Anticoagulant effects of a synthetic peptide containing residues Thr-2253-Gln-2270 within factor VIII C2 domain that selectively inhibits factor Xa-catalysed factor VIII activation.

Factor VIII (FVIII), an essential cofactor that accelerates the generation of factor Xa (FXa) in the tenase complex, is activated by proteolytic cleavage by thrombin or FXa. A strong relationship has been reported between high levels of FVIII activity and thrombosis. We have demonstrated previously that an anti-FVIII C2 antibody (ESH8) with a Val-2248-Gly-2285 epitope inhibited FXa-catalysed FVIII activation, and that a synthetic peptide designated EP-2 (residues 2253-2270) blocked C2 domain binding to FXa. We investigated the inhibitory effect of EP-2 on FXa-catalysed FVIII activation and its anticoagulant effect in the blood coagulation system. EP-2 inhibited FXa-catalysed activation in a clotting assay in a dose-dependent manner and reduced FXa generation in a chromogenic assay using FVIII, factor X, factor IXa and phospholipid. The peptide only inhibited FVIII binding to FXa. We also tested the anticoagulant effect of EP-2 in the plasma milieu. The peptide prolonged the activated partial thromboplastin time and activated clotting time in a dose-dependent manner, but not prothrombin time. Our results indicate that EP-2 mediates the anticoagulant effect by specific inhibition of FVIII and FXa interaction in the intrinsic pathway, and that FXa-catalysed FVIII activation plays a significant role in blood clotting. The peptide may provide the basis for the development of novel anticoagulant therapy.