Activation of prothrombin. Reaction mixtures containing increasing concentrations of prothrombin (0.1-2 µM) with 20 µM phosphatidylcholine-phosphatidylserine were incubated at 25°C in the presence of 20 nM FVa. The reaction was initiated with 0.1 or 0.2 nM wt-FXa (●), FXa-V17M (□), FXa-I16L (▪), FXa-I16M (▼), FXa-V17T (▿), FXa-V17S (△) and FXa-I16T (▲). Aliquots of the reaction mixture were quenched during initial rate of the reaction (0, 0.5, 1, 1.5, and 2 minutes), and thrombin generation was determined by using the chromogenic substrate S-2238. The solid lines were drawn following analysis of all data sets to a rectangular hyperbola with the following fitted parameters: wt-FXa: K_m = 0.4 ± 0.04 µM, k_cat = 2700 ± 75 minutes⁻¹; FXa-V17M: K_m = 0.36 ± 0.04 µM, k_cat = 2300 ± 70 minutes⁻¹; FXa-I16M and FXa-V17T: K_m = 0.38 ± 0.05 µM, k_cat = 2000 ± 80 and 2500 ± 120 minutes⁻¹; FXa-V17S: K_m = 0.2 ± 0.02 µM, k_cat = 1200 ± 30 minutes⁻¹; and FXa-I16T: K_m = 0.12 ± 0.02 µM, k_cat = 340 ± 7 minutes⁻¹. The data are representative of 2 to 3 similar experiments.