Assessing the procoagulant activity of the FVa-Ile359Thr by FXa titration in the PTase assay. The recombinant FV variants (1.25 nM) were incubated with thrombin for 10 minutes at 37°C. After dilution (50 pM final concentration) the FV variants were incubated for 4 minutes with FXa (1-48 000 pM) and 50 μM phospholipids (PS/PC 10:90) at 37°C. Thrombin generation was started by the addition of prothrombin (0.5 μM). After 1 minute, the reactions were stopped by dilution with ice-cold EDTA buffer. The generated thrombin was determined with the chromogenic substrate S-2238. FVa-Ile359Thr, ▴; FVa-WT, □. Each data point represents the mean of 3 independent experiments performed in duplicate. Error bars represent ± SD.