2003; baxter - Supplements - Haematologica

14P. Lollarvation, inhibition of vWf binding would not bedetected by the Bethesda assay. Inhibitors havebeen described that block the binding of FVIII tovWf, and this may be a common property of anti-C2 inhibitors. 36 However, the relative contributionto the bleeding diathesis of inhibition ofbinding of FVIII to vWf versus binding to phospholipidis unknown.High resolution mapping of FVIII inhibitorepitopesHomolog scanning mutagenesis using hybridhuman/porcine, human/canine, or human/murineFVIII molecules has been successful inobtaining higher resolution maps of the B-cellepitopes. 37,38 This strategy is based on the observationthat inhibitors usually have limited crossreactivitywith non-human FVIII. A hybrid FVIIImolecule that is less reactive with an inhibitor tohuman FVIII localizes the inhibitor to the regionin which the non-human substitution has beenmade. Because the hybrids are active, reduction inantigenicity is unlikely to be due to improper foldingof the protein. In contrast, epitope localizationby deletion mapping is complicated by thepossibility that loss of antigenicity can occur bydenaturation of the polypeptide chain.Using this method, the A2 epitope recognizedby most A2-specific antibodies has been localizedto residues Arg 484 -Ile 508 . Analysis of several hybridscontaining A2 substitutions indicates that the A2epitope appears to be confined with the Arg 484 -Ile 508 linear segment. This is consistent with thefact that this segment protrudes as a large loopbetween β-strands in a homology model of the A2domain based on the X-ray structure of ceruloplasmin.The A2 epitope has been mapped further by alanine-scanningmutagenesis. Human A2-specificpolyclonal antibodies recognize several aminoacids in this segment, including Tyr 487 and to alesser extent Ser 488 , Arg 489 , Pro 492 , Val 495 , Phe 501 ,and Ile 508 . 39 There is variability between the antibodies,indicating that although the antibodyfootprint may be relatively invariant, individualamino acid residues recognized by the antibodiesdiffer.The region recognized by several C2-specificinhibitors has been mapped using a panel ofhybrid human/porcine FVIII molecules to residuesGlu 2181 -Val 2243 . 37 Recently, an X-ray structure ofthe human FVIII C2 domain has been solved, providinga more detailed approach to the analysis ofC2 inhibitors. The structure reveals a putativehydrophobic three-prong phospholipid membrane-bindingsite consisting of Met 2199 /Phe 2200 ,Val 2223 , and Leu 2251 /Leu 2252 . 40 Additionally, severalbasic residues contribute to a ring of positivelycharged residues that may contribute to an electrostaticinteraction of FVIII with negativelycharged phosphatidylserine. Several active humanFVIII mutants were constructed based on differencesbetween human, porcine, murine, andcanine FVIII at proposed phospholipid-bindingsites. The antigenicity of the mutants toward C2-specific polyclonal human antibodies, a humanmonoclonal anti-C2 antibody, BO2C11, 41 and amurine C2-specific monoclonal antibody, NMCVIII-5, 42 was measured. The results suggest thatC2 inhibitors frequently target the Met 2199 /Phe 2200and Leu 2251 /Leu 2252 β-hairpins, which is consistentwith the hypothesis that these residues participatein binding to phospholipid membranes. Incontrast, mutations at Val 2223 increased antibodybinding, indicating that Val 2223 may oppose antibodybinding sterically or through stabilization ofa low-affinity membrane-binding conformationof the C2 domain. Subsequently, the X-ray structureof BO2C11 in complex with the FVIII C2domain was solved and revealed contact with theMet 2199 /Phe 2200 and Leu 2251 /Leu 2252 . 43Delineation of the boundaries of an epitopeusing hybrid FVIII molecules can be limited bysequence identity. For example, human andporcine FVIII are identical from residues 2243(the end of the sequence mapped using human/porcinehybrids) through 2252. Thus, identificationof the epitope extending to residue 2252was not possible using hybrid human/porcineFVIII.Subsequently, we identified four C2-specificplasmas that do not appear to recognize the phospholipid-bindingregion (Barrow RT and Lollar P,personal observations). The functional propertiesof these antibodies have not been characterized.Overall, the analysis of C2 inhibitors appearsconsiderably more complex than that of A2inhibitors both with respect to epitope structureand mechanism of inhibition.Regions outside the A2 and C2 domains appearto be minor targets for inhibitory antibodiesbecause the inhibitory activity in most plasmascan be neutralized by the A2 and C2 domains. 28However, three inhibitor IgGs have been identifiedthat are neutralized by a synthetic peptidecorresponding to residues 1804-1819 in the A3domain. 44 This region encompasses a regiondefined by residues 1811-1819 that constitutespart of the factor IXa binding site. 45 Additionally,several inhibitor plasmas have been identified inwhich substitution of the human ap region withthe corresponding porcine segment results in areduction of antigenicity. This indicates that theap segment also contains an inhibitory epitope. 46Unusual FVIII inhibitorsInhibitory antibodies have been described thatrecognize epitopes outside the A2, C2, A3 and apregions. A human monoclonal antibody, LE2E9,is an IgG4k antibody that it is directed against theC1 domain and was derived from a patient withmild hemophilia A who has an Arg 2150 His mutation.47 The antibody recognizes wild-type FVIIIbut not endogenous FVIII, indicating that theantibody recognizes Arg 2150 . The antibody is a type2 inhibitor and blocks FVIII binding to vWf. Similarly,an inhibitor from a patient with mildhemophilia A due to an Arg 593 Cys mutation inhaematologica vol. 88(supplement n. 12):september 2003