Initial rate of PC activation by thrombin. (A) The time course of PC-WT (○) and PC-T315A (●) (1 μM each) activation by thrombin (1 nM) in complex with sTM (10 nM) was monitored in TBS/Ca2+. At indicated time intervals, the activity of thrombin was inhibited by antithrombin and the rate of APC generation was determined by an amidolytic activity as described in “Materials and methods.” (B) The same as panel A except that the concentration dependence of PC activation by the thrombin-sTM complex was carried out in TBS/Ca2+ for 10 minutes and the rate of APC generation was determined as described previously. Data are derived from at 3 independent measurements (± standard deviation). The solid lines in panel A are computer fits of data to a linear equation and those in panel B are fits of data to the Michaelis-Menten equation, yielding Km and kcat values of 2.7 ± 0.2 µM and 7.8 ± 0.3 minutes−1 for APC-WT and 4.2 ± 0.4 µM and 34.5 ± 1.6 minutes−1 for APC-T315A, respectively.
