Pro- and anticoagulant activities of factor V

Abstract: Coagulation factor V (FV) is activated by thrombin through proteolytic cleavage at Arg-709, Arg-1018 and Arg-1545. Upon thrombin activation, the active form of factor V (FVa) is formed. FVa functions as a cofactor to factor Xa in the conversion of prothrombin to thrombin during coagulation. Recently, it was shown that in addition to being a precursor form of the procoagulant FVa, FV functions as a cofactor to activated protein C (APC) and thus also has anticoagulant properties. APC down-regulates coagulation through proteolytic inactivation of FVa and FVIIIa (the cofactor to FIXa in the activation of FX). With this study we have determined the cleavage events that regulate the pro- and anticoagulant activities of FV and have identified a 70 amino acid region essential for the anticoagulant function of FV. Using recombinant FV mutants in which the thrombin cleavage sites were mutated we studied the cleavage requirements for activation of FV by thrombin or FXa. Here we have shown that FXa cleaves FV at all three thrombin cleavage sites, at Arg-709, Arg-1018 and Arg-1545. Thrombin- or FXa-mediated cleavage at Arg-709 was kinetically favoured over cleavage at Arg-1545, but cleavage at both sites was necessary for maximal activation. Recombinant FV cleaved by thrombin at Arg-709 and/or Arg-1018 retained the ability to express APC-cofactor activity in an APC-mediated FVIIIa degradation assay. However, cleavage at Arg-1545 led to a complete loss in APC-cofactor function. The C-terminal region of the FV B-domain (amino acids 1476-1545) was found to be essential for expression of anticoagulant activity. Using FV recombinants mutated at the different APC cleavage sites, we have demonstrated that APC-mediated cleavage at Arg-506 is necessary for the expression of FV anticoagulant activity. Taken together, these results indicate the importance of proteolytic cleavage in regulating the dual functions of FV.