quality controls programsare to be validated. Aim of the study: Set up,run and evaluate a quality control program for INR-POC measurements. Materials and Methods. Ten POCdevices (ProTime, IL, Milan) are routinarily used forINR determination in 350 patients over seven outpatientsservices. Four time a year, five randomlyselected patients from each service gave their consentto undergo both fingerstick and venipuncture.The device was then recalled in the coordinating laboratory,checked with control material (Direct-check,IL, Milan) by a skilled technician, and rotated toanother service following a randomized scheme.Venous PT-INR was performed in duplicate in twodifferent laboratories (using one MLA – Recombiplastinand the other Futura – Recombiplastin, allfrom IL, Milan). Correlation coefficients, Bland andAltman comparison, and ANOVA were used toanalyse the data. Results. Sixty two couples of datawere available at the end of April. No effect of service,instrument and sample was found. Main resultsare shown in the table.Mean Mean SD Agreement Range R 2of means of means limitsProtime vs lab1 2.54±0.91 0.49±0.66 -0.8 to + 1.78 -0.3 to +3.6 0.70Protime vs lab2 2.70±1.03 0.129±0.38 -0.624 to +0.882 -0.9 to +1.9 0.88Lab1 vs lab2 2.47±0.84 0.33±0.56 -0.77 to + 1.42 -0.2 to +2.7 071Mean SD 95%CI Range CV %Control low 1.631 .2496 1.08 to 2.18 1.3 to 1.9 15Control high 3.32 .606 2.12 to 4.51 3.0 to 4.0 18Conclusions. POC results are comparable to thoseof a clinical laboratory. Direct-check showed a CVsuitable for its use as control materials.PO-171DETERMINATION OF THE TECHNIQUE- AND SPECIMEN-SPECIFIC INTERNATIONAL SENSITIVITY INDEX FOR THETHROMBOTESTTM REAGENTChantarangkul V, 1 Frontoni R, 2 Gresele P, 3 Oca G, 4Paniccia R, 5 Pellegrini L, 6 Tripodi A 11A Bianchi Bonomi Hemophilia and ThrombosisCenter, University and IRCCS Maggiore Hospital,Milano; 2 Laboratorio di Coagulazione, Dipartimentodi Patologia Clinica, Ospedale di Macerata; 3 Dipartimentodi Medicina Interna, Sezione di MedicinaInterna e Cardiovascolare, Universita’ di Perugia;4U.O. Angiologia e Malattie della Coagulazione,Policlinico S. Orsola - Malpighi, Bologna; 5 CentroTrombosi, Azienda Ospedaliera Universitaria Careggi,Florence; 6 Servizio Trasfusionale, ASL1-Umbra,Citta’ di Castello, ItalyISI values for 3 different lots of ThrombotestTM,(Axis-Shield PoC, Oslo, Norway) were estimated usingboth the manual (tilt-tube) technique and the Thrombotrackselect 2 (Axis-Shield PoC, Oslo, Norway)coagulometer. The calibration has been divided into2 steps. First, Thrombotest Lot1 was calibratedagainst the WHO-International Standard codedOBT/79 using citrated plasma in 2 expert laboratoriesthat used both the manual technique and Thrombotrack.Second, Thrombotest Lot 2 and 3 were calibratedagainst Thrombotest Lot 1 in other 4 laboratoriesthat used only Thrombotrack. In this secondstep, testing was performed using both citrated plas-haematologica vol. 89(suppl. n. 8):september 2004

182Postersma and native whole blood. Both calibration exerciseshave been performed according to the WHO guidelines(WHO Technical 48th report, 1999). Briefly, prothrombintime for 60 patients on oral anticoagulantsand 20 healthy individuals has been performed in 10different working sessions. ISI-manual obtained inthis study for plasma with Lot 1 (0.99) was similar tothat declared by the manufacturer (0.98), but the ISI-Thrombotrack (0.91) was 8% lower. ISI-Thrombotrackfor Lot 2 and Lot 3 were similar to that of Lot 1, thedifferences being 75 yrs; main indications for OAT: AF 41%, valvularprosthesis 20%, DVT/PE 13%. The table displays theresults comparing pts. < 75 yrs and > 75 yrs (qualityanalysis on 735 pts), mean follow-up 248 days.All pts Pts < 75 yrs Pts ≥ 75 yrs p #Number of pts 925 496 (53.6%) 429 (46.4%)Death due to bleeding complications 0 0 0Major bleeding 4 (0.6%y/pts) 1 (0.3%y/pts) 3 (1.0%y/pts) 0.517Minor bleeding 36 (5.7%y/pts) 14 (4.2%y/pts) 22 (7.5%y/pts) 0.101Thrombotic events 18 (2.8%y/pts) 6 (1.8%y/pts) 12 (4.1%y/pts) 0.132INR within range 74.9% 73.4% 76.6% 0.361INR above range 13.8% 14.1% 13.4%INR below range 11.2% 12.3% 9.9%#between groups (< 75 yrs - ≥ 75 yrs) analysis; %y/pts = % year/patient.Conclusions: The management of patients in a ACimproves the treatment quality also in elderlypatients. The incidence of complications in our AC iscomparable to those of reference trials. In the elderly,OAT quality was similar to the younger but complicationstended to increase (not significantly), suggestinga closer monitoring of these patients.PO-173EXTERNAL QUALITY ASSESSMENT PROGRAM FORCOAGULATION: THE EXPERIENCE OF EMILIA-ROMAGNAREGIONLudovici S, Legnani C,* Poggi M,* Franceschetti F,Incorvaia LR, Maltoni P, Mimmi P, Tancredi MA,Capelli MGruppo Controllo di Qualità Analitico, * U.O. diAngiologia e Malattie della Coagulazione “MarinoGolinelli”, Azienda Ospedaliera di Bologna,Policlinico S. Orsola-Malpighi, Bologna, ItalyThe external quality assessment program of theEmilia-Romagna region was created in 1987 and in1990 was extended to coagulation tests. Currently,244 Italian labs either from national health (n=74)or private (n=170) services are participating in theprogram. The program uses lyophilized human plasmaspecimens locally prepared in large, stable, uniformlots, which are distributed as unknown specimensto participants for analysis and generation ofinter-laboratory data. The participants are asked tohandle the distributed specimens using their routinemethods for the determination of Prothrombin Time,activated Thromboplastin Time, Fibrinogen and Antithrombin.Participants receive 12 specimens/year in6 cycles each of whom include one normal and onepathological specimen. Thrombophilia tests are goingto be included within the end of 2005. Results arereported using standard report forms, includinginformation on the assay principle, method andequipment used. Data are elaborated separately forhaematologica vol. 89(suppl. n. 8):september 2004

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 2004183different combinations of reagent/instrument used.Data are classified as: A if they are between ±1 Z-score, B if between ±2 Z-score, C if between ±3 Z-score and D if above/below 3 Z-score or outliers. Participantsare immediately contacted if results arepersistently (3 consecutive exercises) above the fixedacceptable limits (± 1 Z-score). Elaboration of datafrom 1990 to 2003 shows that labs with consistentparticipation have consistent and statisticallyimprovement in performance. In 2003 80% of allparticipant labs showed results in class A. Data alsosuggest that labs with more experience with proficiencytesting have lower rate of unacceptableresults and that this rate tends to decrease with eachyear of experience. These conclusions support thefindings of other researchers who have documentedthe benefits of inter-laboratory comparison programsand proficiency testing.PO-174ISAM: INTERNATIONAL STUDY OF ANTICOAGULATIONMANAGEMENT. THE ITALIAN EXPERIENCEPengo V, 1 Adriani C, 1 Alatri A, 2 Antonucci E, 3Baudo F, 4 Caimi TM, 4 Cauli C, 5 Lopatriello S, 6Malato A, 7 Manotti C, 8 Marongiu F, 5 Negrini C, 6Poli D, 3 Siragusa S, 7 Tassoni MI, 8 Testa S, 2 Attinà G 91Dip. di Medicina Clinica e Sperimentale, Sezione diCardiologia, Ospedale di Padova; 2 Centro Emostasie Trombosi, Istituti Ospedalieri, Cremona; 3 Dip. delCuore e dei Vasi, Unità di Malattie Aterotrombotiche,Azienda Ospedaliera-Universitaria Careggi,Florence; 4 Strutt. Compl. di Ematologia, OspedaleNiguarda-Cà granda-Milano; 5 Cattedra di MedicinaInterna, Policlinico Universitario di Monserrato,Università di Cagliari; 6 PBE Consulting S.r.L, Milano;7Cattedra di Ematologia dell'Università di Palermo;8Centro Emostasi, Ospedale Maggiore di Parma;9Economic Affairs, AstraZeneca Italia; ItalyObjectives. ISAM, a multicenter, observational, retrospective,cross-sectional study, aimed at describingthe anticoagulation treatment monitoring onpatients with chronic non-valvular atrial fibrillation(CNVAF) for stroke prophylaxis (follow-up: Jan-Dec2002); and estimating the direct and indirect costsassociated with Oral Anticoagulation Therapy (OAT)according to the Italian National Health System perspective.Methods. 7 out of 8 AntiCoagulation Clinics(ACCs), selected to represent the whole Italianterritory, enrolled 23 randomized patients withCNVAF. Results. The total number of patients was177: 102 were males and 75 females (mean age 72years); 90% was affected by chronic Atrial Fibrillationand 10% by paroxysmal. 77% received warfarinand 23% acenocumarol. Only 18% had no risk factors,64% had hypertension, 12% diabetes, 32% previousthromboembolic events, 24% coronary arterydisease and 12% congestive heart failure. Everypatient had on average 19 INR tests per year (meanfrequency: every 20 days); 40% of total tests (3168)required dosage changes and 73% were in the recommendedrange (2.0-3.0) anyway. Quality of OATmonitoring, according to Rosendaal's method, was:67.9% of time was spent in the range 2.0-3.0, 21%below and 10% above this range. Total cost perpatient per year was 943 : 745 ¤ direct costs and198 ¤ indirect ones (productivity loss by patients ortheir caregivers). Medical costs, 525 ¤, included OA Tdrug (5%), INR tests (18%) monitoring visits (44%)and admissions (33%). Non-medical costs (transportation)amounted to 220 . Conclusions. Patientsin ACC management could obtain good level of INRcontrol: Time in Target Range 67.9%, comparablewith other literature findings. ISAM is a first attemptto measured the resource consumption generated bythe monitoring system (ACCs) associated with a goodlevel of control, and whose costs include drugs, INRtests and monitoring visits, which are the majorcomponents of such costs.haematologica vol. 89(suppl. n. 8):september 2004

