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047 COUNTER REGULATION OF THROMBOXANE PRODUCTION IN HUMAN PLATELETS BY PROTEASE-ACTIVATED RECEPTORS BA Parkins, JL Wallace The formation of new blood vessels (angiogenesis) is crucial for ulcer healing and is controlled in part by platelets. Platelets can modulate healing through release of pro- and anti-angiogenic factors. Vascular endothelial growth factor (VEGF) is a pro-angiogenic factor, the release of which can be triggered by activation of protease-activated receptor (PAR)-1. PAR1 agonists also inhibit the release of an anti-angiogenic factor, endostatin. Activation of PAR4 elicits effects opposite to those of PAR1 agonists.
Inflammation Research Network, University of Calgary, Calgary, Alberta
This study was performed to determine if the counter-regulatory effects of PAR1 vs. PAR4 extended to other platelet functions; in particular, we examined effects on thromboxane (TXB2) synthesis. We observed that exposure to a PAR4 agonist 5 min or less before exposure to a PAR1 agonist resulted in a significant decrease in TXB2 synthesis compared to the effects of the PAR1 agonist alone. We then investigated the mechanism underlying the reduction of PAR1-mediated effects observed following treatment with a PAR4 agonist. Initial studies focused on the possibility that the PAR4 agonist interfered with the rapid increase in intracellular calcium that is triggered by activation of PAR1. The PAR4 agonist alone, at 8 µM, did not produce a significant change in intracellular calcium, while a PAR1 agonist triggered a marked influx of calcium into the platelets. Pre-treatment with the PAR4 agonist had negligable effects on the calcium influx triggered by the PAR1 agonist.
We next hypothesized that PAR4 activation may interfere with one or more steps in the signal transduction pathway for PAR1-induced thromboxane synthesis. These studies are ongoing, with a focus on phosphorylation-dephosphorylation of several of the key kinases in this pathway (eg, ERK1/ERK2/MAPK). Pretreatment with inhibitors of several kinases (eg, p42MAPK, ERK2, MEK, p38 MAPK, Src, Raf1 kinase) confirmed that these factors are important in PAR1-induced thromboxanes synthesis in human platelets. Our studies thus far demonstrate an important system of counter-regulation of human platelet function by PAR1 and PAR4. Studies to identify the mechanism underlying this regulation are continuing.