Initial rate of plasma PC and recombinant GDPC activation by the exosite 2 mutant thrombin R93,97,101A in the presence of EDTA. The initial rate of PC (A) or GDPC (B) activation was measured with 10 nmol/L thrombin mutant in the presence (□) or absence (▪) of heparin in TBS buffer containing 100 μmol/L EDTA, 1 mg/mL BSA, and 0.1% PEG 8000. After 15 minutes of incubation at room temperature, the thrombin activity was inhibited by antithrombin and the rate of PC activation was determined by an amidolytic activity assay as described under the Materials and Methods. Less than 5% PC was activated at all substrate concentrations.