184PostersPostersTHERAPY OF HEMOPHILIAPO-175PHYSICIANS’ AND PHARMACISTS’ PREFERENCES FORHEMOPHILIA TREATMENT EVALUATED BY CONJOINT ANALYSISScalone L,* Monzini MS,* Mantovani LG,*Gringeri A,^ Villa M,° Mannucci PM^ for theCONAN Study Group*Center of Pharmacoeconomics, Department ofPharmacological Sciences, University of Milan,Milan, Italy; ^Angelo Bianchi Bonomi Hemophiliaand Thrombosis Center, Department of InternalMedicine and Dermatology, University of Milan andIRCCS Maggiore Hospital, Milan, Italy; °Laboratoryof Epidemiology, Advanced Biotechnology Institute,National Research Council, Milan, ItalyBackground. Hemophilia care entails the absorptionof huge amounts of human and economicresources. The treatment of hemophilia involves acomplex interaction between patients, physicians andpolicy-makers. Aim. To evaluate the preferences ofphysicians and pharmacists toward products used forreplacement therapy. Methods. This study investigatespreferences on hemophilia care in 69 physicians and58 pharmacists using conjoint analysis, a techniquefor establishing the relative importance of differentcharacteristics in the provision of a good or service.Attributes and levels were: perceived viral safety (asthat provided by highly purified double inactivationplasma derived versus recombinant concentrates), riskof inhibitor development (1/4, 1/6, 1/10 PUP’s), factorinfusion frequency on prophylaxis (thrice, twice,once a week), pharmaceutical dosage form(lyophilized material or a ready-to-use solution), wayof distribution (home, office pharmacy, hospital) andprice. Results. Excluding pharmaceutical dosage formfor physicians and office pharmacy delivery for pharmacists,all attributes considered tested important torespondents. Physicians showed a strong preferencetoward both outcome variables (viral safety, risk ofdevelopment of inhibitors) and process variables (distribution,infusion frequency) while pharmacistsshowed a strong preference only for outcome variablesand unexpectedly not toward way of distribution.Conclusions. Our study is the first to apply conjointanalysis to establish preferences of physiciansand pharmacists in hemophilia replacement therapy.This study provided evidence of the usefulness of conjointanalysis in plan optimal health care through theelicitation of physicians’ and pharmacists’ preferencesto health care, even in the particular context of hemophiliamanagement.PO-176CLINICAL AND SOCIO-DEMOGRAPHIC CHARACTERISTICSINFLUENCE PATIENTS’ PREFERENCES TOWARDS HEMOPHILIATREATMENTMantovani LG,* Gringeri A,^ Scalone L,*Monzini MS,* Villa M,° Mannucci PM^ for TheCONAN Study Group*Center of Pharmacoeconomics, Department ofPharmacological Sciences, University of Milan,Milan, Italy; ^Angelo Bianchi Bonomi Hemophiliaand Thrombosis Center, Department of InternalMedicine and Dermatology, University of Milan andIRCCS Maggiore Hospital, Milan, Italy; °Laboratoryof Epidemiology, Advanced Biotechnology Institute,National Research Council, Milan, ItalyBackground. Clinical and sociodemographic characteristicsmight affect patients’ preferences towardshemophilia treatment. Aim. The aim of this work is toevaluate influence of clinical and socio-demographiccharacteristics on the strength of preferencestowards each characteristic. Methods. We analyzeddata originated by CONAN database from 178patients. Patients were asked to make choicesbetween variables of replacement therapy such asperceived viral safety, risk of inhibitor development,infusion frequency on prophylaxis, pharmaceuticaldosage form, way of distribution and price. Results.The value of each characteristic was indicated by itsmonetary value: the monetary value for perceivedviral safety was greater for moderate (2,547€) comparedto severe hemophiliacs (1,810€) and wasgreater for patients in recombinant treatment(2,421€) to plasma derived (1,001€). Compared tomoderate hemophiliacs, severe hemophiliacs placed ahigher value on the decrease in the risk of developinginhibitors (1,033€ vs. 518€) and on the infusion frequency(706€ vs. 144€). The employment statusplayed an important role on monetary values attributedto way of distribution: employed patients placeda higher monetary value to home delivery (1,495 €)and office pharmacy distribution (2589 €) comparedto self-employed patients (541€ and 89€, respectively)and to unemployed patients (1,002€ and928€). Conclusions. We demonstrate that clinical andsocio-demographic characteristics influencedpatients’ preferences. One of the practical utilities ofconjoint analysis consist in the capability of targetdifferent group with different characteristics thatconsequently require different therapeutic decisions.This allow to plan optimal hemophilia care.haematologica vol. 89(suppl. n. 8):september 2004

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 2004185PO-177ESTABLISHING HEMOPHILIA PATIENTS’ PREFERENCES INPROPHYLAXIS: A CONJOINT ANALYSIS PILOT STUDYMonzini MS,* Gringeri A,^ Scalone L,Mantovani LG,* Villa M,° Mannucci PM^ For TheCONAN Study Group*Center of Pharmacoeconomics, Department ofPharmacological Sciences, University of Milan,Milan, Italy; ^Angelo Bianchi Bonomi Hemophiliaand Thrombosis Center, Department of InternalMedicine and Dermatology, University of Milan andIRCCS Maggiore Hospital, Milan, Italy; °Laboratoryof Epidemiology, Advanced Biotechnology Institute,National Research Council, Milan, ItalyBackground. Prophylaxis was shown to be veryeffective in preventing bleeding and haemophilicarthropathy, but high cost often hampers its introductionin hemophilia treatment. Conjoint analysis isa technique to elicit preferences toward hemophiliatreatment and can help planning optimal health careand guiding therapeutic decisions. Aim. To establishingwhich characteristics patients considerimportant in prophylaxis, and if cost of treatmentcould influence their preferences. Methods. A focusgroup identified 5 characteristics: type of product(plasma-derived vs. recombinant), infusions frequency,hemorrhages frequency, possibility to do vigorousphysical activities, possibility to do usual activities.Patients gave their opinion rating each characteristicfrom 0 (not important) to 100 (very important).Scenarios describing hypothetical treatmentmodalities were administered to patients who wereasked to chose one for each couple presented. Thecharacteristic cost, expressed as increase in healthcare taxes, was then added in each scenario andpatients were invited to make again their choices.Results. Fifteen people, 13 adult patients and 2 parents,were enrolled. The characteristic consideredmost important was usual activities (median=95),followed by hemorrhages frequency (86), infusionsfrequency (81), type of product (76), vigorous physicalactivities (69) and finally cost (50). Almost allpatients did not suggest other important characteristics.Eleven patients (73%) changed at least onechoice when cost was present. Conclusions. This pilotstudy allowed us to identify aspects considered byhemophilia patients as important in prophylaxis.These results will help us to design a Conjoint Analysisaimed at evaluating utilities and monetary valueof these aspects.PO-178EVALUATION OF PATIENT PREFERENCES FOR HEMOPHILIACARE BY CONJOINT ANALYSISMantovani LG,* Gringeri A,^ Monzini MS,*Scalone L,* Villa M,° Mannucci PM^ For TheCONAN Study Group*Center of Pharmacoeconomics, Department ofPharmacological Sciences, University of Milan,Milan, Italy; ^Angelo Bianchi Bonomi Hemophiliaand Thrombosis Center, Department of InternalMedicine and Dermatology, University of Milan andIRCCS Maggiore Hospital, Milan, Italy; °Laboratoryof Epidemiology, Advanced Biotechnology Institute,National Research Council, Milan, ItalyBackground. The hemophilia treatment of involvesa complex interaction between patients, physiciansand policy-makers, each carrying their own set ofpreferences. Even if a better understanding of thesepreferences towards the specific attributes of treatmentproducts is of great utility to plan optimal care,this area remains largely unexplored. Aim. To establishpreferences in adult Italians with severe or moderatehemophilia as they relate to hemophilia treatmentand to evaluate the feasibility of conjointanalysis. This technique shows how individuals arewilling to trade between different characteristics ofhealth care and thereby estimates their relativeimportance. Methods. Patients were asked to choosebetween combinations (scenarios) of attributes ofreplacement therapy such as perceived viral safety(as that provided by highly purified double-inactivatedplasma-derived vs. recombinant concentrates),risk of inhibitor (1/4, 1/6 and 1/10 PUPs), infusionfrequency on prophylaxis (3, 2 or 1 a week), pharmaceuticaldosage form (lyophilized material or aready-to-use solution), way of distribution (home,office pharmacy, hospital) and price. Results. Overall178 patients’ responses were evaluated. All attributesconsidered, excluding pharmaceutical dosageform, tested important to respondents, who showeda strong preference toward both outcome variables(viral safety, risk of inhibitors) and process variables(infusion frequency, distribution, price). Conclusions.Our study demonstrates the high utility value attributedto the perceived safety of recombinant productsand to the reduction in inhibitor development risk.Finally, this study provides evidence of the usefulnessof conjoint analysis in planning optimal hemophiliacare through the elicitation of patients’ preferencesand utility values.haematologica vol. 89(suppl. n. 8):september 2004

