Protein S enhancement of APC-mediated cleavage of FVa at Arg306 and of FVIIIa. Protein S was incubated with 0.8nM FVa R506Q/R679Q and phospholipids in the presence or absence of APC. The remaining FVa activity was measured in a prothrombinase assay (A-C). The FVa activity was measured either after incubation with increasing concentrations of protein S (0-100nM) in the presence or absence of 0.25nM APC (n = 3; A) or after incubation with 0 to 4nM APC in the presence or absence of 100nM protein S (n = 2; B). Time course experiments were performed to calculate the pseudo–first-order rate constants of WT protein S and protein S E36A using 100nM protein S and 0.25nM APC (Table 2; n = 4; C). Protein S (0-100nM) was incubated with 280 mU/mL FVIIIa, 5nM FIXa, 2.5nM FV, and phospholipids in the presence or absence of 1nM APC for 2.5 minutes. The remaining FVIIIa activity was measured with an Xase assay (n = 3; D). Data are plotted as mean ± SD.