FX activation by FIXa in the presence or absence of FVIIIa. FX activation by FIXa-WT or FIXa variants was assessed as described in “Methods.” (A) FX activation (300 nM) by 20 nM FIXa and 20 μM PCPS without FVIIIa. Data are presented as mean ± standard deviation (SD) (n = 3). (B) Enzyme kinetics of FX activation by 0.2 nM FIXa, 40 nM FVIIIa, and 20 μM PCPS. Solid lines represent the best fit of the data to the Michaelis-Menten equation (R² ≥ 0.97). (C) FVIIIa binding to 0.2 nM FIXa was kinetically assessed using varying concentrations of FVIIIa, PCPS (20 μM), and FX (200 nM). The solid lines are the fittings of a quadratic binding equation (R² ≥ 0.97). Data are representative of 3 independent experiments. The fitted parameters are listed in Table 2. E, enzyme.