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 (fVDE) along with wild type factor V (fVWT) 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 Kd of fVaDE for human fXa, as well as the kcat and Km values of prothrombinase assembled with fVaDE for human Pro activation were similar to the values obtained following Pro activation by prothrombinase assembled with fVaWT. Surprisingly, SDS-PAGE analyses of prothrombin activation time courses revealed that the overall rate of cleavage of Pro by prothrombinase assembled with fVaDE 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 fVaWT. Two-stage clotting assays (PT times) also revealed that fVaDE had reduced clotting activity when compared to fVaWT. 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 fVaDE 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 fVaWT. These findings were verified by experiments using active-site blocked purified human meizothrombin (FPR-meizo). Prothrombinase assembled with fVaDE was considerably impaired in its ability to cleave FPR-meizo at Arg271 as compared to the ability of prothrombinase assembled with fVaWT for the same cleavage. In fact, gel electrophoresis analyses demonstrated that prothrombinase assembled with fVaDE 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.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.