Effect of oxLDL treatment of SMC on assembly and activity of the Xase complex.

(A) Determination of the apparent affinity of fVIIIa/fIXa interaction. SMCs in the amount of 2 × 10⁵/well were incubated in the absence (○) or presence (●) of 100 μg/mL oxLDL for 12 hours. The initial rates of fXa generation were determined in the chromogenic assay performed at constant concentrations of fVIII (1 nM) and fX (140 nM) and indicated concentrations of fIXa. Each data point represents the mean value ± SD of triplicates. The experimental data were fitted to an equation describing the equilibrium bindingV = V_sat[fIXa]/K_app+ [fIXa], where V is the initial rate of fX activation, [fIXa] is the concentration of fIXa, V_sat is the rate of fX activation at the saturating [fIXa], and K_app is the apparent affinity of fVIIIa for fIXa. The data were fitted to this equation using Marquart algorithm and SigmaPlot 1.02 computer program. (B) Determination of the kinetic parameters of fX activation. FVIII (1 nM), fIXa (1 nM), and indicated concentrations of fX were added to SMCs incubated in the absence (○) or presence (●) of oxLDL, which was followed by determination of the initial rates of fXa generation as in panel A. Each data point represents the mean value ± SD of triplicates. The curves show the best fit of the data to the Michaelis equationV = V_max[fX]/K_m+ [fX], where V is the initial rate of fX activation, [fX] is the concentration of fX, V_max is the rate of fX activation at its saturating concentration, and K_m is the Michaelis constant. The data were fitted to the above equation as in panel A.