Figure 3. Effect of FX on A1 cleavage at Arg³³⁶ by APC.

Mixtures of FVIIIa (130 nM) and various concentrations of FX were incubated with APC (2 nM) and PSPCPE (100 μM) in HBS-buffer containing 2 mM CaCl₂ for 10 min. Samples were run on 8% gels followed by Western blotting using an anti-A1 monoclonal antibody, 58.12. Inset: Lanes 1–6 show the A1 cleavage of FVIIIa in the presence of FX (0, 20, 50, 100, 200, and 400 nM, respectively). The graph shows quantitative densitometry of the A1³³⁶ product. Density values of A1³³⁶ generated by APC cleavage in the absence of FX were used to represent the 100% level. Data were fitted by nonlinear least squares regression using Equation 2 (competitive inhibition model) and shown as a dashed line. Experiments were performed at least three separate times and typical results are shown.