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.
Highlights
Human factor Va is the important regulatory subunit of prothrombinase that controls the rate of human prothrombin activation by prothrombinase during hemostasis.[1]
HfVa has the identical sequence as higher primates and fVa from horse, all other species shown in Figure 11 have different amino acids at these two positions, suggesting an important role of these two residues within fVa
There is no effect of the 700NR701→DE mutation in Human factor Va (hfVa) on the direct binding of hfVa to Human factor Xa (hfXa), there is a significant effect of the mutation on human prothrombin (hPro) activation by prothrombinase reconstituted with the mutated cofactor molecule, which is translated by hindered cleavage at both Arg320/Arg[271]
Summary
Human factor Va (hfVa) is the important regulatory subunit of prothrombinase that controls the rate of human prothrombin (hPro) activation by prothrombinase during hemostasis.[1]. To determine the effect of the 700NR701→DE substitutions within the factor Va heavy chain on the cleavage of hPro at Arg[320] alone when the substrate is not associated with the membrane surface, we compared the rates of activation of Pre[1] by prothrombinase made with rhfVaWT and rhfVaNR→DE (Figure 9). We have shown repeatedly that Asp[695] and Tyr[698] are a part of a peptide portion of the hfVa heavy chain that represents a control switch for the activity of prothrombinase.[27,28] Overall our experimental data together with the recent computational model of hfVa clearly establish a prolific interaction between the acidic carboxyl-terminus of the hfVa heavy chain and several residues adjacent or nearby to the crucial cleavage site at Arg[320] of hPro for timely thrombin production. For easy reading of the article, the rhfVa species used for the reconstitution of prothrombinase is shown under each panel
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