Titrating FXa and prothrombin in the prothrombinase assay. Wild-type FV and the Ala221Val FV variant were incubated with thrombin (0.5 NIH units/mL) for 10 minutes at 37°C. (A) FVa was diluted to 50 pM (final concentration) and incubated for 4 minutes with FXa (1 to 50 000 pM) and 10:90 PS/PC phospholipid vesicles (50 μM) at 37°C. Thrombin generation was started by the addition of prothrombin (0.5 μM). (B) FVa was diluted to 100 pM (final concentration) and incubated for 4 minutes with prothrombin (PT) (1 to 2500 pM) and 10:90 PS/PC phospholipid vesicles (50 μM) at 37°C. Thrombin generation was started by the addition of FXa (5 nM). The reactions were stopped after 1 minute by dilution with ice-cold EDTA buffer. The generated thrombin was determined with the chromogenic substrate S-2238. The activity was expressed as percentage of maximum activity generated in the presence of WT FVa. WT FVa (▪) and Ala221Val (□). Each data point represents the mean of 3 independent experiments performed in duplicate. Error bars represent ± SDs.