The permeability barrier enhancing and proapoptotic effects of thrombin are reversed by the ligand occupancy of EPCR. (A) EA.hy926 cells were incubated with increasing concentrations of thrombin (○), thrombin + anti-PAR-1 antibody (●), meizothrombin (□), PC-Gla/meizothrombin (■), thrombin + PC-S195A (50 nmol/L) (▴), and APC (▵) for 3 hours before inducing permeability with 5 nmol/L thrombin for 10 minutes. (B) The same as (A) except that after incubation of endothelial cells with increasing concentrations of thrombin (○), thrombin + PC-S195A (▴), and APC (▵), the permeability was induced with LPS (10 ng/mL) for 4 hour. (C) EA.hy926 cells were treated with increasing concentrations of thrombin (○), thrombin + PC-S195A (▴), and APC (▵) for 24 hours, followed by induction of apoptosis with staurosporine (5 μmol/L) for 4 hours. The number of apoptotic cells is expressed as the percentage of TUNEL-positive cells of the total number of nuclei. The number of TUNEL-positive cells in the absence of staurosporine was 10%-15%. (D) The same as (A) except that the concentration dependence of the protective effect of PC-S195A in the presence of 2 nmol/L thrombin (○) is measured in the permeability assay. The extent of the protective effect of 10 nmol/L APC (▵) is also presented. OD, optical density. Error bars represent SD.