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Presenter: H.S., Smith-Hurst, Sydney
, Australia
Authors: H.S. Smith-Hurst, W.J. Hawthorne, P.J. O'Connell
Protease-activated recetor-1 (PAR-1) activation by thrombin is protective in human islets if the endothelial protein C receptor (ECPR) is occupied
H.S. Smith-Hurst, W.J. Hawthorne, P.J. O'Connell
University of Sydney (Westmead Hospital), Centre for Transplant and Renal Research, Sydney, Australia
Objectives: Our previous studies have indicated that Activated Protein C (APC) exerts anti-inflammatory activities during the Instant Blood Mediated Inflammatory Reaction (IBMIR). The mechanisms for the anti-inflammatory response are unclear. The aim of this study was to determine if APC leads to recruitment of PAR1 to protective signalling pathways through coupling of PAR1 to the Gi-protein.
Methods: Human Islet cells were exposed to specific PAR1 agonist peptides (PAR-APs). Receptor and protein expression was determined by RT-PCR and western blot assays analysis and measurement of pro-coagulant activity was performed by clotting assays.
Results: When protein C is bound to ECPR, PAR1 cleavage dependent protective signalling responses in islets can be mediated by thrombin or APC. To confirm that the occupancy of EPCR by its natural ligand protein C switches the signalling specificity of thrombin from a pro-inflammatory to anti-inflammatory response, we monitored the clotting of islets which were treated with PAR1-agonist peptides (PAR1-APs). Prior treatment of islets to block ECPR via APC with thrombin resulted in a protective effect in a concentration dependent manner by reducing clot formation 5-fold with optimal effect at 50nM. Clotting was also significantly reduced 4-fold when islets treated with PAR1-AP were reversed by the addition of APC. Thrombin in the presence of EPCR blocking inhibited clot formation with an optimal concentration of 20nM thrombin. The protective effects were PAR-1 and ECPR dependent since functional-blocking antibodies to either receptor abrogated the protective response of APC as well as thrombin with ECPR blocking. Islets treated with TNF-α induced apoptosis. APC inhibited cell death by a concentration dependent manner.
Conclusions: These results suggest that cleavage of PAR-1 by thrombin in human islets elicits protective responses if EPCR is occupied by protein C. The results provide new understanding of how PAR1 and APC participate in protective signalling events in IBMIR.
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