Antigenicity of putative phospholipid membrane-binding residues in factor VIII

Most inhibitory antibodies to human factor VIII (fVIII) bind to epitopes in the A2, ap-A3, or C2 domains. The anticoagulant action of antibodies to th e C2 domain is due to inhibition of binding of fVIII to phospholipid. The x -ray structure of the human fVIII C2 domain shows a putative hydrophobic, 3 -prong, phospholipid membrane-binding site consisting of Met2199/Phe2200, V al2223, and Leu2251/Leu2252. Additionally, Lys2227, near Val2223, is part o f a ring of positively charged residues that may contribute to electrostati c interaction of fVIII with negatively charged phosphatidylserine. In this study, 8 active mutants of human fVIII (Met2199lle, Leu2252Phe, Phe220Leu, Val2223Ala, Lys2227Glu, Met2199lle/Phe2200Leu, Val2223Ala/Lys2227Glu, and M et2199lle/Phe2200Leu/Val2223Ala/Lys2227Glu), which were constructed on the basis of differences between human, porcine, murine, end canine fVIII at pr oposed phospholipid binding sites, were expressed. The antigenicity of the mutants toward 5 C2-specific polyclonal human antibodies was measured by us ing the Bethesda assay, A human monoclonal anti-C2 antibody, BO2C11, and a murine CS-specific monoclonal antibody, NMC VIII-5, were also included in t he analysis. In comparison with wild-type, B-domainless fVIII, the Met2199l le, Phe2200Leu, and Leu2252 single mutants had lower antigenicity toward mo st of the inhibitors. In contrast, the Val2223Ala and Lys2227Glu mutants us ually showed increased antigenicity. These results suggest that C2 inhibito rs frequently target the Met2199/Phe2200 and Leu2251/Leu2252 beta -hairpins and are consistent with the hypothesis that these residues participate in binding to phospholipid membranes. In contrast, Val2223Ala and Lys2227 may oppose antibody binding sterically or through stabilization of a low-affini ty membrane-binding conformation of the C2 domain. (C) 2001 by The American Society of Hematology.