Evidence That the COOH-Terminal Region of Factor Va Heavy Chain Is Involved in the Regulation of Prothrombinase Activity.

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 divalent metal ions. Incorporation of fVa into the prothrombinase complex results in a 300,000-fold increase in the catalytic efficiency of fXa for thrombin generation. A first cleavage of prothrombin by prothrombinase at Arg³²⁰ produces the active intermediate meizothrombin, while the second cleavage at Arg²⁷¹ produces thrombin. It has been demonstrated that elimination of the carboxyl terminal portion of the heavy chain of fVa by proteolytic enzymes results in a cofactor molecule with decreased clotting activity and slightly increased to normal chromogenic activity. In addition, we have previously shown that the carboxyl terminal portion of the heavy chain of fVa is involved in the interaction of the cofactor with prothrombin. To further ascertain the importance of this region of the molecule for cofactor activity we used PCR based methods to produce recombinant fVa molecules with several portions of the COOH-terminus deleted. Recombinant fV⁶⁵³ has amino acids 653–709 deleted, recombinant fV⁶⁹⁶ has amino acid residues 680–696 deleted, recombinant fV⁶⁸⁰ has amino acid residues 653–680 deleted, while recombinant fV⁷⁰⁹ has amino acid residues 680–709 missing. These recombinant molecules along with wild type fV (fV^WT) were transiently expressed in COS7L cells and assessed for their capability to promote prothrombin activation following activation by Russell’s Viper Venom factor V activator (RVV-V activator). Thrombin generation was evaluated by SDS-PAGE and the kinetic parameters of the reactions were determined. While fVa⁶⁵³ and fVa⁶⁸⁰ were devoid of clotting activity, fVa⁶⁹⁶ and fVa⁷⁰⁹ had reduced clotting activities compared to fVa^WT and plasma-derived fVa. This level of clotting activity was similar to the clotting activity of a fV molecule that was treated with thrombin and human neutrophil elastase (HNE) resulting in fVa^HNE. fVa^HNE is cleaved at Ala⁶⁷⁷/Thr⁶⁷⁸ resulting in a cofactor with a shorter heavy chain. Further analyses revealed that all mutant recombinant molecules as well as fVa^HNE have similar K_D values for fXa when compared to plasma fVa and fVa^WT. SDS-PAGE analyses of prothrombin activation time courses revealed that the overall cleavage of prothrombin by prothrombinase assembled with fVa⁶⁹⁶, fVa⁷⁰⁹, or fVa^HNE was slower resulting in accumulation of meizothrombin. This data confirm our previous findings and suggest that this region on the heavy chain of fVa contribute to cofactor function. A logical explanation for these findings is that the COOH-terminus of the heavy chain of fVa participates in the regulation of the rates of appearance/disappearance of meizothrombin. Increased persistence of meizothrombin in the reaction mixture can explain the slower clotting times since it is well known that meizothrombin has poor clotting activity. Thus at a given time point there will be more meizothrombin present in a sample where prothrombinase was assembled with fVa⁷⁰⁹, or fVa⁶⁹⁶, or fVa^HNE than in a sample where prothrombinase was formed with fVa^WT. Overall the data suggests that the COOH-terminal portion of the factor Va heavy chain contributes to the appropriate orientation of prothrombin with respect to the catalytic site of fXa resulting in efficient cleavages at Arg³²⁰ /Arg²⁷¹ and competent thrombin formation.