EPCR occupancy regulates β-arrestin-2 biased PAR1 signaling by recruiting GRK5. Confluent EA.hy926 endothelial cells were transfected with scrambled shRNA (A) or shRNA specific for GRK5 (B) and GRK6 (C) before treating cells with the PAR1 agonists (2 nM thrombin, 20 nM APC, or 2 nM thrombin + 50 nM PC-S195A for 3 hours) and monitoring cell permeability in response to either thrombin (10 nM for 10 minutes in panels A-C) or to LPS (10 ng/mL for 4 hours in panels D-F) as described in “Materials and methods.” (G) Western blot analysis of the efficiency of silencing by the scrambled control shRNA or shRNAs specific for GRK5 and GRK6. (H-I) Western blot analysis of Dvl-2 in cell lysates using an anti-Dvl-2 antibody in the control shRNA (H) or GRK5 shRNA silenced endothelial cells treated with thrombin (2 nM) in the absence and presence of PC-S195A (50 nM). All results are shown as means ± standard deviation of 3 different experiments. **P < .01; ***P < .001.