Concentration dependence of wild-type and mutant protein C and prethrombin-2 activation by the activation complexes. (A) The activation of both wild-type (○) and mutant protein C (•) by thrombin (1 nM) in complex with saturating concentrations of TM456 (100 nM) was monitored in TBS/Ca²⁺. Following 8 minutes of activation at room temperature, thrombin activity was neutralized by hirudin and the rate of APC generation was determined by an amidolytic activity assay described in “Materials and methods.” (B) The activation of both wild-type (○) and mutant prethrombin-2 (•) by fXa (0.1 nM) in complex with saturating concentrations of factor Va (20 nM) on PC/PS vesicles was monitored in TBS/Ca²⁺. Following 2 to 5 minutes of activation at room temperature, EDTA was added to a final concentration of 20 mM and the rate of thrombin generation was determined by an amidolytic activity assay described in “Materials and methods.” Solid lines in both panels are nonlinear regression analyses of kinetic data according to the Michaelis-Menten equation.