Impact of PL, FXa, and prothrombin on prothrombinase activity reacted with FVa-A2086D. FV-WT and FV-A2086D (100 pM) was activated by thrombin (5 nM) for 1 minute, before the addition of hirudin (2.5 U/mL). FVa was mixed, (A) with FXa (1 nM) and prothrombin (1 μM) and various concentrations of PL (0-40 μM), (B) with various concentrations of FXa (0-2 nM) and prothrombin (1 μM) and PL (10 μM), or (C) with FXa (1 nM) and various concentrations of prothrombin (0-4 μM) and PL (10 μM). The reactions were quenched with EDTA (f.c. 50 mM). Rates of thrombin generation were determined after the addition of S-2238. Thrombin generation was quantified by extrapolation from a standard curve prepared using known amounts of thrombin. The plotted data were fitted using the Michaelis-Menten equation (dashed line) and the K_m and V_max values for FVa-dependent FXa-catalyzed prothrombin activation were calculated. Experiments were performed at least 3 times, and the average values ± standard deviations are shown.