186PostersPO-179INHIBITOR DEVELOPEMENT IN PREVIOUSLY UNTREATED ORMINIMALLY TREATED PATIENTS WITH HEMOFILIA A TREATEDWITH PLASMA DERIVED FACTOR VIII VIRUS-INACTIVATED BYSOLVENT-DETERGENT METHODMonzini MS,* Gringeri A,° Tagariello G, §Scaraggi FA,^ Di Stasi F* and The EMOCLOT 15Study Group*Center of Pharmacoeconomics, Department ofPharmacological Sciences, University of Milan,Milan, Italy; °Angelo Bianchi Bonomi Haemophiliaand Thrombosis Centre and Department of InternalMedicine and Dermatology, IRCCS MaggioreHospital and University of Milan, Milan, Italy;§Haemophilia Centre, Ospedale Civile, CastelfrancoVeneto (TV), Italy; ^Haemophilia Centre, PoliclinicoII, Bari, ItalyBackground. A solvent-detergent virus-inactivatedplasma-derived FVIII concentrate has been introducedin the treatment of Italian patients withhemophilia A since 15 years ago. Methods. A retrospectivesurvey was carried out with an electronicform in Italy in order to evaluate the immunogenicityof a solvent-detergent virus-inactivated plasmaderivedFVIII concentrate in previously untreatedpatients (PUPs) never exposed to other concentratesor in minimally treated patients (MTPs) previouslyexposed for up to 5 days to other concentrates.Results. The investigated population was representedby 78 patients (87% severe and 12% moderatehemophiliacs) with ages ranging from 6 to 64 years(mean=30.1, median=27.9) of whom 18 (23%) PUPsand 60 MTPs (77%). Surveyed patients had beenexposed to a solvent-detergent virus-inactivatedplasma-derived FVIII concentrate for 52 to 392 days(mean=178.0 and median=64.5). Six out of 78patients (2 PUPs and 4 MTPs) developed inhibitors(7.7%, 95% C.I. ) after 4 to 20 exposure days (median=8.0,mean=10.4 days). Of these, 5 were lowresponders (less than 5 BU/mL) and 1 was a highresponder (peak inhibitor titer=99 BU/mL, currentinhibitor titer=43 BU/mL). Two inhibitor patientscleared inhibitors spontaneously. Conclusions. Thesefindings suggest that the solvent-detergent plasmaderivedFVIII concentrate that has been used in Italyfor 15 years is at low risk of inhibitor developmentin PUPs and MTPs with hemophilia.PO-180DEVELOPMENT OF A DISEASE-SPECIFIC QUALITY OF LIFEQUESTIONNAIRE FOR ADULT PATIENTS WITH HEMOPHILIA(HAEM-A-QOL)Mackensen SV, 1 Gringeri A, 2 Santoni L, 3Tagliaferri A, 4 Tagariello G, 5 Ciavarella N, 6 Iorio A, 7Molinari C 81Institute and Policlinics for Medical Psychology,UKE Hamburg, Germany; 2 A. Bianchi BonomiHaemophilia & Thrombosis Center, IRCCS MaggioreHospital and University of Milan, Italy; 3 Centre ofPharmacoeconomics, University of Milan, Italy;4Haemophilia and Thrombosis Centre, Ospedale diParma, Parma, Italy; 5 Haemophilia Centre, OspedaleCivile, Castelfranco Veneto (TV), Italy; 6 OspedalePoliclinico I, Bari, Italy; 7 Haemophilia Centre,University Hospital, Perugia, Italy; 8 Gaslini Hospital,Genova, ItalyBackground: Hemophilia and its treatment influencethe every-day-life of patients and impact ontheir quality of life. For the adequate assessment ofquality of life validated instruments are necessary.While haemophilia-specific questionnaires for childrenare existing, up to now no validated instrumentfor adults is available. In a multi-centre validationstudy in Italy a health-related quality of life questionnairefor adults with haemophilia is in development.Aim: This study was designed to develop andvalidate a haemophilia-specific quality of life measurementfor adults (Haem-A-QoL) in Italy. Methods:The study consists of three phases: a) a qualitativestudy including focus groups, expert ratings anditem formulation, b) a psychometric study includingcognitive debriefing and feasibility testing and c)data analysis. The qualitative study has been performedby the University of Hamburg in cooperationwith the University of Milan. Results: Focus groupswith semi-structured interviews were performed in32 patients from 6 Italian haemophilia centres(Milan, Perugia, Genova, Bari, Castelfranco, Parma).14 physicians and 8 nurses were asked the samequestions. Patients and physicians/nurses considersimilar dimensions important for quality of life. Butthe order of importance is different for patients(physical health, social interaction, dependence,future, work) and physicians/nurses (dependence,work, social interaction, treatment, physical health).Based on the focus group results a draft version ofthe questionnaire was formulated and given topatients and physicians in order to evaluate the itemsconcerning importance for hemophilia and comprehensibility.A revised version of the Haem-A-QoL willbe psychometrically tested now in 10 Italian centres.Conclusions: The development of a disease-specificquality of life instrument requires multiple steps andhaematologica vol. 89(suppl. n. 8):september 2004

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 2004187the involvement of different experts (patients, physicians,nurses) in order to have a standardised andvalid questionnaire. The results of the qualitativestudy show already that the Haem-A-QoL is wellacceptedby adult haemophiliacs.PO-181SEVERE BLEEDING TENDENCY IN A PATIENT WITH MILDHEMOPHILIA B, FACTOR X DEFICIENCY AND LIVER CIRRHOSIS:SUCCESSFUL PROPHYLAXIS WITH PROTHROMBIN COMPLEXCONCENTRATECimino E, Coppola A, Sorrentino P,* Varricchione N,Di Minno M, Lombardini D, Tufano A, Di Minno GRegional Reference Centre for Coagulation Disease,and *Hepatology Unit, Dep. of Clinical and ExperimentalMedicine, “Federico II” University, Naples,ItalyA 59 yr-old man with a history of mild hemophiliaB was referred to our Institution because ofincreasing severity of a bleeding tendency, includingtwo hemorrhages from gastro-esophageal varices.On admission the patient, who had a long-lastinghistory of chronic hepatitis C, showed a clear-cutclinical picture of cirrhosis with severe portal hypertension.An unusual prolonged PT and aPTT andreduced Factor (F) X levels (10%) led to the diagnosisof combined mild FX deficiency. This was confirmedin his two sons. In several occasions FIX concentrateinfusions were unable to stop joint bleedingand the patient needed hospitalization and freshfrozen plasma or prothrombin complex concentrate(PCC) infusions. Prophylaxis with 40 IU/Kg FIX concentratetwice or thrice weekly did not reduce thepatient’s bleeding tendency: this led to progressivejoint disability. A prophylaxis regimen with 30 IU/KgPCC (Uman Complex D.I, Kedrion, Italy) was startedon April 2003, after a new episode of gastric bleeding.The patient did not show any symptom or signof cardiovascular disease nor significant risk factorsand thrombophilic abnormalities. To reduce bleedingand hospitalization frequency and to enable physicaltherapy for joint function recovery, a similarschedule was also prescribed at home. Clinical andlaboratory follow-up was carried out weekly andthen monthly; mild increase of D-Dimer was detectedbetween 4 and 24 hours after PCC infusion. However,no adverse event was documented over 1-yrtreatment. To date no further significant bleedingepisode occurred and the patient’s quality of life significantlyimproved. Thus, PCC prophylaxis has beenhelpful to manage the bleeding tendency in thispatient whose severity had been increased by thecoexistence of more genetic and acquired coagulationabnormalities.PO-182TGF-ETA TO EVALUATE ANTIVIRAL THERAPY IN HEMOPHILICPATIENTS WITH HCV-RELATED CHRONIC HEPATITISCoppola A, Tarantino G,* Conca P,* Cimino E,Sorrentino P,* Ariello M,* Scopacasa F,^Grimaldi E,^ Di Minno GRegional Reference Centre for Coagulation Disease,and *Sector of Hepatology in Internal Medicine,Dep. of Clinical and Experimental Medicine; ^Dep.Of Biochemistry and Medical Biotechnology;Federico II University, Naples, ItalyChronic hepatitis C (CHC) and its complicationsare a major problem in young-adult hemophiliacs.Treatment with interferon and ribavirin has beenshown effective in reducing hepatic fibrosis (HF). Dueto the limitations of ordinarily performing liver biopsiesin hemophiliacs, few data are available in thissetting. Recently, various serum substances reflectingliver fibrogenesis have been discovered. Amongthem, transforming growth factor-β (TGF-β) plays apivotal role, by stimulating the expression of extracellularmatrix components, inhibiting collagenasecomponents, and promoting the activation of fatstoringhepatic stellate cells towards a myofibroblastphenotype. We evaluated serum levels of this surrogatemarker of HF (ELISA) in 11 patients with hemophiliaA, aged 29.1±6.3 yrs, with CHC (genotypes1/4), comparable for clinical features and duration ofinfection (25.5±4.4 yrs), undergoing treatment (allnaïve) with peg-interferon α2b+ribavirin (canonicaldose) at baseline, after 6 mo. and 12 mo. (end) oftreatment. As expected, patients showed significantlyhigher TGF-β levels than age-matched controls(44.5 ng/mL, 28.4-48.1 vs. 28.3, 23.2-34.1,median and 95%CI; p=0.004). When compared tobaseline, irrespective of treatment outcome (3patients were non-responders/breakthrough, 8 weresustained responders–SR; for the latter, samples 6mo. after the end of treatment were also collected),TGF-β was significantly reduced after 6 mo. and at12 mo. (37.0 ng/mL, 22.6-44.1 and 27.0 ng/mL, 23.9-43.6, respectively; p

