title: Factor VIII C2 domain contains the thrombin-binding site responsible for thrombin-catalyzed cleavage at Arg1689.
abstract: Thrombin-catalyzed factor VIII activation is an essential positive feedback mechanism regulating intrinsic blood coagulation. A factor VIII human antibody, A-FF, with C2 epitope, exclusively inhibited factor VIII activation and cleavage at Arg(1689) by thrombin. The results suggested that A-FF prevented the interaction of thrombin with factor VIII and that the C2 domain was involved in the interaction with thrombin. We performed direct binding assays using anhydro-thrombin, a catalytically inactive derivative of thrombin in which the active-site serine is converted to dehydroalanine. Intact factor VIII, 80-kDa light chain, 72-kDa light chain, and heavy chain fragments bound dose-dependently to anhydro-thrombin, and the K(d) values were 48, 150, 106, and 180 nm, respectively. The C2 and A2 domains also dose-dependently bound to anhydro-thrombin, and the K(d) values were 440 and 488 nm, respectively. The A1 domain did not bind to anhydro-thrombin. A-FF completely inhibited C2 domain binding to anhydro-thrombin (IC(50), 18 nm), whereas it did not inhibit A2 domain binding. Furthermore, C2-specific affinity purified F(ab)'(2) of A-FF, and the recombinant C2 domain inhibited thrombin cleavage at Arg(1689). Our results indicate that the C2 domain contains the thrombin-binding site responsible for the cleavage at Arg(1689).
authors.affiliation: Department of Pediatrics, Nara Medical University, Kashihara City, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Nogami
authors.forename: M
authors.initials: M
authors.lastname: Shima
authors.forename: K
authors.initials: K
authors.lastname: Hosokawa
authors.forename: M
authors.initials: M
authors.lastname: Nagata
authors.forename: T
authors.initials: T
authors.lastname: Koide
authors.forename: E L
authors.initials: EL
authors.lastname: Saenko
authors.forename: I
authors.initials: I
authors.lastname: Tanaka
authors.forename: M
authors.initials: M
authors.lastname: Shibata
authors.forename: A
authors.initials: A
authors.lastname: Yoshioka
mesh_terms: D000911:Antibodies, Monoclonal / Q000378:metabolism; D001120:Arginine / Q000378:metabolism; D001665:Binding Sites; D001667:Binding, Competitive; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000378:metabolism; D005169:Factor VIII / Q000737:chemistry* / Q000276:immunology; D006801:Humans; D007700:Kinetics; D011233:Precipitin Tests; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D013917:Thrombin / Q000276:immunology / Q000378:metabolism*; D013997:Time Factors
chemical_list: D000911:Antibodies, Monoclonal; D000939:Epitopes; D011994:Recombinant Proteins; D005169:Factor VIII; D001120:Arginine; D013917:Thrombin