Prothrombin (1.4μM) was incubated with either human or bovine FVa (10nM), CaCl2 (2.0mM), PCPS vesicles (20μM) and DAPA (3.0μM) in 0.02M HEPES, 0.15M NaCl, pH 7.4 and0.01% (v/v) Tween-80, and its activation was initiated by the addition of FXa (1.0nM). Samples were withdrawn at various times and subjected to 10–14.5% SDS-PAGE. The gels were fixed in 50% methanol, 20% ethanol, and 6% trichloroacetic acid for at least 2 hours and were stained with Coomassie Blue and destained. The time courses were determined by quantitative densitometry.