PAR-1 and PAR-2 activation mediates tissue factor induction in endothelial cells through a redox-sensitive signaling pathway

Objective.To elucidate the mechanisms whereby activation of proteaseproteaseactivated receptor 1 and 2 (PAR1 and PAR2) stimulates tissue factor (TF) induction in human endothelial cells (HUVEC). Methods: TF was evaluated as procoagulant activity in HUVEC lysates. PAR1, PAR2 and TF mRNA levels were determined by RT-PCR. Intracellular reactive oxygen species (ROS) generation was detected by flow cytometry. Tyrosine phosphorylation, MAPK activation, Cox-2 and eNOS protein levels was assessed by Western analysis. Results. HUVEC express both PAR1 and PAR2 mRNA. Receptor occupancy by PAR1 and PAR2 agonist peptides resulted in TF expression and ROS overproduction. Increased TF activity was prevented by antioxidants through a mechanism that involves tyrosine phosphorylation. TF induction by agonists for both PAR1 and PAR2 is mediated by ERK1/2, p38 MAPK and PI-3K activation. PAR1 and PAR2 agonists also up-regulated eNOS and Cox-2 enzymes, whose metabolites do not participate in PAR-induced TF expression. A divergence in the signaling pathway downstream PAR1 and PAR2 was found in the coupling of PAR activation to the G protein subunit Gai/o. Conclusions. Results from this study entail common and divergent signaling pathways emanating from PAR1 and PAR2 activation that lead to TF induction and underline a role of ROS as key mediators in the appearance of an immediate procoagulant response by endothelial cells.