The effect of thrombin on the permeability of endothelial cells in the presence of protein C S195A and siRNA for PAR-4. (A) EA.hy926 cells were transiently transfected with siRNA for PAR-4 or control siRNA (1 μg each). After 3 days of incubation at 37°C, total cellular proteins were extracted, separated on 10% SDS-PAGE, and Western-blotted with antibodies directed to either PAR-4 or PAR-1. An antibody against actin was used as internal control. (B) EA.hy926 cells were incubated with increasing concentrations of thrombin (○), thrombin with prior treatment of cells for 3 days with control siRNA (●), thrombin with prior treatment of cells with siRNA for PAR-4 (□), thrombin + PC-S195A (■), thrombin + PC-S195A with prior treatment of cells with control siRNA (▵), and thrombin + PC-S195A with prior treatment of cells with siRNA for PAR-4 (▴) for 3 hours before inducing permeability with 5 nmol/L thrombin for 10 minutes. (C) The same as (B) except that the barrier-protective effect of APC was monitored in the absence (▵) and presence of control siRNA (●) or siRNA for PAR-4 (□). (D) The barrier-protective effect of thrombin + PC-S195A in endothelial cells is sensitive to PTX. EA.hy926 cells were incubated with increasing concentrations of thrombin (○), thrombin with prior treatment of cells with 100 ng/mL PTX for 16 hours (●), APC (□), APC with prior treatment of cells with 100 ng/mL PTX for 16 hours (■), thrombin + PC-S195A (▵), and thrombin + PC-S195A with prior treatment of cells with PTX (▴) for 3 hours before inducing permeability with 5 nmol/L thrombin. Error bars represent SD.