Role of the Acidic Hirudin-Like COOH-Terminal Amino Acid Region of Factor VA Heavy Chain in the Enhanced Function of Prothrombinase

Jamila Hirbawi, Department of Chemistry, Cleveland State University, Cleveland, Ohio 44115, USA.
Michael A. Bukys
Melissa A. Barhoover
Evrim Erdogan
Michael Kalafatis


Prothrombinase activates prothrombin through initial cleavage at Arg(320) followed by cleavage at Arg(271). This pathway is characterized by the generation of an enzymatically active, transient intermediate, meizothrombin, that has increased chromogenic substrate activity but poor clotting activity. The heavy chain of factor Va contains an acidic region at the COOH terminus (residues 680-709). We have shown that a pentapeptide from this region (DYDYQ) inhibits prothrombin activation by prothrombinase by inhibiting meizothrombin generation. To ascertain the function of these regions, we have created a mutant recombinant factor V molecule that is missing the last 30 amino acids from the heavy chain (factor V(Delta680-709)) and a mutant molecule with the (695)DYDY (698) --> AAAA substitutions (factor V(4A)). The clotting activities of both recombinant mutant factor Va molecules were impaired compared to the clotting activity of wild-type factor Va (factor Va (Wt)). Using an assay employing purified reagents, we found that prothrombinase assembled with factor Va(Delta680-709) displayed an approximately 39% increase in k cat, while prothrombinase assembled with factor Va(4A) exhibited an approximately 20% increase in k cat for the activation of prothrombin as compared to prothrombinase assembled with factor Va(Wt). Gel electrophoresis analyzing prothrombin activation by prothrombinase assembled with the mutant molecules revealed a delay in prothrombin activation with persistence of meizothrombin. Our data demonstrate that the COOH-terminal region of factor Va heavy chain is indeed crucial for coordinated prothrombin activation by prothrombinase because it regulates meizothrombin cleavage at Arg(271) and suggest that this portion of factor Va is partially responsible for the enhanced procoagulant function of prothrombinase.