Concluding Summary

Background—Activated protein C in complex with endothelial protein C receptor (EPCR) can reverse barrier disruptive and cytotoxic effects of proinflammatory cytokines by cleaving protease-activated receptor 1 (PAR-1). Recently, it was reported that the PAR-1-dependent vascular barrier-protective effect of APC is mediated through transactivation of the angiopoietin (Ang)/Tie2 signaling pathway. The antagonist of this pathway, Ang2, is stored in Weibel-Palade bodies within endothelial cells. Objectives—The objective of this study was to determine whether the occupancy of EPCR by its ligand can switch the PAR-1-dependent signaling specificity of thrombin through the Ang/Tie2 axis. Methods—We activated endothelial cells with thrombin before and after treating them with the catalytically inactive Ser-195 to Ala substitution mutant of protein C. The expression levels of Ang1, Ang2 and Tie2 were measured by both an ELISA and cell permeability assay in response to thrombin in the absence and presence of siRNA and a blocking antibody to Tie2. Results—We discovered that thrombin up-regulates the expression of both Ang1 and Tie2, but down-regulates Ang2 when EPCR is occupied by its ligand. The Ang1/Tie2-dependent protective effect of thrombin was initiated through protein C inhibiting the rapid mobilization of Ang2 from Weibel-Palade bodies. Interestingly, the protein C mutant also inhibited the thrombin mobilization of P-selectin. Conclusions—These results suggest a physiological role for the low concentration of thrombin in maintaining the integrity of the EPCR-containing vasculature through the PAR-1-dependent inhibition of Ang2 and P-selectin release from Weibel-Palade bodies