title: High-resolution mapping of epitopes on the C2 domain of factor VIII by analysis of point mutants using surface plasmon resonance.
abstract: Neutralizing anti-factor VIII (FVIII) antibodies that develop in patients with hemophilia A and in murine hemophilia A models, clinically termed "inhibitors," bind to several distinct surfaces on the FVIII-C2 domain. To map these epitopes at high resolution, 60 recombinant FVIII-C2 proteins were generated, each having a single surface-exposed residue mutated to alanine or a conservative substitution. The binding kinetics of these muteins to 11 monoclonal, inhibitory anti-FVIII-C2 antibodies were evaluated by surface plasmon resonance and the results compared with those obtained for wild-type FVIII-C2. Clusters of residues with significantly altered binding kinetics identified "functional" B-cell epitopes, defined as those residues contributing appreciable antigen-antibody avidity. These antibodies were previously shown to neutralize FVIII activity by interfering with proteolytic activation of FVIII by thrombin or factor Xa, or with its binding to phospholipid surfaces, von Willebrand factor, or other components of the intrinsic tenase complex. Fine mapping of epitopes by surface plasmon resonance also indicated surfaces through which FVIII interacts with proteins and phospholipids as it participates in coagulation. Mutations that significantly altered the dissociation times/half-lives identified functionally important interactions within antigen-antibody interfaces and suggested specific sequence modifications to generate novel, less antigenic FVIII proteins with possible therapeutic potential for treatment of inhibitor patients.
authors.affiliation: Puget Sound Blood Center Research Institute, Seattle, WA;
authors.forename: Phuong-Cac T
authors.initials: PC
authors.lastname: Nguyen
authors.forename: Kenneth B
authors.initials: KB
authors.lastname: Lewis
authors.forename: Ruth A
authors.initials: RA
authors.lastname: Ettinger
authors.forename: Jason T
authors.initials: JT
authors.lastname: Schuman
authors.forename: Jasper C
authors.initials: JC
authors.lastname: Lin
authors.forename: John F
authors.initials: JF
authors.lastname: Healey
authors.forename: Shannon L
authors.initials: SL
authors.lastname: Meeks
authors.forename: Pete
authors.initials: P
authors.lastname: Lollar
authors.forename: Kathleen P
authors.initials: KP
authors.lastname: Pratt
mesh_terms: D000409:Alanine / Q000737:chemistry; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D000941:Antigens / Q000737:chemistry; D001777:Blood Coagulation; D018360:Crystallography, X-Ray; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry; D018985:Epitopes, B-Lymphocyte / Q000737:chemistry; D005169:Factor VIII / Q000737:chemistry*; D006801:Humans; D051379:Mice; D016296:Mutagenesis; D009154:Mutation; D017354:Point Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D020349:Surface Plasmon Resonance; D013552:Swine
chemical_list: D000596:Amino Acids; D000911:Antibodies, Monoclonal; D000941:Antigens; D000939:Epitopes; D018985:Epitopes, B-Lymphocyte; D011994:Recombinant Proteins; D005169:Factor VIII; D000409:Alanine