Contributions of PAR3 and Tie2 to FXa-induced endothelial barrier protection. (A) Protection of endothelial barrier function by FXa (25 nM; 3 hours) in the presence of blocking antibodies against Tie2 (anti-hTie2; 25 µg/mL). The maximal permeability induced by thrombin in nontreated cells was considered as 100%. (B) Upregulation of ZO-1 compared with β-actin in time after treatment of EA.hy926 cells with FXa (50 nM) or P3R (50 µM). (C) Quantification of the time-dependent ZO-1 upregulation by FXa (50 nM). (D) Quantification of P3R (50 µM) induced ZO-1 upregulation after 3 hours. (E) Effect of blocking Tie2 (anti-hTie2; 25 µg/mL) and PAR3 (Moab19b; 25 µg/mL) antibodies on FXa-induced (50 nM; 3 hours) ZO-1 upregulation. (F) Immunofluorescent staining for ZO-1 and 4,6-diamidino-2-phenylindole (DAPI) in EA.hy926 cells incubated for 3 hours in the absence or presence of FXa (50 nM) or P3R (50 µM). Images were acquired with a fluorescence microscope system (Axio Carl Zeiss Imager.M1) with a ×40/0.75 EC Plan-NEOFLUAR objective and a Zeiss Axiocam MRm black-and-white camera and AxioVision 4.8 (Zeiss) acquisition software (exposure time: DAPI, 2 milliseconds; Alexa488, 200 milliseconds). Images were processed in Adobe Photoshop CS6, image size was adjusted to 300 dpi, contrast and brightness were increased to 100 and 80, respectively, and images were pseudo-colored by compiling the individual grayscale images into the corresponding red-green-blue layers. (A, C, and D) Data points represent the mean ± SD of at least 3 individual experiments. (B,E-F) Shown are typical images of representative experiments. *P < .05. ns, not significant.