Factor Va inactivation by APC is inhibited by prothrombin in the absence of phospholipid. Factor Va (100 nmol/L) was incubated at 37°C in 0.15 mol/L NaCl, 5 mmol/L CaCl₂, 20 mmol/L HEPES, pH 7.5, 0.2 mg/mL BSA with 10 nmol/L APC in the absence or presence of different prothrombin concentrations. The reaction was stopped at the times indicated by addition of 10 mmol/L benzamidine HCl. The residual factor Va was measured with a 1-stage clotting assay using factor V–deficient plasma. The prothrombin concentrations present were: •, no prothrombin; □, 1.4 μmol/L prothrombin; ▵, 5 μmol/L prothrombin; and ○, 10 μmol/L prothrombin. Bars represent the standard deviation of 2 experiments, duplicate samples.