188PostersPO-183PROPOSAL OF IMMUNOTOLERANCE INDUCTION INHEMOPHILIACS A WITH INHIBITORS UNDERGOINGURGENT ELECTIVE SURGERY BY USING FVIII/VWF PUREPLASMADERIVATESMusso R, Cultrera D,* Motta S,** La Greca C,**Cipolla A, Giustolisi R*Regional Reference Haemophilia and ThrombosisCenter; *Division of Clinical Haematology, Universityof Catania; **V th Division of General Surgery,Catania, ItalyTo date, an urgent elective surgery in haemophiliacsA (HA) with inhibitor even in those low respondersor with long-standing inhibitor (LSI), not undergonein the past to immunotolerance treatment (ITT),remains a big concern. In fact, even if ITT widelyadvised from physicians is often refused. In our opinion,ITT may be really performed in expectation of apressing elective surgery. On the basis of our previouspositive ITT result in an adolescent HA with LSIhighresponder type treated with pure FVIII/vWFplasma concentrate (Fanhdi®, Grifols, Italy), we herereport a further success of ITT in an adult severehaemophiliac (43 yrs old), with low responderinhibitor (4.7 UB/mL, lasting 15 yrs) (HCV + and HIV −) and recurrent hemartroses, undergoing to urgentelective surgery. In August 2003 the patient experiencedfrequent painful colics for congenital retentionof the right testicle in the abdomen. The computeredtomography confirmed his pathology. The videolaparoscopiclaser-surgery was successfully performedby bolus infusion of Fanhdi (8.000 UI). Duringthe post-operative period the concentrate wasinfused (2.000 UI, twice/day) for one week. No bleedingwas observed and he was discharged. The patiententhusiastically continued ITT (Fanhdi, 8.000UI/week) at his home. The inhibitor progressivelysloped and the in vivo recovery (IVR)was normal (>6h) 15 days after the operation. At present, theinhibitor remains negative; the IVR is normal. Nohemartroses have been seen in these last 8 months;he actually gained his articular motions. On the basisof recent reports regarding the favourable achievementof ITT by using FVIII/vWF plasma pure derivates,we suggest that ITT induction with these productscan be considered in haemophiliacs with inhibitorundergoing to an urgent elective surgery.PO-184COST OF CARE AND QUALITY OF LIFE IN HEMOPHILIACSDEVELOPING INHIBITORS AGAINST PRODUCTS OFCOAGULATION: THE COCIS STUDYGringeri A,* Mantovani LG, # Scalone L, #Mannucci PM* and The COCIS Study Group*A. Bianchi Bonomi Hemophilia and ThrombosisCentre, IRCCS Maggiore Hospital and University ofMilan, Milan, Italy; # Center of Pharmacoeconomics,Dpt. of Pharmacological Sciences, University ofMilan, ItalyBackground. Bleeding and its complications causepain, disability and lead to impairment of hemophiliacs’quality of life (QoL). Problems become extremewhen hemophiliacs develop antibodies compromisingeffectiveness of treatment: modern strategies arelikely to have improved this situation, with a sensitiveincrease of health care cost. Aim. To estimate theeconomic burden and QoL of hemophiliacs withinhibitors. Methods. We conducted a longitudinal,prospective, multicentre Cost Of Care InhibitorsStudy (COCIS), observing inhibitory patients for 18months. Costs were evaluated from the point of viewof the Italian National Health Service (NHS). QoL wasmeasured by means of EQ-5D and SF-36 questionnaires.Results. Fifty-two hemophiliacs (median age:34.8; range:15-64) were enrolled, almost all highresponders. The orthopedic functioning was impairedin 98% of patients, 81% had at least one bleedingevent, with an average of 0.60/patient/month. Elevensurgical procedures (6 for joint replacements), 30hospitalizations, 712 out-patients visits and 702physiotherapy sessions occurred. Cost of care to theNHS was 18,000/patient/month, 99% due to treatmentproducts. Recombinant activated factor VIIrepresented 50% of total medical cost, mostly usedfor surgery. Patients showed an important impairmentof physical health perception, while psycologicalQoL was comparable to that of the Italian generalmale population. QoL of inhibitor patients wasnot different from that in other moderate and severehemophiliacs as reported by other Authors. Conclusions.Hemophilia with inhibitors represents anexample of a rare disease that requires high cost.Effective and expensive care of hemophilia withinhibitors provides a satisfactory QoL.haematologica vol. 89(suppl. n. 8):september 2004

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 2004189PO-185QUALITY OF LIFE IS ASSOCIATED TO ORTHOPEDIC STATUS INHEMOPHILIACS WITH INHIBITORSScalone L,* Gringeri A,° Mannucci PM,°Mantovani LG* for the COCIS Investigators*Center of Pharmacoeconomics, University of Milan,Italy; °IRCCS Maggiore Hospital, Milan, ItalyAim. We evaluated Quality of Life (QoL) in hemophiliapeople with inhibitors and investigated if QoLis associated to patients’ clinical status. Methods: TheCOCIS (Cost Of Care of InhibitorS) study was a longitudinalstudy involving 52 hemophiliacs (median age35, 15-64) with inhibitors, 98% high responders(>5BU), sequentially enrolled at 11 Italian HemophiliaCare Centers. Information on demographics, clinicalstatus, health-care resources consumption wasprospectively collected. QoL was investigated bymeans of generic instruments: results from EuroQol-5D are reported. Results. The majority of patientsreported some/moderate problems in domainspain/discomfort (72%), mobility (66%) and usualactivities (54%). Around one third declaredsome/moderate problems with anxiety/depression(34%) and self-care (32%). No more than 6% ofpatients reported severe problems in any of thedomains. The EQ-VAS was normally distributed, witha median value of 66 (30-95). Patients with moresevere orthopedic impairment perceived more problemswith physical component of QoL, while no associationwas found with anxiety or depression. TheOrthopedic Joint Score, a measure of the orthopedicfunctioning, significantly correlated with EQ-VAS(Pearson’s R is -0.57, p

190PostersAt the last follow-up, 11 patients with severe hemophilia,treated on demand, showed a mean orthopedicjoint score of 4 and a mean number of hemartrhosesper year of 2.3; 9 patients with moderatehaemophilia showed a mean orthopedic joint scoreof 1.1 and a mean number of haemartrhoses per yearof 1.5. Sixteen patients began prophylaxis at a medianage of 6.5 (range 2.1-15.6). Nine out of 16received prophylaxis for a period 45 weeks per year.Before starting prophylaxis and at last control, meanorthopaedic joint score was 3.9 and 0.66 respectively(p=0.0069), and mean number of haemartrhosesper year was 3.5 and 0.47 respectively (p=0.00035).Seven/16 received prophylaxis for a period < 45weeks per year; they showed at last control a meanorthopaedic joint score of 1.2 and a mean number ofhaemarthroses per year of 2.5. Two patients are notevaluable because in immunotolerance program.Prophylaxis in severe haemophilia patients results ina significantly better orthopaedic outcome than theone observed in severe and moderate hemophiliapatients on demand treatment. In order to obtaingreatest efficacy, patients on prophylaxis must betreated without discontinuation.PO-188KIDNEY AND BLADDER STONES IN HAEMOPHILIA PATIENTSMusso R, Cultrera D,* Chiarenza A,* D'Arpa S,Cipolla N, Burgio N, Giustolisi R*Haemophilia and Thrombosis Regional ReferenceCenter, *Division of Clinical Haematology, Universityof Catania, ItalyTo date, there are few data about kidney stones(KS) and bladder stones (BS) in haemophilia patientsand it is still unknown if these complications fall inthe general population prevalence or should be considereda new emergent concern in haemophilia. Westudied our large cohort of haemophiliacs to collectin an uniform way clinical data regarding KS and BSprevalence. One hundred and twenty two [males 109,females 15 including severe Factor XI:C deficiency(n=8) and obligate symptomatic carriers (n=5)patients, with a mean age 27.07 years] were followed.KS (n=16) and BS (n=7) were found in 19 (13males, 6 females) for a total prevalence of 15.8% vs5-10% in the general population. We observed ayounger age (23.4 years) in hemophiliac patientscompared with normal population (peak of frequencybetween 40-70 years). From our observation, it isnecessary to evaluate a larger haemophilia patientnumber of different Centres to confirm our data. Amore dept research on this pathology will be usefulto know which other factors among the anamnesticand/or recurrent hematuria, substitution therapiesor antiphibrinolitics agents may play a key role inthe pathogenesis of these complications. If thesedata would be confirm, a new chapter will be addedin hemophilia related pathologies.PO-189IN VIVO RECOVERY (IVR), EFFICACY AND SAFETY OFSOLVENT/DETERGENT (S/D) TREATED PLASMA IN PATIENTSWITH RARE INHERITED COAGULATION DISORDERS (RICDs)Mancuso ME,* Santagostino E,* Morfini M,^Schiavoni M,° Tagliaferri A, # Barillari G,°°Mannucci PM**Angelo Bianchi Bonomi Hemophilia and ThrombosisCenter, IRCCS Maggiore Hospital, Milan, Italy;^Hemophilia Center, Careggi Hospital, Florence,Italy; °Hemophilia & Thrombosis Center, PoliclinicoHospital, Bari, Italy; # Hemophilia Center, ParmaHospital, Parma, Italy; °°Hemophilia Center, S. Mariadella Misericordia Hospital, Udine, ItalyVirus-inactivated concentrates are not availablefor most RICDs and virus-inactivated plasma is recommendedas therapeutic alternative. This prospectivemulticenter study evaluated the efficacy andsafety of S/D plasma (Octaplas, Kedrion, Italy), theIVR of the deficient factors and the dose regimensrequired for RICDs. Seven-teen patients wereenrolled (age: 13-65 yr; M/F: 6/11): 8 had severedeficiencies (1 afibrinogenemia, 2 FV, 1 FX, 4 FXI)and 9 had mild deficiencies (2 FV:20-27%; 6 FV/FVI-II:12-20/6-47%; 1 FXI:30%). Seronegative patientsfor HIV, HCV, HBV, HAV and parvovirus B19 were 17,14, 7, 5, and 1, respectively. Reasons for treatment:delivery (2), minor surgery (5) and major surgery (10).Octaplas (6-29 mL/Kg) was given at 6-96h intervals,according to factor half-lives and clinical features fora mean treatment duration of 5 days. FVIII and/orDDAVP were associated for replacement of FV/FVIIIdeficiency. Mean IVRs: FV 1.8 dL/Kg (0.4-2.9), FVIII0.8 dL/Kg (0.4-1.5), FXI 1.3 dL/Kg (0.6-1.8), FX 1.8dL/Kg and fibrinogen 1.7 dL/Kg. Two perioperativehaemorrhages, in a patient maintaining FXI levels20-41% and in a patient with FV 43%, were controlledby surgical haemostasis and further Octaplasadministrations. Postoperative bleeding in a FV/FVI-II deficient patient occurred when FVIII was 18% andit was controlled by giving FVIII instead of DDAVP.Rash during Octaplas infusion occurred in 1/3patients who had had reactions to FFP. No viral seroconversionwas observed during one-year follow-up.Octaplas was safe and effective in patients withRICDs and it should be preferred when virus-inactivatedconcentrates are not available.haematologica vol. 89(suppl. n. 8):september 2004

