APC-protamine anticoagulant synergy is not mediated by enhanced substrate proteolysis. (A) FVa proteolysis by APC was assessed in both the absence (▲) and presence (○) of 3 μg/mL protamine. Purified FVa (1 nM final concentration) was incubated with phospholipid vesicles (PC:PS:PE, 60%:20%:20%, 20 μM final concentration) in a buffer containing 175 mM NaCl, 3 mM CaCl₂, and 25 mM HEPES (pH 7.4) at 37°C. APC (2 nM final concentration) was added to initiate FVa inactivation. At specific time points, aliquots of the reaction mix were added to a separate prothrombinase mixture and thrombin generated was measured, as described in “Methods.” (B) To assess FVIIIa proteolysis by APC, thrombin generation was initiated with TF, phospholipid vesicles, and CaCl₂ in FVIII-deficient plasma to which 1.0 U/mL wild-type (□) or APC-resistant FVIII R336Q/R562Q (■), respectively, had been added. ETP was determined for each FVIII variant in the presence and absence of protamine (3 μg/mL) and also in the presence of APC (10 nM).