The barrier-enhancing effect of TRAP and its reversal by the ligand occupancy of EPCR. (A) EA.hy926 cells were incubated with increasing concentrations of thrombin (○), thrombin + thrombomodulin fragments 456 (TM456) (●), TRAP (□), TRAP + wild-type protein C (■), TRAP + PC-S195A (▵), TRAP + wild-type protein C + blocking anti-PAR-1 (H-111) (▴), and TRAP + PC-S195A + anti-PAR-1 (H-111) (▿) for 3 hours before inducing permeability with 5 nmol/L thrombin for 10 minutes. (B) The same as (A) except that the permeability was directly monitored as a function increasing concentrations of thrombin (○), thrombin + PC-S195A (▴), TRAP (□), and TRAP + PC-S195A (■) by incubation of EA.hy926 cells with different agonists for 3 hours (the 5 nmol/L thrombin step for inducing permeability has been omitted in this assay). The first data point on the y-axis represents the baseline permeability level. OD, optical density. Error bars represent SD.