Activation of prothrombin on stimulated platelets. Thrombin-activated human platelets (1 × 10⁸/mL; 20nM thrombin, 3 minutes followed by the addition of 30nM hirudin) were incubated with increasing concentrations of prothrombin (0.1-1.4μM) either in the presence (closed symbols) or absence (open symbols) of 10nM FVa at 25°C. The reaction was initiated with 0.1nM rFXa (squares, wt-FXa; circles, FXa^I16L), and aliquots of the reaction mixture were quenched during the initial rate of the reaction (0, 0.5, 1, 1.5, and 2 minutes), and thrombin generation was measured 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-Xa +FVa: K_m, 0.18 ± 0.03μM, k_cat, 450 ± 21 minutes⁻¹; wt-Xa: K_m, 0.17 ± 0.06μM, k_cat, 440 ± 36 minutes⁻¹; FXa^I16L +FVa: K_m, 0.22 ± 0.02μM, k_cat, 530 ± 15 minutes⁻¹; FXa^I16L: K_m, 0.15 ± 0.03μM, k_cat, 370 ± 18 minutes⁻¹. The data are representative of 4 similar experiments.