APC cofactor activity of FV-W1920R assessed by the degradation of FVIIIa. (A) FVIIIa inactivation. FVIII (10 nM) with PL (20 μM) was activated by thrombin (5 nM), followed by the addition of hirudin (2.5 U/mL). Generated FVIIIa was incubated either with mixtures of APC (0.5 nM), PS (5 nM), and FV variants (0.5 nM) for the indicated times (a) or with various concentrations of FV variants for 20 minutes (b). FXa generation was initiated by the addition of FIXa (2 nM) and FX (200 nM) for 1 minute. The symbols used are as follows: open circles, WT; closed circles, W1920R; open squares, R506Q; closed squares, no FV. Values of FXa generation at the initial time (a) or in the absence of FV variants (b) were regarded as 100%. The data in (a) were fitted on an equation of single exponential decay (dashed lines). All experiments were performed at least 3 separate times, and the average values are shown. (B) A1 cleavage at Arg336; FVIII (10 nM) with PL (20 μM) was activated by thrombin (5 nM) for 30 second, followed by the addition of hirudin (2.5 U/mL). Generated FVIIIa was incubated with mixtures of APC (0.5 nM), PS (5 nM), and FV variants (0.5 nM) for the indicated times. Samples were analyzed on 8% gels, followed by western blotting using an anti-A1 C5 IgG, as described in “Materials and methods” (a). Band densities of intact A1^1-372 observed from panel a were measured by quantitative densitometry. Density before the addition of APC was regarded as 100% (b). The plotted data were fitted in an equation of single exponential decay (dashed lines). The symbols used are as follows: open circles, WT; closed circles, W1920R; open squares, R506Q. All experiments were performed at least 3 separate times, and the average values are shown.