Time-dependent decay of factor Xa generating activity by factor Xase composed of R372H and wild-type factor VIIIa forms. Factor VIII (300 nM) was reacted with thrombin (50 nM) for 18 hours at 37°C. After the addition of hirudin and a 30-fold dilution of reactant, aliquots were removed at the indicated times and reacted with factor IXa (20 nM) and phospholipid vesicles (10 μM), and factor Xa generation was initiated with the addition of factor X (300 nM) as described in “Materials and methods.” ○ indicates wild type; •, R372H. The initial activity of factor Xa generated (100% level) for factor VIII wild type and R372H was 13.0 and 6.1 nM/min, respectively. Initial rates of factor Xa generation were plotted as a function of incubation time and fitted to a single exponential decay curve. Experiments were performed at least 3 separate times and average values are shown.