Cleavage at Both Arg306 and Arg506 Is Required and Sufficient for Timely and Efficient Inactivation of Factor VA by Activated Protein C
Activated protein C (APC) inactivates membrane-bound factor Va following cleavages of the heavy chain at Arg, Arg, and Arg. The objective of this study is to examine which cleavage is most important for inactivation. The recombinant factor V molecules were constructed as follows: factor V (mutations R→Q), factor V (mutations R→Q), and factor V (mutations R→Q and R→Q). The recombinant molecules were expressed in mammalian cells, purified, and assayed prior and after incubation with APC and lipids for 30 min (factor Vai) in clotting assays and in an assay using purified reagents and saturating concentrations of factor Va. Clotting assays demonstrated that wild-type factor Vai (Vai), factor Vai, and factor Vai were devoid of activity, whereas factor Vai maintained approximately 70% activity following a 30 min incubation with APC. Prothrombinase assembled with all mutant cofactor molecules before and after treatment with APC had kinetic constant (Km) values similar to values found with prothrombinase assembled with factor Va. Prothrombinase assembled with factor Vai demonstrated a 20-fold reduction in kcat, whereas prothrombinase assembled with factor Vai had a two-fold reduction in kcat as compared with prothrombinase assembled with factor Va. In contrast, factor Vai and factor Vai did not show any loss in kcat under similar experimental conditions. In conclusion, our data demonstrate that the activity of an APC-treated factor Va molecule bearing a single mutation at Arg or Arg depends on the assay used; and regardless of the assay employed, in the absence of the APC-cleavage sites at Arg and Arg, the active cofactor is unable to be significantly inactivated by APC in the presence of a membrane surface.