The Role of Amino Acids 700–701 of the Factor Va Heavy Chain During Prothrombin Activation by Factor Xa

Abstract 2203

Blood coagulation is initiated after vascular injury, promoting formation of the fibrin plug. The prothrombinase complex plays a crucial role during activation of prothrombin (Pro) to thrombin. The complex is composed of the enzyme, factor Xa (fXa), along with its non-enzymatic cofactor, factor Va (fVa), in the presence of calcium on a phospholipid surface. The incorporation of fVa into the prothrombinase complex results in a 300,000-fold increase in the catalytic efficiency of fXa for thrombin generation. Prothrombinase activates prothrombin through initial cleavage at Arg³²⁰ followed by cleavage at Arg²⁷¹ to yield human alpha-thrombin. This pathway is responsible for the generation of a transient catalytically active intermediate, meizothrombin. Recent data has suggested a differential effect of bovine and human factor Va on prothrombin-1 (Pre-1) activation by prothrombinase. This difference was localized within the last ten amino acids from the carboxyl-terminal region of fVa heavy chain. The only amino acid difference between the two cofactor molecules is localized at position 700–701 where the Asn-Arg dipeptide in the fVa of human origin is replaced by the Asp-Glu sequence in the carboxyl-terminal region of the cofactor of bovine origin. We have therefore constructed a recombinant human mutant fVa molecule with these amino acids mutated to their bovine counterpart. We have created a recombinant fVa molecule with the mutation⁷⁰⁰NR⁷⁰¹ →DE. This recombinant cofactor molecule (fV^DE) along with wild type factor V (fV^WT) were transiently expressed in COS7 cells, purified to homogeneity, and assessed for their capability to by assembled in prothrombinase and promote Pro activation. Thrombin generation was evaluated by SDS-PAGE in a system using all proteins of human origin and the kinetic parameters of the reactions were determined using a chromogenic substrate to assess for thrombin activity. Kinetic analyses revealed that the K_d of fVa^DE for human fXa, as well as the k_cat and Km values of prothrombinase assembled with fVa^DE for human Pro activation were similar to the values obtained following Pro activation by prothrombinase assembled with fVa^WT. Surprisingly, SDS-PAGE analyses of prothrombin activation time courses revealed that the overall rate of cleavage of Pro by prothrombinase assembled with fVa^DE was significantly delayed with significant accumulation of the intermediate meizothrombin, and delayed thrombin generation when compared to the rate of activation of Pro by prothrombinase assembled with fVa^WT. Two-stage clotting assays (PT times) also revealed that fVa^DE had reduced clotting activity when compared to fVa^WT. Comparison of the rate of cleavage of two recombinant Pro mutant molecules, rMZ-II a recombinant Pro molecule that cannot be cleaved at Arg²⁷¹ and rP2-II a recombinant Pro molecule that cannot be cleaved at Arg³²⁰, by prothrombinase assembled with fVa^DE demonstrated impaired rate of cleavage of both substrates when compared to the rate of cleavage of the mutant recombinant Pro molecules by prothrombinase assembled with fVa^WT. These findings were verified by experiments using active-site blocked purified human meizothrombin (FPR-meizo). Prothrombinase assembled with fVa^DE was considerably impaired in its ability to cleave FPR-meizo at Arg²⁷¹ as compared to the ability of prothrombinase assembled with fVa^WT for the same cleavage. In fact, gel electrophoresis analyses demonstrated that prothrombinase assembled with fVa^DE cleaves FPR-meizo with a rate similar to the cleavage of FPR-meizo by fXa alone. All these data together strongly suggest that the ⁷⁰⁰NR⁷⁰¹ portion of the COOH-terminus of the fVa heavy chain plays a significant role in enzyme-substrate recognition/interaction during Pro activation by prothrombinase and thus regulates the rates of thrombin formation locally at the place of vascular injury.