Factor VIIIC2 domain contains the thrombin-binding site responsible for thrombin-catalyzed cleavage at Arg1689

Thrombin-catalyzed factor WI 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 Vm 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 Al 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, CS-specific affinity purified F(ab)'(2) of A-FF, and the recombinant Ca 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).