XVIII Congress of the Italian Society for Hemostasis and Thrombosis Research, Rome, Sept. 30-Oct. 3, 2004191PO-190FAILURE OF UMBILICAL-RELATED CORD BLOOD STEMTRANSPLANTATION TO CORRECT HEMOPHILIA ABruno G, Cerneca F, Andolina MIstituto per l'Infanzia IRCCS Burlo Garofolo, Trieste,ItalyIf the hematopoietic stem cells or the mesenchimalof bone marrow were able to mature intoendothelial cells, a bone marrow transplantationcould theoretically cure haemophilia A. We reportthe case of a child whose outcome seems to rule outthis hypothesis. A 6 year old child, suffering fromboth hemophilia A (Factor VIII:c

192PostersPO-192SENSIBILITY OF THE PROTHROMBIN TIME (PT) ANDACTIVATED PARTIAL THROMBOPLASTIN TIME (APTT)IN THE EVALUATION OF COAGULATION FACTOR DEFICIENCYCau C, Rolla R, Albertario M, Bellomo G,Pergolini PLaboratorio di Ricerche Chimico-Cliniche,Università del Piemonte Orientale, ASO Maggioredella Carità, Novara, ItalyCongenital or acquired coagulation factor deficiencypromotes hemorrhagic disorders. The prothrombintime (PT) and activated partial thromboplastintime (APTT) are global screening proceduresprimarily employed to evaluate coagulation abnormalitiesin the intrinsic or extrinsic pathway respectively.Deficiency in any of the factors involved inthe extrinsic or intrinsic pathways results in a prolongedthromboplastin time (PT) or in a prolongedpartial thromboplatin time (APTT). Aim of the presentstudy was to investigate the sensibility of bothThromborel S reagent/BCS instrument and Actin/FSreagent/BCS instrument (Dade Behring) in the evaluationof coagulation factor deficiency using PT andAPTT as revealing tests. A pool human plasmaobtained from 20 healthy donors was diluted in commerciallyavailable plasma deficient in factors V, VII,X, VIII and IX, in order to obtain 10, 15, 20, 25, 30,35, 40, 50, 60, 70, 80 90% decrease of the originalactivity (100%). PT (INR) or APTT (sec) determinationswere performed at any reported dilution usingThromborel S Reagent (factors V, VII and X) or ActinFS reagent (factors VIII, IX). A prolongation of the PT(increase in INR) or APTT above the normal rangewas considered as the lower detection limit for possiblecoagulation factor deficiency. The resultsobtained are reported in the following tableCoagulation factor Normal range APTT (22-33 sec) PT (0.8-1.2 INR) Residual activityFV (70-120%) 1.22 55%FVII (70-120%) 1.22 60%FX (70-120%) 1.22 55%FVIII (70-150%) 33.5 69%FIX (70-120%) 33.5 60%Dade Thromborel S reagent, Actin FS reagent andBCS intrument exhibit a high degree of sensibilityfor the investigated factors of the extrinsic (V, VII, X)and intrinsic (VIII, IX) coagulation pathway. Thedetection of abnormally high PT-INR or APTT mightresult from the occurrence of as low as 45% decreasein single factor activity.haematologica vol. 89(suppl. n. 8):september 2004

Index of authorsauthor, pageAbate C 84Abbate R 14, 24, 25, 26, 31, 38, 43, 50, 62, 69,71, 72, 73, 74, 94, 95, 96, 97, 112, 114,116, 120, 122, 124, 140, 142, 152, 153,163, 165, 167, 178, 179Abbracchio MP 15Adamo S 133, 151Adriani C 183Agangi A 189Ageno W 35, 36, 67, 87, 89, 160, 168, 169, 173Agnelli G 35, 82, 88, 89, 132, 160, 162, 163,168Agnes C 104, 107, 149Agnes R 28Agosti M 86Agrifoglio M 154Aiosa A 129Aito S 167Akhavan S 47Alamanni F 154Alatri A 63, 172, 183Albanese P 13Albertario M 156, 192Albiero E 110Albisinni R 103, 110, 162Alessandrello Liotta A 43, 69, 74, 96, 114, 116,122, 167Alessandrì A 172Alessandri C 70Allegra C 82Allegrone J 169Allemand E 100Altomonte F 152Altomonte M 152Amato V 128Ambaglio C 77Ambrosio Rl 176Ameglio F 158Amendola A 78Amendola G 100Amore C 85, 134Anastasio R 36, 67, 102, 126, 159Ancora MA 76Anderson FA Jr 38, 163, 169Andolina M 191Andreana L 167Angelico F 11Angelucci PA 158Antonelli MI 160Antonucci E 14, 178, 179, 183Aprile C 42Arca M 11Arcara M 102Ardissimo D 61Arduini D 158Arezzi N 77, 78Argento A 177Ariello M 187Armani U 41, 76, 110, 141Arnout J 17, 31, 72Arquati M 115, 153Arrigoni L 148Arrotta M 127Artoni A 99Asti D 59Attanasio AF 121Attanasio M 25, 43, 69, 95, 96, 97, 178Atterritano M 123Attinà G 183Averna M 11Baccaglini U 164Badolati G 41Bagatella P 82Bagni D 110Baldi G 61Baldi M 31, 120Balducci D 22, 86Balduini CL 15, 77, 78, 107Ballerio R 154Banas A 96, 154Bandinelli B 74, 95, 114, 153Bandinelli S 94Bandini S 124Banfi C 1Barbati G 158Barbieri SS 25Barbui T 22, 86, 139Barcellona D 40, 59, 160Barillari G 30, 190Barisone E 137Baronci C 78Baronciani L 90, 92, 93Barone M 37, 42, 81, 176Barone S 24Barsotti G 124Bartoli MG 129Barulli S 161Basili S 117, 130Bassi P 179Basta G 32, 123, 124, 143Batlle J 92, 104, 105Battaglia D 124Battaglioli T 66, 83, 160Battistelli S 131, 132Battiston M 98Baudo F 177, 183Bazzan M 87, 89, 104, 107, 149Becattini C 35, 132Beck-peccoz P 121Beconcini S 38, 73Bellomo G 156, 192Benevolo G 112Berardinelli L 28Beretta A 60Beretta C 28Beretta M 90Bergesio F 124Bergmann JF 38Berloco P 43Bernacchi M 133, 151Bernardi E 36, 82Bernardi F 56, 58Bernardi M 104, 105Bernasconi S 78, 189Bertoglio S 89Bertola A 87Bertolini S 41Bertoncello K 104, 105Bertorello N 136, 137Besada P 55Bettini F 84Bianchi M 36, 60Bianchini C 88Bianchini D 148Biasiolo A 13Biasoli C 27Bifarini B 81Biggeri A 24Biglioli P 15, 20, 154Biguzzi E 51Bilora F 83Binetti BM 59Biondi A 93Birolini A 148Bischofberger N 55Blagojevich J 38, 163, 165Blangiardo M 24Bo M 136Boccadoro M 87Bocchi L 107Boddi M 69Boddi W 144Bodzenta-Lukaszyk A 49Boemi M 109Boeri E 30Bogatti P 148Boggian O 65Boido V 141Bolis G 88Bolli P 72, 142Bologna G 152Bonamassa B 128Bonanno E 8Bonanno G 22Bonfigli AR 109Bonifacio G 67, 102, 126, 159Bonifati C 80Bonini A 138Bonizzoni E 88Borchiellini A 104Bordone S 129Bosari S 51Boschi L 153Boseggia C 99Bossone A 46, 95Bottasso B 115Brambilla M 154Brambilla P 179, 180Brancaccio V 176Brandi ML 165Braschi A 42Bringhen S 87Brogi D 114, 122, 153Bronzi C 98Brun P 120Brunati AM 19Bruno G 145, 148, 191Bruschettini A 26, 43, 74, 152, 179Brusi C 66Bucciarelli P 62, 66, 80, 102, 160Bulato C 97Büller HR 166Bulotta G 147Buonanno MT 145Burgio N 35, 46, 121, 170, 190Busani C 58Busani S 109Buttiglieri R 75, 112Buzzalino M 152Caciolli S 62Caiazza A 152Caimi L 174Caimi TM 183Cajani A 75Calabria G 76Calabrò G 129Calevo MG 76, 110Calori G 12Camaiora A 63Camera M 1, 15, 20, 25, 96, 153, 154Camicione P 76, 110

