Spellbinding Effects of the Acidic COOH-Terminus of Factor Va Heavy Chain on Prothrombinase Activity and Function

Human factor Va (hfVa) is the important regulatory subunit of prothrombinase. Recent modeling data have suggested a critical role for amino acid Arg⁷⁰¹ of hfVa for human prothrombin (hPro) activation by prothrombinase. Furthermore, it has also been demonstrated that hfVa has a different effect than that of bovine fVa on prethrombin-1 activation by prothrombinase. The difference between the two cofactor molecules was also found within the Asn⁷⁰⁰-Arg⁷⁰¹ dipeptide in the human factor V (hfV) molecule, which is replaced by the Asp-Glu sequence in bfV. As a consequence, we produced a recombinant hfV (rhfV) molecule with the substitution ⁷⁰⁰NR⁷⁰¹→DE. rhfV^NR→DE together with the wild-type molecule (rhfV^WT) were expressed in COS7 cells, purified, and tested for their capability to function within prothrombinase. Kinetic studies showed that the K_d of rhfVa^NR→DE for human fXa as well as the k_cat and K_m of prothrombinase made with rhfVa^NR→DE for hPro activation were similar to the values obtained following hPro activation by prothrombinase made with rhfVa^WT. Remarkably, sodium dodecyl sulfate polyacrylamide gel electrophoresis analyses of hPro activation time courses demonstrated that the rate of cleavage of hPro by prothrombinase reconstituted with rhfVa^NR→DE was significantly delayed with substantial accumulation of meizothrombin, and delayed thrombin generation, when compared to activation of hPro by prothrombinase made with rhfVa^WT. These unanticipated results provide significant insights on the role of the carboxyl-terminal end of the heavy chain of hfVa for hPro cleavage and activation by prothrombinase and show that residues ⁷⁰⁰NR⁷⁰¹ regulate at least in part the enzyme-substrate/product interaction during fibrin clot formation.