Figure 1.
Effects of FVIII K1813A, K1818A, and K1813A/K1818A mutants on the kinetics of FIXa-catalyzed FX activation. (A) FIXa association. FVIII WT or mutants (1 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 (0-40 nM). (B) FX association. The WT 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). WT, open circles; K1813A, closed circles; K1818A, open squares; and K1813A/K1818A, closed squares. Experiments were performed 3 separate 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 using nonlinear least-squares regression.

Effects of FVIII K1813A, K1818A, and K1813A/K1818A mutants on the kinetics of FIXa-catalyzed FX activation. (A) FIXa association. FVIII WT or mutants (1 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 (0-40 nM). (B) FX association. The WT 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). WT, open circles; K1813A, closed circles; K1818A, open squares; and K1813A/K1818A, closed squares. Experiments were performed 3 separate 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 using nonlinear least-squares regression.

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