APC-mediated inactivation of the Ala221Val FVa variant. Recombinant FV variants (0.8 nM) were incubated with thrombin (0.5 U/mL) for 10 minutes in 37°C. In panel A, 0.05 nM APC was added together with 10:20:70 PS/PE/PC phospholipids at a final concentration of 25 μM, and the FVa degradation was followed for 5 minutes. The subsamples withdrawn at different time points were stopped by 5-fold dilution in ice-cold HNBSACa buffer. The residual activity was measured with the prothrombinase assay using suboptimal FXa concentrations (0.05 nM). In panel B, 0.1 nM APC was added together with 100 nM protein S and 10:20:70 wt/wt/wt PS/PE/PC phospholipids (final concentration of 25 μM). The FVa degradation was followed for 20 minutes. The subsamples were stopped as in panel A. The residual activity was determined with the prothrombinase assay using saturated FXa concentrations (5 nM). The remaining activity was expressed as percentage of the activity generated by each FVa variant in the absence of APC. WT FVa (▪) and Ala221Val (□). Each data point represents the mean of 3 independent experiments performed in duplicate. Error bars represent ±SDs.