Protamine inhibits TF-initiated thrombin generation in normal plasma. Coagulation was initiated in pooled normal plasma with 5 pM TF, 4 μM phospholipid vesicles (PC:PS:PE 60%:20%:20%), and 6.67 mM CaCl₂, and thrombin generation was followed using a fluorogenic substrate, as described in “Methods.” (A) Thrombin generation in the absence of protamine (●) was compared with 3 μg/mL (■) and 30 μg/mL (○) of protamine, respectively. (B) The effect of protamine (3-80 μg/mL) on individual thrombin generation curve parameters was determined. Parameters measured (top to bottom) were ETP (% of normal pooled plasma), TTP (minutes), lag time (minutes), and peak thrombin (nM). Unpaired 2-tailed t tests were performed to determine significance (***P < .0001 compared with plasma in the absence of protamine). All experiments were performed in triplicate. Results are expressed as mean ± SEM.