Campagna F 11Campagnoli MF 137Campana C 42Campo G, 70Campobasso N 80Canciani MT 16, 90, 92, 93Candolfi R 106Cangemi R 11, 75, 112Cangialosi C 87Cannata A 154Cannistraro D 144Cantini R 144Capani F 118Capasso F 129, 133, 146Capelli M 182Caponi C 82, 132Caponi C, 168Caporrella A 152Cappelletti A 92, 105Cappelli R 118, 131, 132, 144Cappucci F 43, 57, 84, 85, 114, 138, 176Cappuccio FP 17, 31, 71, 72Capussotti L 88Caracchini R 54Carbone C 78Carboni A 61Carboni E 38, 116Carloni D 58Carluccio M 76Carluccio MA 49, 76Carnevale R 11Carpani G 57Carraro MC 148Caruso A 128Caruso S 151Casali A 138Casali L 12Casazza F 35Casini A 31, 116, 120Castaldo G 157Castaldo M 93Castaman G 16, 44, 92, 99, 104, 105Castori L 58Catalano L 145Catalano V 134Cati G 144Catozzi L 70Cattabiani L 161Cattaneo M 2, 6, 15, 21, 51, 55, 56, 57, 79, 80,115, 164, 165Cattaneo S 152Cau C 156, 192Cauli C 183Cavalca V 25Cavallaroni K 65Cavallo F 87Cavo M 135Ceccarelli E 118Cecchi E 14, 26, 50, 152, 178, 179Cecconi N 144Cefis M 148Celentano A 9Celestini A 33, 53Celikel R 91Cella G 136Cellai AP 31, 69, 71, 116, 120, 163Cellerino L 79Cellini C 135Cenerelli S 109Cerasuolo L 129Cerbone AM 103, 110, 145, 158, 162, 189Cerè E 182Ceresa I 77Ceresa IF 78Cerletti C 9, 23, 143Cerneca F 145, 148, 191Cerone R 76, 110Cerullo M 43Cervellin G 152Cerveri I 42Cesari F 24, 73, 95, 116, 163, 167Cesaro S 136Chantarangkul V 62, 156, 181Chellini B 69Chiarelli F 117Chiarenza A 35, 46, 121, 190Chinni E 95, 138Christensen JH 32, 123Ciabattoni G 11, 117Ciampa A 176Ciarafoni I 113Ciavarella N 28, 186, 191Ciccarone E 118Cicogna L 127Cimino E 28, 103, 145, 187Cini M 14, 56, 65, 66, 68, 84, 126, 135Cipolla A 188Cipolla N 35, 46, 121, 170, 190Cipollone F 11Cipriano M 61Cirese E 158Cirillo F 28, 110, 121, 158, 162Cittadini e 85Ciulla M 51Ciuti G 38, 165Ciuti R 124Cocomazzi N 43, 85, 95, 114Colaizzo D 43, 57, 84, 85, 95, 114, 138Colla G 41Colli S 25, 96Collura M 52Colombo M 30, 102Colonna G 122Colucci M 5, 18, 23, 54, 59, 119Comeglio M 122, 153Conca P 103, 187Concolato A 83, 171Consiglio P 129Consoli A 34Conti AA 94Conti E 50Conti F 149Conti S 37, 133Conti V 78Contino L 79Contu P 40, 59Coppo M 62, 122Coppo M, 178Coppola A 9, 28, 30, 75, 103, 110, 145, 158,162, 187, 189Coppola D 153Coppola R 93Corazzi T 119, 141Cordero Di Montezemolo L 137Corsi AM 73, 74, 94, 114Cortelezzi A 88Cortellaro M 115, 153Cosmi B 14, 50, 65, 66, 68, 135Costamagna G 93Costanzi S 21Costanzo M 26, 38, 62, 140, 142, 152Coviello M 134Cozzi G 90, 92, 93Cozzi MG 151Cozzi MR 16, 98Crescenzio N 136Crippa L 146Cristalli G 21Cristiani P 176Cristofano C 124Crivelli S 106Croce AI 114Crowther M 173Cuccurullo C 11Cuccurullo F 11Cultrera D 35, 46, 121, 170, 188, 190Cupisti A 124Cuppini S 137D'Amico M 166D'Andrea G 46, 57D'Angelo A 6, 12, 75, 77, 112, 116, 125, 146,149, 176D'Armini AM 42D'Arpa S 35, 46, 121, 190D’Ecclesia A 43D'Incà M 27, 138D'Orazio A 13, 65, 127, 128D'Uva M 128Da Lio L 161Daddi G 162Dalla Giovanna S 16Dallagiovanna S 93Daming T 26Danna P 101Davì G 52, 117, 130Davidson B 13de Bellis G 146De Candia E 15, 22De Carolis S 128de Caterina A 26De Caterina R 26, 27, 32, 34, 49, 76, 123, 143De Ceglie A 134De Cristofaro R 15, 16, 22, 47, 91, 93De Curtis A 32, 33De Dominicis C 120De Filippi F 30, 102De Filippis V 91De Franceschi M 25De Francesco F 133, 151De Gaetano G 9, 23, 143de Giorgi F 152de Iaco F 152de Lucia D 133De Lucia D 129, 131, 151De Marco L 10, 16, 98de Marino C 128De Mattia D 108De Michele S 129De Micheli V 179De Palma A 152De Placido G 128De Renzo A 157De Robertis E 189De Rossi G 78De Simone C 28, 103, 145, 158, 162, 189De Stefano V 13, 65, 127, 128De Vicariis A 107Deana R 19, 97Debole S 107Decousus H 169Del Ben M 11, 70, 145Del Bono R 174Del Maschio A 142, 148Del Prato C 152Del Prete SM 151Del Rio P 146Del Rosso M 25Del Turco S 32, 123, 124, 143Del Vecchio GC 136Dell’ Elba G 54Della Valle P 6, 77, 146Demicheli M 79Dentali F 67, 87, 160, 168, 169Denti N 63Depunzio I 58Devoto GL 41di Bari F 77

Di Bernardo C 166Di Castelnuovo A 17, 31, 49, 72, 118Di Fiore R 157Di Lecce VN 70, 75, 112Di Maggio F 129Di Maio C 129Di Maira G 173Di Micco G 122Di Micco P 122, 128, 157Di Minno G 9, 28, 75, 103, 110, 121, 145, 164,187, 189Di Minno M 28, 103, 187di Napoli F 28Di Napoli P 26Di Pasquale G 182Di Perna P 28, 57Di Pierro G 158Di Prima S 127Di Pumpo M 77, 78Di Renzo L 53Di Santo A 54, 85, 134Di Santo S 99Di Stasi F 186Di Vita G 102Didona B 113Dircks C 17, 31, 72Distante A 49, 76Donati C 118Donati G 153Donati MB 9, 17, 23, 31, 32, 33, 49, 71, 72, 85,118, 134, 143Donella Deana A 19, 97Donvito V 127Dore R 42Dorigo W 69Dragani A 30Dragoni S 153Duca E 180Duca MI 151Eichinger S 164Eikenboom J 92, 104, 105Elia L 147Eligini S 25, 96Emiliani C 20Emmi V 42Erba N 179, 180Esmon CT 77Esmon NL 77Ettorre CP 28Evangelista M 32Evangelista V 24, 54Evangelisti L 95, 96, 97, 142, 165, 167Fabris F 100, 140, 160Facchini L 109Fadin M 18, 41, 64Faioni EM 6, 51, 57, 165Falanga A 19, 22, 86, 88, 139Falciani M 114, 167, 178Falcinelli E 20, 53, 119Falco A 52, 117, 130Falco P 87Faranna C 129Farina A 36Farinaro E 71Farinasso L 136, 137Fatini C 26, 72, 74, 114, 165, 178Fattorini A 77Fazi P 78Fazia M 11Febo F 166Federici AB 90, 92, 93, 101, 102, 104, 105Federico L 24Fedi S 31, 69, 74, 114, 167Felaco M 26Felici A 132, 168Feliciani M 166Fenu L 40, 59Fermo I 75, 116, 125Ferrandi P 142Ferrante F 37, 58, 62, 63, 81, 119, 180, 181Ferrara R 70Ferrari R 70Ferrazzani S 128Ferrero S 6, 51Ferrini S 24, 73, 95, 97, 167, 178Ferro D 70, 98, 99, 145, 149Ferrucci L 74, 94, 114Ferrucci LC 73Fibbi G 25Filippucci E 163Filoni C 38, 95Fimognari F 70, 75, 112Finardi A 61Finazzi G 7Finocchi A 98, 99Fioritoni G 78Florioi A 167Folco G 75Folda A 19, 97Foli C 87, 107Fontana G 51, 57, 165Forconi S 118, 153Fortuna S 41Fra GP 112Franceschetti F 182Franceschi C 109Franchi F 51Franzone G 166Franzoni C 62Frascaro M 126Fratoni S 8Freson K 4Frigerio G 60Frigerio L 60, 179Frigerio M 15, 20, 154Frisina N 123Froehlich J 38Frontoni R 181Frulla M 36, 171Frumento F 152Gachet C 21Gai V 152Gaidano G 112Gaijno TM 137Galante M 133, 151Galanti A 154Galimberti D 138Galletti M 177Galli F 119Galli M 67Gallini C 79Gallo L 142Gallo M 124Gallo S 140Gallucci M 124Gallus A 81, 163Gamba A 78, 107Gamba G 107Gambardella A 131Garagiola I 99Garancini P 12Garbarini L 100, 137Garghetti E 151Gatti S 56Gaudio A 123Gavasso S 18, 41, 64Gazzini A 74, 96, 97, 165, 167, 178Gelosa P 150Gemmati D 70Geng YJ 26, 27Gensini F 24, 50, 72, 74, 112, 178Gensini GF 14, 24, 26, 31, 38, 50, 62, 69, 71, 72,73, 74, 94, 95, 96, 97, 112, 114, 116, 120,122, 140, 142, 152, 153, 163, 165, 178,179Gentili M 166Gerdes VEA 166Germagnoli F 176Ghio S 42Ghiotto R 44Ghirarduzzi A 61, 62, 152, 160, 161, 177Giacomelli S 39Giacomelli SH 44Gianazza E 150Gianchecchi D 144Gianella A 150Gianelli U 51Gianniello F 92Giannini S 12, 20, 53Giglioli C 140, 142, 153Gioffrè M 152Giordano G 119Giordano L 32, 33Giordano P 100, 108, 136Giovanardi D 152Girardis M 109Girelli D 58Girolami A 36, 82Giudici GA 164Giuello A 112Giuliani G 31, 116Giuliani M 120Giusti B 24, 25, 73, 95, 112Giustolisi R 35, 46, 121, 170, 188, 190Golinelli MP 152Gonzi G 61Goodeve A 92, 104, 105Gori AM 31, 50, 69, 73, 74, 94, 96, 97, 114,116, 122, 153, 163Gozzini A 165Granata G 128Grandini A 58Grandone E 43, 46, 57, 84, 85, 95, 114, 138,176Grassilli S 56Grazziosetto R 166Gresele P 3, 12, 20, 23, 48, 52, 53, 54, 119, 141,181Grilli A 26Grimaldi E 187Grimaudo S 126, 159Grimwood R 87Gringeri A 28, 29, 184, 185, 186, 188, 189, 191Guagnano MT 130Gualdoni A 115Guasparini A 124Guazzaloca G 14, 50, 65, 66, 68Guercini F 133Guerrini U 150Guglielmini G 54Gugliotta L 78, 138Guidetti G 78Gussoni G 88Harden Tk 55Hartman L 83, 171Hill F 92, 104, 105Huang W 38Iacone R 71Iacoviello L 9, 17, 31, 32, 33, 49, 71, 72, 118Iannaccaro P 42, 147, 155Iapichino L 52Iavarone A 136Ieran M 161

