Mitochondrial reactive oxygen species: A common pathway for PAR1- and PAR2-mediated tissue factor induction in human endothelial cells

Background: Protease-activated receptors (PARs) comprise a family of G-protein-coupled receptors with a unique proteolytic activation mechanism. PARs regulate a broad range of cellular functions and are involved in the pathogenesis of inflammatory disorders. Moreover, PAR1 and PAR2 activation in the endothelium shifts it toward a prothrombotic condition. Objectives: To assess the relevance of intracellular reactive oxygen species (ROS) in the signaling events underlying tissue factor (TF) expression elicited by PAR1 and PAR2 occupancy in endothelial cells, and to investigate their source. Methods: Human umbilical vein endothelial cells (HUVEC) were exposed to specific PAR1 and PAR2 agonist peptides. TF expression was determined by real-time reverse transcription polymerase chain reaction analysis and measurement of procoagulant activity. ROS generation was determined by a fluorometric assay after cell loading with 2′-7′-dichlorofluorescein diacetate. Results: ROS generatedby the mitochondrial chain, mostly from complex III, provide a pathway through which PAR1 and PAR2 occupancy induces TF. Other sources of ROS do not participate in TF induction. Activation of both ERK1/2 and p38 MAPK is critical for mitochondrial ROS generation. In addition to these pathways shared by the two PARs, mechanisms downstream from PAR1 and PAR2 activation, different for the two receptors, also induced TF. A module that sensitively regulates PAR1 signaling and ultimately involves NF-κB activation has been identified. Conclusions: Our data identify ROS originating in mitochondria as key mediators of the signaling pathways triggered by PAR1 and PAR2 engagement in endothelial cells and show that downstream from receptor activation occur cascades that are mechanistically coupled to procoagulant activity.