Figure 3.

Initial rate of protein C activation by thrombin in the absence and presence of TM and interaction with EPCR. (A) Time course of activation of protein C zymogens (O, WT; ●, G74S) (1 μM) by thrombin (1 nM) in the presence of sTM (10 nM) was monitored in TBS/Ca²⁺. At indicated time intervals, the activity of thrombin was inhibited by antithrombin (20 nM) and heparin (100 nM) and the rate of APC generation was determined by an amidolytic activity using SpPCa as described in Materials and methods. (B) The same as A, except that in the absence of TM, the time course of protein C activation by 10 nM thrombin was carried out in TBS containing 1.0 mM EDTA. (C) The same as B, except that the zymogens were activated by thrombin (50 nM) in the absence of TM in TBS/Ca²⁺. (D) The affinity of APC-WT (O) and APC-G74S (●) for interaction with the HPC4-tagged soluble EPCR (0.5 μM) was measured by an ELISA-based binding assay using the monoclonal antibody, HPC4 (1 μg/mL), and a goat polyclonal anti-protein C antibody as described in Materials and methods. (E) The inhibition of thrombin-induced hyperpermeability (10 nM for 10 min) by APC derivatives (20 nM each) in the absence and presence of protein S (110 nM) was monitored from the flux of Evans blue-bound albumin across endothelial cells as described under Materials and methods. Data are derived from two independent measurements.