The kinetics of FIXa-catalyzed FX activation with D519V/E665V/K1813A, D519V/E665V, and K1813A FVIII mutants. (A) FIXa association; WT-FVIII or FVIII mutants (0.5 nM) were activated by thrombin (30 nM) for 30 seconds in the presence of PL vesicles (20 μM). FXa generation was initiated by the addition of FX (300 nM) and various concentrations of FIXa (0-5 nM) as described in “Methods.” (B) FX association; WT-FVIII or mutant FVIII (1 nM) was activated by thrombin (30 nM) for 30 seconds in the presence of PL vesicles (20 μM). FXa generation was initiated by the addition of FIXa (40 nM) and various concentrations of FX (0-400 nM). The symbols used are: open circles, WT; closed circles, D519V/E665V/K1813A; open squares, D519V/E665V; and closed squares, K1813A. Experiments were performed 3 times, and the average and standard deviation values are shown. The initial rates of FXa generation were plotted as a function of FIXa or FX concentration and fitted to Equation 1 by nonlinear least squares regression.