Coagulation proteases can modulate intracellular signaling events by activating a subfamily of G-protein coupled receptors named protease-activated receptors (PARs) expressed on the cell surface of various organs. In vitro studies have indicated that while activated protein C (APC) in complex with endothelial protein C receptor (EPCR) elicits anti-inflammatory responses via activation of PAR1, thrombin elicits proinflammatory responses via the activation of the same receptor. We have provided some insight into the basis for this paradoxical effect by these proteases and showed that activation of PAR1 by thrombin can also elicit protective cellular responses if EPCR is occupied by the Gla-domain of protein C. Thus, we demonstrated that the occupancy of EPCR by protein C switches the PAR1 signaling specificity of thrombin from a proinflammatory to an anti-inflammatory response. Recent data indicates that a ?rrestin2-dependent biased PAR1 signaling accounts for the protective signaling of APC. We hypothesize that occupancy of EPCR induces ?arrestin2 biased PAR1 signaling independent of the protease activating PAR1. In a recent study, we also demonstrated that APC inhibits the secretion and proinflammatory signaling of high mobility group box 1 (HMGB1) protein in endothelial cells through activation of PAR1. We further demonstrated inorganic polyphosphate (similar to the size in platelets) dramatically up-regulates proinflammatory signaling responses of HMGB1 though interaction with receptor for advanced glycation end products (RAGE) and the purinergic P2Y1 receptor. In an in vivo study, we investigated the mechanism of the cytoprotective activity of APC in an ischemia/reperfusion (I/R) injury model and showed that APC elicits a cardioprotective response through activation of the AMPK signaling during I/R. Further studies revealed that APC inhibits production of reactive oxygen species (ROS) in cardiac tissues. Based on our preliminary data, we hypothesize that EPCR/PAR1 dependent signaling by APC results in epigenetic regulation and suppression of the proinflammatory, redox modulating protein, p66shc, thereby inhibiting ROS-mediated cellular injury. We also hypothesize that APC, through epigenetic regulation, down- regulates HMGB1 release, which has emerged as a key nuclear cytokine involved in the pathogenesis of inflammatory disorders including severe sepsis. We propose to investigate these important questions in four Specific Aims:
Aim 1 will investigate the mechanism by which the activation of PAR1 by APC and thrombin elicits paradoxical signaling responses in endothelial cells.
Aim 2 will investigate the mechanism by which polyphosphate (by itself or in complex with HMGB1) elicits proinflammatory signaling responses that are counteracted by APC.
Aim 3 will investigate the mechanism of the protective activity of APC in a mouse model of injury-mediated peritoneal adhesion band formation.
Aim 4 will investigate the mechanism by which the APC activation of AMPK limits cardiac damage caused by ischemia and reperfusion injury.
The studies of this application will employ cellular and animal models to investigate mechanisms through which coagulation proteases, in particular APC and thrombin, activate the G-protein coupled cell surface receptor PAR1 to elicit intracellular signaling responses. The activation of PAR1 by coagulation proteases is known to be important for the regulation of a range of (patho)physiological processes including coagulation and inflammation. Understanding the PAR1-dependent role of coagulation proteases can lead to development of a new generation of therapeutic drugs that can potentially be useful for controlling thrombotic and inflammatory disorders, including heart attack and severe sepsis.
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