Protamine inhibits activation of intact FV by thrombin and by FXa. (A) The rate of activation of 100 nM purified FV by 1 nM thrombin was measured in a PT-based clotting assay using FV-deficient plasma (as described in “Methods”) in the absence (○) and presence (▼) of 50 μg/mL protamine. (B) To determine the 50% inhibitory concentration for the observed effect, the activity of FV was measured after incubation with thrombin and increasing (0.25-12.5 μg/mL) concentrations of protamine for 60 seconds. Activity was expressed as a percentage of activity in the absence of protamine. (C) To assess whether FXa-mediated FV activation was affected by protamine, the rate of activation of 100 nM purified FV by 20 nM FXa in the presence of phospholipid vesicles (PC:PS:PE 60%:20%:20%, 80 μM final concentration) was assessed in the absence (●) and presence (□) of 50 μg/mL protamine in a PT-based clotting assay.