Ieri M 142Iezzi A 11Ilari I 26, 167, 178Iliceto S 13Imberti D 35, 36, 89, 160, 168, 169Incorvaia LR 182Ingerslev J 92, 104, 105Innocenti D 50, 142Insidioso M 129Intra E 41Introcaso G 179Iori I 138, 161Iorio A 30, 37, 58, 61, 62, 63, 81, 119, 133, 152,161, 180, 181, 186, 191Iotti M 61, 138, 160, 161Izzo T 157Jacobson KA 15, 55Joshi Bv 55Kassack MU 55Kidona C 113, 157Kluijtmans LAJ 115Kowal K 49Kunicki Tj 92Kuznetsova P 163Kyrle PA 164La Greca C 188La Rosa L 148Laglia G 166Lai V 93Lamagna R 148Lamberti S 152Landini G 172Landolfi R 15, 22Lanza F 189Lapini I 14, 25, 73, 95, 97, 112Larciprete G 158Lari B 14, 63, 69, 71, 72, 74, 97Lasco A 123Latella C 111Lattuada A 101, 106Laudati G 96Laureano R 172Lavoretano S 16Lazzeri C 140Lazzerini G 32, 123, 124Lecchi A 21, 55, 79, 80, 164Ledro S 19Lee K 81Lee LH 81Lee WC 81Legnani C 14, 50, 56, 58, 65, 66, 68, 84, 126,135, 182Lemmi A 162Lensing A 13, 36, 82Lenti L 11, 98, 99Lenti M 72, 74, 96, 163, 165Leo F 42Leonardi A 120Leoncini G 41Leone G 13, 22, 65, 78, 127, 128Leone M 23, 52, 141Leoni P 161Leprini E 26, 43Lessiani G 166Lethagen S 92, 104, 105Levis A 79Libera L 106Librè L 37, 42, 81, 176Licciardello M 100Limbiati S 87Linari S 30Livadiotaki A 113, 157Lo Bue A 85Lo Manto G 126Lo Pinto G 41, 148Loffredo l 11, 70, 75, 112, 149Logullo F 161Lombardi A 122, 140, 142, 182Lombardi G 121Lombardi MR 175Lombardi R 55, 56, 164Lombardini D 103, 158, 162, 187lomonaco A 162Longoni R 107Lopatriello S 183Lorecchio G 75Lorenzet R 54, 85, 134Lorenzi B 131, 132Lorenzi C 152Lotti L 98Lu H 81Lucania A 157Lucarini L 25, 73, 95, 97, 112, 178Luciani M 30Luciano C 136Ludovici S 182Luise F 111Lunghi B 56, 58lupattelli L 162Lupo E 159Lupone MR 46, 151Lussana F 51, 79, 80, 115, 165Luzzatto G 100Macarone Palmieri N 158Macchi C 94Machetti S 144Mackensen SV 186, 191Madeo D 39, 44, 99, 110Madonna P 110Madonna R 26, 27, 34Magaldi L 43Maggiano N 15Magi A 24Maina A 127Maisto G 46, 151Malatesti V 173Malato A 67, 102, 126, 159, 183Maltoni P 182Mameli G 40Manarini S 24, 54Mancino A 27Mancuso G 30Mancuso ME 29, 30, 190Mandelli F 78Mandich P 41, 110Manfredi E 67, 173Manigrasso MR 130Mannini L 26, 43, 114, 152, 178, 179Manno C 80Mannucci PM 4, 16, 28, 29, 30, 46, 47, 62, 66,83, 90, 99, 102, 160, 164, 184, 185, 188,189, 190Manoni M 96Manotti C 61, 175, 183Mantovani l 191Mantovani LG 184, 185, 188, 189Marchese M 93Marchese P 21Marchetti M 19, 22, 86, 139Marchiori A 13, 36, 64, 82, 171Marcucci R 14, 31, 43, 69, 71, 94, 120, 122,124, 153, 163, 165, 167Margaglione M 28, 43, 45, 46, 57, 84, 85, 95,114, 138, 176Margheri M 122, 153Mari D 109Mariani G 102, 159Marietta M 27, 109Marinopiccoli M 130Marongiu F 40, 59, 160, 183Marotta G 144Marra M 109Marroni M 119Martellenghi E 63Martelli A 182Martelli N 54, 85Martinelli I 62, 66, 83, 160Martinelli N 58Martinelli P 95, 189Martini G 174Martino D 134Martinotti S 11Masotti L 118, 144Massaro M 26, 34, 49, 76, 143Massi Benedetti M 119Mastronuzzi VMA 153Matarese MR 100Matera R 189Mattarozzi S 65, 68, 84Matteucci A 144Mattiello T 33Matucci-Cerinic M 25Maugeri N 23, 143Mauriello A 8Mavilia C 165Mazo S 131Mazzei G 63Mazzola G 12, 75, 112, 116, 125Mazzucato M 16, 98Mazzucconi MG 30, 78, 189Meis S 55Melillo L 138Mencarelli S 20Mendolicchio GL 91Menegatti M 47, 156Menozzi I 129Meo A 35, 121Meola M 162Mera V 67Merola B 121Messeri G 38Messina C 136Messina M 30Metafora S 122Meyer D 92, 104, 105Mezzasoma Am 52Mezzetti A 11Micelli M 80Michelucci A 50Micieli E 173Milillo G 162Milio G 67Milite MT 70, 75Mimmi P 182Mina L 141Minciotti A 63Mingozzi F 56Miniati B 94Minniti G 76, 110Mirone P 79Misso M 133, 151Mistretta C 92, 93, 102Mochetti F 179Mogni S 102Moia M 82, 88, 164Molinari A 41Molinari AC 136Molinari C 186Molinatti M 177Mollo A 128Momi S 12, 23, 48, 54Monaco M 114Moneti G 116Moniello G 131

