Factor VIIIC2 domain contains the thrombin-binding site responsible for thrombin-catalyzed cleavage at Arg(1689)

Thrombin-catalyzed factor WI activation is an essential positive feedback m echanism regulating intrinsic blood coagulation. A factor VIII human. antib ody, A-FF, with C2 epitope, exclusively inhibited factor VIII activation an d cleavage at Arg(1689) by thrombin. The results suggested that A-FF preven ted the interaction of thrombin with factor Vm and that the C2 domain was i nvolved in the interaction with thrombin. We performed direct binding assay s using anhydro-thrombin, a catalytically inactive derivative of thrombin i n which the active-site serine is converted to dehydroalanine. Intact facto r VIII, 80-kDa light chain, 72-kDa light chain, and heavy chain fragments b ound 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, respect ively. The Al domain did not bind to anhydro-thrombin. A-FF completely inhi bited C2 domain binding to anhydro-thrombin (IC50, 18 nM), whereas it did n ot 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).