Kinetics of recombinant factor VIII activation by thrombin. Variable amounts of factor VIII were reacted with thrombin (0.04 nM) for 1 minute. Thrombin was inactivated by addition of hirudin, and factor VIIIa was reacted with factor IXa (20 nM) in the presence of phospholipid vesicles (10 μM). Factor Xa generation was initiated by addition of factor X (300 nM) as described in “Materials and methods.” ○ indicates wild type; •, R372H. Initial rates of factor Xa generation are plotted as a function of factor VIII concentration and fitted to the Michaelis-Menten equation by nonlinear least squares regression. The inset shows the rate of factor Xa generation using factor VIII R372H activated by thrombin. All experiments were performed at least 3 separate times and average values are shown.