APC-catalyzed proteolytic cleavage of the HCh of FVa-W1920R. FV variants (8 nM) were incubated with thrombin (100 nM) for 5 minutes, and the reaction was terminated by the addition of hirudin (25 U/mL). FVa variant samples (0.5 nM) were incubated with APC (1 nM) and PS (30 nM) in the presence of PL (20 μM) for the indicated times. Samples were analyzed on 8% gels, followed by western blotting using an anti-FV HCh mAb 5146 IgG, as described in “Materials and methods.” A vertical line is inserted to indicate a repositioned gel lane.