2003; baxter - Supplements - Haematologica

IV International Workshop on Immune Tolerance in Hemophilia 13hemophilia and the frequency of FVIII infusionsis less common. Occasionally, patients with missensemutations develop inhibitors in responseto increased FVIII usage.Additionally, FVIII is the most likely coagulationprotein to be targeted in autoimmune reactions,producing a condition termed acquiredhemophilia or acquired hemophilia A. The incidenceof this condition is very low, being approximately1 in 5 million people. It usually manifestsas a serious bleeding disorder, which anecdotally,and for unknown reasons, is more severethan when inhibitory antibodies develop inpatients with hemophilia A. FVIII autoantibodiesdevelop in a variety of clinical settings, includingthe postpartum period, systemic lupus erythematosus,and chronic lymphocytic leukemia. Inapproximately 50% of the cases, acquired hemophiliais idiopathic. 19In both hemophilia A and acquired hemophilia,FVIII inhibitors are polyclonal IgG populationsthat are usually directed toward more than oneepitope. For unknown reasons, the IgG4 subclassis more common in plasma of inhibitor patientsthan in the normal population. Inhibitors areclassified into two types. 20,21 Type I inhibitorsinactivate FVIII completely with second-orderkinetics, which would be expected for a simplebimolecular antigen-antibody reaction. Type IIinhibitors inactivate FVIII incompletely and displaymore complex kinetics of inhibition. Mostautoantibody patients have type II inhibitors. Inmost cases, type II inhibitors behave like type Iinhibitors when they are tested against FVIII inthe absence of vWf, indicating that inhibition bythe type II antibodies is blocked due to competitionfor binding by vWf. 22FVIII domains recognized by inhibitoryantibodiesA comprehensive analysis of the antibodyresponse to FVIII in either human disease or inanimal models does not yet exist. A hierarchicalanalysis would answer whether: 1) the humoralimmune response is restricted to only some of theFVIII domains; 2) antibody binding to epitopesin different domains is independent or co-operative;3) there are single or multiple immunodominantepitopes within a domain; 4) andwhether there is variation in the amino acidswithin a single FVIII epitope (corresponding toan antibody footprint) that contribute significantlyto antibody binding energy.SDS-polyacrylamide gel electrophoretic analysisof FVIII reveals bands corresponding to FVIIIheavy chain (A1-A2-B) and light chain (ap-A3-C1-C2). After exposure to thrombin, the threeheterotrimer bands, A1, A2, and A3-C1-C2, areapparent. Initial epitope mapping studies byWestern blotting of purified FVIII revealed thatmost patients’ antibodies bound to the A2 andA3-C1-C2 domains. 23 The relative lack of anti-A1 reactivity was an early indicator that the B-cell response to FVIII was restricted. Subsequently,the C2 domain was identified as the mostcommon target for anti-light chain antibodies byWestern blotting of defined domain fragments ofrecombinant FVIII produced in E. coli. 24,25 Anti-A3antibodies were also detected, although less frequently.Some inhibitory antibodies do not binddenatured FVIII in Western blotting experimentsand can be detected by immunoprecipitationusing soluble FVIII fragments. 26An important complementary method to Westernblotting analysis is antibody neutralization, inwhich recombinant or plasma-derived fragmentsof FVIII are used to block inhibitory antibodies.25,27,28 The sum of neutralization of inhibitoryactivity by the A2 domain and FVIII light chain(ap-A3-C1-C2) fragment approaches 100% inmost cases, 28 indicating that antibodies to the A1domain do not contribute significantly to mostinhibitor titers. By comparing antibody neutralizationby the ap-A3-C1-C2 fragment to that bythe C2 fragment, the relative contribution of anti-C2 antibodies can be assessed. Most anti-lightchain activity is against the C2 domain, althoughin some plasmas there is significant activityagainst ap-A3-C1. The dominant inhibitors inmost autoantibody plasmas are directed onlyagainst C2 or A2, but not both. In contrast, mosthemophilia A inhibitor plasmas recognize multipleepitopes. However, the similarity betweenalloantibodies and autoantibodies is perhaps morestriking than the difference, because the A2 andC2 domains are immunodominant in both settings,despite the different immunologic backgroundsfrom which they arise.Mechanism of action of FVIII inhibitorsFVIIIa binds factor IXa, 29 phospholipid 30 andfactor X 31 in the intrinsic FXase complex. Thus,disruptions of any of these interactions by anantibody could be inhibitory and all these possibilitieshave been observed. Additionally,inhibitors could function by binding to sites thatare recognized by thrombin or factor Xa. However,this mechanism of action has not been convincinglydemonstrated.Anti-A2 inhibitors inhibit intrinsic FXase noncompetitively,i.e., most likely by blocking thebinding of FVIIIa to factor X. 31 However, inhibitionof binding of FVIIIa to factor IXa by anti-A2inhibitors also has been observed. 32 Many, if notall, anti-C2 antibodies inhibit the binding of FVIIIto phospholipid. 33 However, the C2 domain alsobinds vWf, 34,35 at or near the phospholipid bindingsite. Thus, vWF could interfere with the bindingof anti-C2 antibodies, producing the type IIantibody pattern described above. Consistentwith this, polyclonal patients’ antibodies that recognizeonly the C2 domain are much more commonin autoantibody patients, 28 in whom mosttype II antibodies are found. 20,22Thus, another possible mechanism of action ofinhibitors could be to block the binding of FVIIIto vWf. In contrast to disruption of the intrinsicFXase complex or inhibition of proteolytic acti-haematologica vol. 88(supplement n. 12):september 2003