Proteolytic processing of factor Va by APC in the presence and absence of prothrombin. A total of 500 nmol/L factor Va, 120 μg/mL PE/PS/PC with or without 1.4 μmol/L prothrombin was incubated in 150 NaCl, 20 mmol/L HEPES, pH 7.4, 5 mmol/L CaCl₂, 0.2% BSA at 37°C for the times indicated. The reaction was stopped by addition of 10 mmol/L benzamidine HCl. At this point, prothrombin was added to the samples from the time course performed in the absence of prothrombin. This was to insure that the differences observed in the blots were not due to a prothrombin effect on the electrophoretic pattern. Gradient 5% to 15% SDS-PAGE gels were run, blotted, and probed with an affinity-purified, goat anti–factor Va heavy chain antibody. The blot was developed as described in Materials and Methods. Residual Va activity is indicated along the bottom of the gel. Molecular weight marker locations are indicated on the left of the figure. The locations of the factor Va heavy chain (HC), the product of cleavage at Arg506 (506), and the product of cleavage at Arg306 (306) recognized by this antiserum are indicated along the right side of the figure.