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(fVa^DFDY), KFDY(fVa^KFDY),DEDE(fVa^DEDE),DFDF(fVa^DFDF) substitutions were constructed. These recombinant molecules along with wild type factor V (fV^WT) 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 fVa^KFDY and fVa^DFDY was delayed, while prothrombinase assembled with fVa^DEDE and fVa^DFDF had no significant effects when compared to fVa^WT. Two- stage clotting assays (PT times) revealed that fVa^KFDY and fVa^DFDY both had reduced clotting activity when compared to fVa^WT, while fVa^DEDE and fVa^DFDF gave similar clotting results as fVa^WT. Determination of k_cat values for prothrombinase assembled with the various recombinant molecules revealed that prothrombinase assembled fVa^KFDY and fVa^DFDY had a 20% increase catalytic efficiency as compared with prothrombinase assembled with fVa^WT, while fVa^DEDE and fVa^DFDF gave values that were comparable to prothrombinase assembled with fVa^WT. 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 fVa^KFDY and fVa^DFDY 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.