Amino Acids Asp695 and Tyr696 of Factor Va Are Essential for Optimal Rate of Prothrombin Activation by Prothrombinase.

Abstract 3163 Poster Board III-101 Blood coagulation is initiated after vascular injury, promoting formation of the fibrin clot. Without the proper regulation of this process, serious life threatening conditions, such as DVT (deep vein thrombosis), can occur. The proteolytic conversion of prothrombin to thrombin is catalyzed by the prothrombinase complex composed of the enzyme, factor Xa (fXa), the cofactor, factor Va (fVa), assembled on a membrane surface in the presence of Ca2+. 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 intermediate, meizothrombin, that is enzymatically active with increased chromogenic substrate activity, but yields poor clotting activity. Factor Va is composed of heavy and light chains that play a crucial role during thrombin formation. Portions of the fVa heavy chain have been found to interact with proexosite 1 (pro1) of prothrombin and influence prothrombinase activity. It has been recently demonstrated that deletion of the COOH-terminal region of the factor Va heavy chain causes accumulation of meizothrombin due to delayed cleavage of prothrombin at Arg271. Site-directed mutagenesis was performed to generate recombinant mutant molecules in order to identify the specific amino acids of this COOH-terminal region that regulate cleavage. Mutants with the 695DYDY698→DFDY(fVaDFDY), KFDY(fVaKFDY),DEDE(fVaDEDE),DFDF(fVaDFDF) substitutions were constructed. These recombinant molecules along with wild type factor V (fVWT) were transiently expressed in COS7 cells purified to homogeneity and assessed for their capability to promote prothrombin activation. Prothrombin activation was evaluated by SDS-PAGE and the kinetic parameters of the reactions were determined. SDS-PAGE analyses of prothrombin activation time courses revealed that the overall cleavage of prothrombin by prothrombinase assembled with fVaKFDY and fVaDFDY was delayed, while prothrombinase assembled with fVaDEDE and fVaDFDF had no significant effects when compared to fVaWT. Two- stage clotting assays (PT times) revealed that fVaKFDY and fVaDFDY both had reduced clotting activity when compared to fVaWT, while fVaDEDE and fVaDFDF gave similar clotting results as fVaWT. Determination of kcat values for prothrombinase assembled with the various recombinant molecules revealed that prothrombinase assembled fVaKFDY and fVaDFDY had a 20% increase catalytic efficiency as compared with prothrombinase assembled with fVaWT, while fVaDEDE and fVaDFDF gave values that were comparable to prothrombinase assembled with fVaWT. Comparison of the rate of cleavage of two recombinant prothrombin mutant molecules, rMZ that can't be cleaved at Arg271 and rPII that can't be cleaved at Arg320, by prothrombinase assembled with the mutant fVa molecules resulted in normal cleavage of rMZ by all the mutants. Cleavage of rPII, however, was impaired when fVaKFDY and fVaDFDY were incorporated into prothrombinase. The data presented suggests that the 695DY696 portion of the acidic cluster found in the COOH-terminus of the fVa heavy chain plays a significant role in enzyme-substrate interaction during thrombus formation. Disclosures No relevant conflicts of interest to declare.