Cleavage At Arg1018 During Thrombin-Mediated Activation of Coagulation Factor V Is Dispensable

Abstract 1186 Blood clotting results in the proteolytic conversion of prothrombin (Pro) to thrombin which in turn will produce the fibrin clot. The proteolytic conversion of Pro to thrombin is catalyzed by the prothrombinase complex which is composed of the enzyme, factor Xa (FXa), the cofactor, factor Va (FVa), assembled on a membrane surface in the presence of divalent metal ions. Factor V (FV), is a multidomain protein (A1-A2-B-A3-C1-C2) with nominal procoagulant activity and is activated by thrombin to FVa through three sequential proteolytic cleavages at Arg 709 , Arg 1018 and Arg 1545 . To understand the significance of each cleavage for active cofactor formation and prothrombinase function, recombinant factor V molecules were created by site-directed mutagenesis with two out of three cleavage sites mutated simultaneously (to glutamine). We have generated a FV molecule mutated at the Arg 709 / 1018 cleavage sites (FV QQR ), a FV molecule mutated at the Arg 709 / 1545 cleavage sites (FV QRQ ), a FV molecule mutated at the Arg 1018 / 1545 cleavage sites (FV RQQ ), and a FV molecule that is mutated at all three cleavage sites (FV QQQ ). These recombinant FV molecules along with wild type FV (FV WT ) were transiently expressed in COS7L cells, purified to homogeneity and assessed for their capability to interact with factor Xa following activation by thrombin, and participate in prothrombinase. Pro activation by prothrombinase assembled with the mutant molecules was evaluated by SDS-PAGE and the kinetic parameters of the reactions in the presence of saturating concentrations of FXa were determined. Two-stage clotting assays revealed that while FV QQQ was devoid of clotting activity following incubation with thrombin, FVa QQR , FVa QRQ and FVa RQQ all had impaired clotting activities compared to FVa WT and plasma derived FVa (FVa PLASMA ). Kinetic analyses demonstrated that FVa WT had a K d of 0.25nM for FXa while all other mutant molecules had impaired binding capabilities for FXa. FVa QQQ was severely impaired in its ability to interact with FXa. The k cat value for prothrombinase assembled with FVa QQR was approximately 50% lower than the k cat obtained with prothrombinase assembled with FVa WT , while prothrombinase assembled with FVa QRQ and FVa RQQ had approximately 3-fold reduced catalytic efficiency when compared to the values obtained with prothrombinase assembled with FVa WT . Following incubation with thrombin prothrombinase assembled with FVa QQQ had no cofactor activity. To determine the importance of the cleavage site at Arg 1018 for procofactor activation and the function of amino acid region 1000–1008 during proteolysis, several other recombinant molecules were generated. FV RQR is a FV molecule with the mutation Arg 1018 →Gln, and FV Δ1000-1008 is a mutant FV molecule with region 1000–1008 deleted. We have also generated FV Δ1000-1008/RQR and FV Δ1000-1008/QRQ . Two-stage clotting assays revealed that FVa RQR and FVa Δ1000-1008/RQR have similar clotting activities as FVa WT, whereas FVa QRQ , FVa Δ1000-1008/QRQ are impaired in their clotting activities. Kinetic analyses demonstrated that FVa RQR and FVa Δ1000-1008/RQR have similar affinity for FXa as FVa WT while FVa QRQ and FVa Δ1000-1008/QRQ were impaired in their interaction with factor Xa. The k cat values for prothrombinase assembled with FVa RQR and FVa Δ1000-1008/RQR were similar to the k cat obtained with prothrombinase assembled with FVa WT , while prothrombinase assembled with FVa QRQ and FVa Δ1000-1008/QRQ had 2-fold and 7-fold reduced catalytic efficiency respectively, when compared to the k cat values obtained with prothrombinase assembled with FVa WT . Overall, the data demonstrate that cleavage at both Arg 709 and Arg 1545 are a prerequisite for expression of optimum cofactor activity. Our data also suggests that cleavage at Arg 1018 is redundant for cofactor activity. The role of cleavage at this site by thrombin during procofactor activation remains to be determined. Disclosures: No relevant conflicts of interest to declare.