We recently demonstrated that the occupancy of endothelial protein C receptor (EPCR) by protein C switches the PAR-1-dependent signaling specificity of thrombin from a permeability-enhancing to a barrier-protective response in endothelial cells. To determine whether the occupancy of EPCR by protein C renders thrombin a protective enzyme, thus up-regulating the expression of signaling molecules in the antiinflammatory pathways, we investigated the effects of thrombin and thrombin receptor agonist peptides (TRAP) on TNF-a-stimulated HUVECs in the absence and presence of the catalytically inactive protein C-S195A by monitoring the expression of cell surface adhesion molecules (VCAM- 1, ICAM-1 and E-selectin), adhesion of neutrophils to cytokine-stimulated endothelial cells, regulation of the Rho family of small GTPases and the activation of nuclear factorkB (NF-kB) pathway. Analysis of the results indicates that both thrombin and TRAP initiate proinflammatory responses in endothelial cells, thus neither thrombin nor TRAP influenced the proinflammatory effects of TNF-a in the absence of the protein C mutant. Interestingly, however, the occupancy of EPCR by the protein C mutant switched the PAR-1-dependent signaling specificity of thrombin and TRAP, thus leading to inhibition of the expression of all three adhesion molecules as well as the binding of neutrophils to TNFa-stimulated endothelial cells. Furthermore, similar to activated protein C, both thrombin and TRAP activated Rac1 and inhibited the activation of RhoA and NF-kB pathways in response to TNF-a in cells pretreated with protein C-S195A. Based on these results we conclude that when EPCR is bound by its natural ligand protein C, the cleavage of PAR-1 by thrombin initiates antiinflammatory responses in vascular endothelial cells.
Disclosures: No relevant conflicts of interest to declare.