title: Antigenicity of putative phospholipid membrane-binding residues in factor VIII.
abstract: 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 the 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, Val2223, and Leu2251/Leu2252. Additionally, Lys2227, near Val2223, is part of a ring of positively charged residues that may contribute to electrostatic interaction of fVIII with negatively charged phosphatidylserine. In this study, 8 active mutants of human fVIII (Met2199Ile, Leu2252Phe, Phe2200Leu, Val2223Ala, Lys2227Glu, Met2199Ile/Phe2200Leu, Val2223Ala/Lys2227Glu, and Met2199Ile/Phe2200Leu/Val2223Ala/Lys2227Glu), which were constructed on the basis of differences between human, porcine, murine, and canine fVIII at proposed phospholipid binding sites, were expressed. The antigenicity of the mutants toward 5 C2-specific polyclonal human antibodies was measured by using the Bethesda assay. A human monoclonal anti-C2 antibody, BO2C11, and a murine C2-specific monoclonal antibody, NMC VIII-5, were also included in the analysis. In comparison with wild-type, B-domainless fVIII, the Met2199Ile, Phe2200Leu, and Leu2252 single mutants had lower antigenicity toward most of the inhibitors. In contrast, the Val2223Ala and Lys2227Glu mutants usually showed increased antigenicity. These results suggest that C2 inhibitors 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, Val2223 and Lys2227 may oppose antibody binding sterically or through stabilization of a low-affinity membrane-binding conformation of the C2 domain.
authors.affiliation: Winship Cancer Institute, Emory University, Atlanta, GA 30322, USA.
authors.forename: R T
authors.initials: RT
authors.lastname: Barrow
authors.forename: J F
authors.initials: JF
authors.lastname: Healey
authors.forename: M G
authors.initials: MG
authors.lastname: Jacquemin
authors.forename: J M
authors.initials: JM
authors.lastname: Saint-Remy
authors.forename: P
authors.initials: P
authors.lastname: Lollar
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D000941:Antigens / Q000276:immunology; D001665:Binding Sites / Q000235:genetics / Q000276:immunology; D005169:Factor VIII / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D006467:Hemophilia A / Q000097:blood / Q000276:immunology; D006801:Humans; D008563:Membrane Lipids / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010743:Phospholipids / Q000378:metabolism; D011485:Protein Binding; D016415:Sequence Alignment
chemical_list: D000911:Antibodies, Monoclonal; D000941:Antigens; D008563:Membrane Lipids; D010743:Phospholipids; D005169:Factor VIII