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 Arg⁷⁰⁹, Arg¹⁰¹⁸ and Arg¹⁵⁴⁵. 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 Arg⁷⁰⁹/¹⁰¹⁸ cleavage sites (FV^QQR), a FV molecule mutated at the Arg⁷⁰⁹/¹⁵⁴⁵ cleavage sites (FV^QRQ), a FV molecule mutated at the Arg¹⁰¹⁸/¹⁵⁴⁵ 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 Arg¹⁰¹⁸ 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 Arg¹⁰¹⁸→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 Arg⁷⁰⁹ and Arg¹⁵⁴⁵ are a prerequisite for expression of optimum cofactor activity. Our data also suggests that cleavage at Arg¹⁰¹⁸ is redundant for cofactor activity. The role of cleavage at this site by thrombin during procofactor activation remains to be determined.