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Protease-activated receptors (PARs), MAPK and NFκB signalling in response to reactive oxygen species (ROS) and apoptosis in human pancreatic islets 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: Protease-activated receptors (PARs) are involved in inflammatory disorders and are expressed on human islets. Thrombin is the main activator of these receptors but several other proteases can contribute to their function. We assessed the effects of PAR1 activation in human islets and the resulting down stream effects of NFκB, MAPK signalling, on ROS production and apoptotic genes expression. Methods: Human islets prepared for clinical transplantation were exposed to specific PAR1 agonist peptides (PAR-APs). NFκB, MAPK (ERK,p38,Jun) expression, apoptotic genes (Bax,Bcl-2,Bad,Bcl-xL) and transcription factors Pdx-1 and insulin were determined by RT-PCR and western blotting. ROS production was measure using DCF fluorescence. Results: PAR1-AP increased NFκB protein in a time and concentration dependent manner. PAR1-AP increased ROS production and Islets exposure to H2O2 (10-100μmol/L) increased NFκB expression. ROS up-regulation was mediated by ERK1/2 and p38 MAPK activation, and MAPK inhibitors and antioxidants significantly reduced NFκB expression induced by PAR1-AP. Islets exposed to PAR1-AP showed diverse changes of mRNA overtime. Early on (at 1 hr) the anti-apoptotic genes Bcl-2 and Bcl-X­L were increased and the pro-apoptotic gene Bax was reduced as a protective measure against apoptosis. However after 6 hours exposure to PAR-APs a pro-apoptotic environment dominated with reduction in Bcl-2 and Bcl-XL expression, and consistent up-regulation of Bad. The transcription factors PDX-1 and insulin were both significantly reduced at 6 hours incubation. Conclusions: Our data indentify NFκB and MAPK as key signalling pathways triggered by PAR1-AP engagement in human islets and show that receptor activation leads to cascades of mechanistically coupled signalling events resulting in increased pro-coagulant activity and apoptosis. The elucidation of pathways downstream from PAR1 and PAR2 activation may be helpful in the design of pharmacological strategies to control the pro-coagulant and inflammatory effect of IBMIR.