Human factor Va (hfVa) is the important regulatory subunit of prothrombinase. Recent modeling data have suggested a critical role for amino acid Arg 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 Asn-Arg 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 NR→DE. rhfV together with the wild-type molecule (rhfV) were expressed in COS7 cells, purified, and tested for their capability to function within prothrombinase. Kinetic studies showed that the of rhfVa for human fXa as well as the and of prothrombinase made with rhfVa for hPro activation were similar to the values obtained following hPro activation by prothrombinase made with rhfVa. 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 was significantly delayed with substantial accumulation of meizothrombin, and delayed thrombin generation, when compared to activation of hPro by prothrombinase made with rhfVa. 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 NR regulate at least in part the enzyme-substrate/product interaction during fibrin clot formation.