EPCR occupancy inhibits the adhesion of THP-1 cells to cytokine-activated EA.hy926 endothelial cells through Dvl-2 and β-arrestin-2 biased signaling. (A-C) TNF-α (10 ng/mL)–mediated adherence of THP-1 cells to EA.hy926 cell monolayers was analyzed after treating monolayers with APC (20 nM) or thrombin (2 nM) + PC-S195A (50 nM) after transfecting cells with control siRNA (A), β-arrestin-2 siRNA (B), or Dvl-2 siRNA (C). (D) Western blot analysis of the efficiency of silencing by the control siRNA or the siRNA specific for Dvl-2. (E-G) The same as panels A-C except that the adherence of THP-1 cells to LPS (10 ng/mL)–stimulated endothelial cells were monitored. (H) Western blot analysis of the efficiency of silencing by the control siRNA or the siRNA specific for β-arrestin-2. (I-J) Analysis of EPCR-dependent cytoprotective activity of APC and thrombin by the clonogenic assay. Following transfection of EA.hy926 cells with control siRNA of siRNAs specific for β-arrestin-2 (I) or Dvl-2 (J), cells were treated with thrombin (2 nM), APC (20 nM), or thrombin (2 nM) + PC-S195A (50 nM) for 3 hours. Next, cells were treated with the apoptosis inducer staurosporine (1 µM) for 8 hours. Attached and viable cells were stained with crystal violet. *P < .05 as compared with TNF-α in panel A and LPS in panel E.