Factor VIII C2 Domain Contains the Thrombin-binding Site Responsible for Thrombin-catalyzed Cleavage at Arg1689 *

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 Arg1689 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 theK 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 (IC50, 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 Arg1689. Our results indicate that the C2 domain contains the thrombin-binding site responsible for the cleavage at Arg1689.

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