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

Abstract

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. rhfVNR→DE together with the wild-type molecule (rhfVWT) were expressed in COS7 cells, purified, and tested for their capability to function within prothrombinase. Kinetic studies showed that the Kd of rhfVaNR→DE for human fXa as well as the kcat and Km of prothrombinase made with rhfVaNR→DE for hPro activation were similar to the values obtained following hPro activation by prothrombinase made with rhfVaWT. 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 rhfVaNR→DE was significantly delayed with substantial accumulation of meizothrombin, and delayed thrombin generation, when compared to activation of hPro by prothrombinase made with rhfVaWT. 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.

Blood coagulation factor Va's key interactive residues and regions for prothrombinase assembly and prothrombin binding

“…This notion was further supported by alanine substitution of residues Asp695‐Gln698, which resulted in a surprising ~20% increase in k cat for the activation of prothrombin, while, in contrast, an accumulation of meizothrombin and subsequent delay in prothrombin activation were observed . Similar results were obtained for FVa variants in which residues Asp659‐Asp663 were either mutated or deleted, or in which residues Asn700‐Arg701 were replaced for the bovine homologs Asp‐Glu . These results indicate that the A2T plays a prominent role in both the incorporation and catalysis of prothrombin.…”