APC-mediated inactivation of FVa-Asn357Gln/Ile359Thr. (A) The recombinant FV variants (2 μg/mL) were incubated with thrombin (0.05 U/mL) for 30 minutes at 37°C and then incubated with 0.25 nM APC and 100 nM protein S in the presence of 25 μM phospholipids (PS/PE/PC 20:20:60). Both FVa and APC-cleaved FVa were analyzed by Western blot (12% SDS-PAGE) under reducing conditions. The FVa was detected using a monoclonal against the heavy chain (AHV-5146), and Vectastain Elite ABC kit was used for detection. (B) The FV variants (0.8 nM) were incubated with thrombin (0.5 U/mL) for 10 minutes at 37°C. APC was subsequently added (final concentration, 0.050 nM) together with protein S (final concentration, 100 nM) and 10:20:70 PS/PE/PC phospholipids (final concentration, 25 μM). Sub-samples were withdrawn at different time points, and activity was measured as in Figure 3. The values were normalized to the value of FVa activity at time zero for each reaction. FVa-WT, □; FVa-Ile359Thr, ▴; FVa-Asn357Gln/Ile359Thr, ○. Each data point represents the mean of 3 independent experiments performed in duplicate. Error bars represent ± SD.