SOME FACTOR-VIII INHIBITOR ANTIBODIES RECOGNIZE A COMMON EPITOPE CORRESPONDING TO C2 DOMAIN AMINO-ACIDS-2248 THROUGH AMINO-ACIDS-2312, WHICH OVERLAP A PHOSPHOLIPID-BINDING SITE

The finding that human factor VIII (fVIII) inhibitor antibodies with C 2 domain epitopes interfere with the binding of fVIII to phosphatidyls erine (PS) suggested that this is the mechanism by which they inactiva te fVIII. We constructed a recombinant C2 domain polypeptide and demon strated that it bound to all six human inhibitors with fVIII light cha in specificity. Thus, some antibodies within the polyclonal anti-light chain population require only amino acids within C2 for binding. Reco mbinant C2 also partially or completely neutralized the inhibitor tite r of these plasmas, demonstrating that anti-C2 antibodies inhibit fVII I activity. Immunoblotting of a series of C2 deletion polypeptides, ex pressed in Escherichia coli, with inhibitor plasmas showed that the ep itopes for human inhibitors consist of a common core of amino acid res idues 2248 through 2312 with differing extensions for individual inhib itors. The epitope of inhibitory monoclonal antibody (MoAb) ESH8 was l ocalized to residues 2248 through 2285. Three human antibodies and ant i-C2 MoAb NMC-VIII/5 bound to a synthetic peptide consisting of amino acids 2303 through 2332, a PS-binding site, but MoAb ESH8 did not. The se antibodies also inhibited the binding of fVIII to synthetic phospho lipid membranes of PS and phosphatidylcholine, confirming that the blo cked epitopes contribute to membrane binding as well as binding to PS. In contrast, MoAb ESH8 did not inhibit binding. As the maximal functi on of activated fVIII in the intrinsic factor Xase complex requires it s binding to a phospholipid membrane, we propose that fVIII inhibition by anti-C2 antibodies is related to the overlap of their epitopes wit h the PS-binding site. MoAb ESH8 did not inhibit fVIII binding to PS-c ontaining membranes, suggesting the existence of a second mechanism of fVIII inhibition by anti-C2 antibodies. (C) 1995 by The American Soci ety of Hematology.