Figure 3

Inactivation of Factor Va by Activated Protein C. Empirical: FVa (20 nM, Panels A-E) or FVa_i⁵⁰⁶ (20 nM, Panel F) was incubated with PC:PS vesicles (20 μM) and APC (0.5 or 2.0 nM) at 37°C. All data points are presented as averages ± S.D. of 4–6 experiments for each time point. (A) Time course of APC (0.5 nM) inactivation of FVa. Cofactor activity is presented relative to activity present at zero point (black circles). (B) Representative Western blot of time course of APC (2.0 nM) inactivation of FVa. (C) Densitometric analyses of the disappearance of FVa^HC at 0.5 or 2.0 nM APC (black and red circles, respectively). (D) Densitometric analyses of the time course of FVa^1-506 at 0.5 or 2.0 nM APC (black and red circles, respectively). (E) Densitometric analyses of the time course of FVa^307-506 at 0.5 or 2.0 nM APC (black and red circles, respectively). (F) Densitometric analyses of the inactivation fragment FVa^307-506 generated during the APC (0.5nM) inactivation of FVa_i⁵⁰⁶ (open circles). Inset displays a representative Western blot. Mathematical: Mathematically derived time courses of FVa inactivation by APC (0.5 nM - black lines and 2.0 nM - red lines) for the corresponding analytes are also presented in Panels A and C-F. Simulated time courses were generated using two sets of rate constants: Hockin et al. [33] – dashed lines; and current study – solid lines.