Monopoli A 54Monreal M 38Monsù R 152Montali A 11Montani N 107Montaruli B 104, 107, 149Montella M 146Monzani G 124Monzini MS 184, 185, 186Morabito N 123Moratelli S 27Morelli B 151Morello M 120Morfini M 29, 190Mori M 41, 173Moroni B 93Moroni G 51Morra E 177Morstabilini G 63, 172Morstabilini P 174Moschini L 61Mosena L 36, 171Mostarda G 177Motta S 188Muça-perja M 28Mugelli A 120Murgi E 189Murzilli S 32, 33Musso G 152Musso R 35, 46, 121, 170, 188, 190Mussoni L 1, 96, 154Mutignani M 93Naglieri E 89Naliato M 154Napoleone E 54, 85, 134Nardone G 103Natalizi A 37Navarra T 124Negri D 63Negri EA 138Negrini C 183Nenci GG 37, 63, 180Nicolin R 30Nicolini A 61, 138Niglio A 122, 128, 157Nigro A 100, 108, 136Nobili B 100Nobili E 150Nocenti F 152Noris P 77, 78, 107Notarangelo A 114Notarbartolo A 11Oca G 126, 181Ohno M 55Ongini E 54Orefice G 110Oriana V 111Orlacchio A 20Orsini Federici M 119Paccamiccio E 180, 181Pacini G 130Padeletti L 50Page C 12, 48Pagnan A 18, 36, 41, 82, 83Pagnini P 26Pagnoncelli M 19Palaia R 146Palareti G 8, 14, 50, 56, 58, 65, 66, 68, 82, 84,126, 135, 160, 164Palla R 47Palmarini MFG 62Palmieri G 8Palmieri V 9Palumbo A 87Pampuch A 49Pancaldi LG 182Pandolfi A 34Pani A 63Paniccia R 38, 43, 62, 140, 142, 179, 181Panigada G 172Paoletti R 150Paolini R 137Papa A 123Papa ML 129, 133, 146Papa R 129Papaleo G 155Papi M 113Parazzini F 88Paris M 136Parise P 89Parisi V 146Parodi E 100Parolari A 154Parolari A, 154Pasetto A 109Pasi J 92, 104, 105Pasquini M 180Passamonti SM 66Passariello N 131Passera R 81Pastorello M 152Patrono C 117Patruno R 134Pattacini C 27, 61, 175Pattarini E 146Patti R 102Pavanetto M 97Peake I 92, 104, 105Peake Ir 92Pecce R 24Pecci A 15, 77, 78Pecoraro M 95Pedrazzoli F 107Peduto F 81Pegoraro C 13Pellati D 120, 136Pellecchia A 134Pellegrini L 181Pellegrino M 67Pengo V 5, 13, 177, 183Pepe G 24, 25, 62, 73, 95, 112Perdriset G 81Pergolini P 156, 192Perlati M 18, 64Perricone F 46, 133, 151Perrone L 41, 76, 110Petrobelli F 83Petronella K 41Petruzzellis S 80Petruzziello L 93Peyvandi F 16, 45, 46, 47, 99, 156Piana A 76, 110, 141Piccioli A 82Piccoli A 24Pignalosa O 133, 151Pignatelli P 11, 53, 98, 99, 145, 149Pili C 14, 50Pini M 38, 169Piovella C 37, 42, 81, 176Piovella F 37, 38, 42, 81, 169, 176Piraino L 129Piro D 18Piromalli A 111Pistelli R 82Pitchford SC 12, 48Pizzoleo MA 123Planes A 81Podda G 56Podda Gm 21, 51, 80, 115Poggi F 24, 25, 74, 112, 179Poggi M 68, 126, 182Poggini L 189Poggio R 35Pogliani E 35Pogliani EM 88Pola E 79Poli D 14, 50, 112, 152, 177, 178, 179, 183Pollio G 145Pomilio M 117Ponticelli C 51Porciani MC 50Porro F 35Porta B 115, 153Pozzoli D 27Prandoni P 1, 13, 18, 35, 36, 41, 64, 82, 83,160, 171Pratesi C 71, 72Pratesi G 69, 71, 72Preda L 106Pretelli P 62Primignani M 160Prins M 13, 36, 82Prisco D 14, 26, 38, 43, 50, 62, 69, 71, 97, 122,140, 142, 152, 153, 163, 165, 178, 179Pudore L 129, 133, 146Pulcinelli FM 3, 33, 53, 98Pulini S 161Pulli R 71, 72Puma F 162Quaranta M 134Quattrin S 78Quintavalla R 89, 160, 175Ragusa M 162Raimondi L 120Rainaldi G 32Ramazzina E 137Ramenghi U 100Rampinini F 151Randi Ml 100, 140Ranieri G 134Ranieri P 80Ravaglia F 161Ravera GB 41Re G 152Reati R 160Remollino V 152Restifo D 148, 179Ria L 160Ricci L 167Ricciardi A 152Rigo F 41Riondino S 53, 98Roberti S 92Rocino A 29, 30Rodeghiero F 16, 39, 44, 45, 92, 99, 104, 105,110Rogolino A 43, 69, 71, 74, 96, 163Rolla R 156, 192Romano M 52, 130Romualdi E 169Ronconi P 93Rosafalco M 180Rosana A 148Rossetto V 18Rossi A 27Rossi C 54Rossi E 13, 16, 65, 101, 106, 115, 127, 128,142, 148Rossi F 106Rossi L 24, 25, 74, 97, 112, 179Rossi P 98, 99Rossi R 20, 53, 82, 88Rossiello MR 18, 54

Rotilio D 32, 33Rotoli B 145Rotunno C 18Rouillon A 81Rovinetti C 182Ruffolo F 146Ruggeri ZM 21, 91Rumi MG 30, 102Ruotolo G 12Rupoli S 161Rus C 87, 107Russo G 100, 136Russo L 86Russo T 133, 151Russo U 101Russo V 129, 133, 146Rutella S 22Ruzzi L 113Ruzzon E 140Sabatini F 176Sabatini M 160Sabbà C 108Sacchi E 101, 109Safa O 77Saggiorato G 120, 136Saibeni S 51Saitta M 104, 107, 149Sala A 75Saliola M 145Salomon Dr 92Salvadori M 14, 124Salvi R 35Salvia M 129Samiotaki-Bisceglia F 113, 157Sammicheli L 144Sampietro F 12, 75, 112, 116, 125Sangiorgi G 8Sanguigni V 98, 99, 145Santacroce R 28, 45, 57, 84Santagostino E 28, 29, 30, 45, 102, 190Santi R 79Santilli f 117Santoni L 186, 191Santoro A 89Santoro BM 137Santoro C 189Santoro R 42, 147, 155Santozzi M 166Saporito A 152Saracco P 108, 136, 137Saracini C 25, 178Sardone V 80Sardu C 59Sartor D 82, 83, 171Sartori MT 120, 136Savi P 54Savoia A 77, 78Sbrighi P 70Scalone L 184, 185, 188, 189Scamardi R 123Scambia G 22Scanavini D 56, 58Scandellari R 100Scannapieco G 160Scapoli GL 70Scaraggi FA 186Scarpa RM 88Schena Fp 80Schenone A 148Schiavoni M 28, 190Schmidt EB 32, 123Schneppenheim R 92, 104, 105Schunemann H 2Sciannamè N 84, 114Sciolla A 152Scirelli T 104Scoditti E 49Scoditti U 160Scognamiglio F 99Scopacasa F 187Scortechini AR 161Scotto F 134Scudiero O 157Seghi F 96Semeraro N 5, 18, 54, 59Sensini A 119Serafini F 182Seregni R 142Serino Ml 70Servettini I 58, 181Sessa F 57Sessa M 133, 151Sestini I 24, 69, 71, 112, 163, 179Sfiridaki A 113, 157Shamasbloo M 166Siani A 31, 71, 72SianiA 17Sidiropulos M 103, 162Sidoni A 119Silingardi M 35, 36, 62, 138, 161Silvestri A 131Silvestri L 113Simioni M 39, 110Simioni P 18, 41, 64, 82Siragusa S 67, 102, 126, 159, 183Sirolla C 109Sironi L 150Skordilaki A 113, 157Skovlund S 191Socci M 43Sodi N 144Soffredini R 102Sofi F 31, 69, 71, 72, 74, 116, 120, 122, 153,165Solazzo V 69Sonaglia F 88Sorarù M 89Sorge R 145Sorrentino P 187Sottile L 123Sottilotta G 42, 111, 147, 155Spada C 93Spagnoli Lg 8Sparatore F 141Spiezia L 120, 136Spigonardo F 11Spisni L 152Squizzato A 166Stabile A 119Stefanin C 151Stefano Pl 62Stefanoni M 131, 132Steffan A 16Steidl L 67, 87Stella M 170Sticchi E 26, 50, 72, 74, 165Stoppini A 176Storelli C 49, 76Strazzullo P 71Strina I 128Strippoli Gfm 80Stroppolo A 158Suffritti C 109Suno A 156Surrenti C 31, 116, 120Taddei S 11Tagariello G 29, 30, 186, 191Tagliabue L 45, 156Tagliaferri A 27, 29, 30, 61, 133, 151, 175, 186,190, 191Taliani Mr 35, 132, 160Tancredi MA 182Tanganelli P 32Tapson VF 169Tarantino G 187Tassetti A 161Tassoni MI 175, 183Tatini S 172Tchilibon S 55Tellini I 62, 140, 142Terranova E 141Terranova L 56Tesoro S 81Tesorone M 129Testa I 109Testa R 109Testa S 63, 172, 174, 183Tezza F 140Tini N 145Tiozzo F 13Tiraferri E 177Tirelli P 131Tiscia G 114Tiscione V 152Tognazzo S 70Tognin G 18, 41Toldo C 140Tonelli F 88Toniato E 11Toogeh G 99Torelli G 109Tormene D 18, 36, 41, 64, 171Torre S 129, 133, 146Torti M 78Tosetto A 39, 44, 99, 104, 105, 110, 160, 177Tosi PL 124Totani L 24Trabalzini L 153Trapani Lombardo V 111Trapani MR 129Tremoli E 1, 15, 20, 25, 75, 96, 150, 153, 154Trenti C 138Trifiletti A 123Trifirò E 53Tringali A 93Tripodi A 59, 62, 156, 181Tripodi MF 167Tronchin M 38Trottini S 180Tsagarakis N 113, 157Tufano A 9, 28, 103, 110, 145, 158, 162, 164,187, 189Tufano MA 122Turchetti V 153Turetta L 97Turpie AG 81Uccellini M 148Ucchino S 11Ulivi M 110Ullmann H 55Ursi S 11Vaccarino A 87, 104, 107, 149Valdrè L 14, 27, 66Vaghi U 106Valarani B 88Valdrè L 135Valente S 140, 142, 153Valerio P 134Valesini G 149Valle D 121Valori A 137Valpreda A 104Van Geet C 4Vandelli A 152Vannini Ml 40

Varricchione N 110, 158, 162, 187Varughese KI 91Varvarikis C 136Vecce R 144Vecchi M 51Vecchio S 31, 116, 120Vecchioli M 180Vecchione G 43, 85, 95, 114Vedovati C 132Veglia F 154Venco A 67Vendramin C 112Vercelloni S 180Vergura P 43, 84, 85Vermylen J 4Vernillo R 131Verrastro G 76Versiero M 71Verso M 82, 89Vianelli N 78Viganò D'Angelo S 6, 146Viganò M 42Viggiano G 157Vigliaroli L 43Vigneri S 117, 130Vignoli A 19, 22, 86, 139Villa A 152Villa I 180Villa M 184, 185Villalta S 82Vincenzi D 27Violi F 3, 11, 33, 53, 70, 75, 98, 99, 112, 145,149Virdis A 11Vitale G 129Vittori S 21Vitullo G 11Volpi R 118, 174Vorlova Z 92, 104, 105Wang CJ 81Wells P 87Weltermann A 164Willerson JT 27Za T 13, 65, 127, 128Zamagni E 135Zamboni S 137Zampolli A 49, 124, 143Zanazzi M 14Zanna M 152Zappalà D 147Zerbinati P 18, 41Zighetti Ml 51, 55, 56, 79, 115Zignego Al 69Zito F 17, 31, 72, 118Zocca N 100Zoratti R 152Zotz RB 169Zurlini C 175