Importance of protein S and phospholipid for activated protein C-mediated cleavages in factor Va

The procoagulant function of activated factor V (FVa) is inhibited by activated protein C (APC) through proteolytic cleavages at Arg306, Arg506, and Arg679. The effect of APC is potentiated by negatively charged phospholipid membranes and the APC cofactor protein S. Protein S has been reported to selectively stimulate cleavage at Arg306, an effect hypothesized to be related to reorientation of the active site of APC closer to the phospholipid membrane. To investigate the importance of protein S and phospholipid in the APC-mediated cleavages of individual sites, recombinant FV variants FV(R306Q/R679Q) and FV(R506Q/R679Q) (can be cleaved only at Arg506 and Arg306, respectively) were created. The cleavage rate was determined for each cleavage site in the presence of varied protein S concentrations and phospholipid compositions. In contrast to results on record, we found that protein S stimulated both APC cleavages in a phospholipid composition-dependent manner. Thus, on vesicles containing both phosphatidylserine and phosphatidylethanolamine, protein S increased the rate of Arg306 cleavage 27-fold and that of Arg506 cleavage 5-fold. Half-maximal stimulation was obtained at approximately 30 nm protein S for both cleavages. In conclusion, we demonstrate that APC-mediated cleavages at both Arg306 and Arg506 in FVa are stimulated by protein S in a phospholipid composition-dependent manner. These results provide new insights into the mechanism of APC cofactor activity of protein S and the importance of phospholipid composition.