Inhibiting protease-activated receptor 4 limits myocardial ischemia/reperfusion injury in rat hearts by unmasking adenosine signaling

Harnessing endogenous cardioprotectants is a novel therapeutic strategy to combat ischemia/reperfusion (I/R) injury. Thrombin causes I/R injury, whereas exogenous adenosine prevents I/R injury. We hypothesized that blocking thrombin receptor activation with a protease-activated receptor ( PAR) 4 antagonist would unmask the cardioprotective effects of endogenous adenosine. The protective role of two structurally unrelated PAR4 antagonists, trans-cinnamoyl-YPGKF-amide (tc-Y-NH2) and palmitoyl-SGRRYGHALR-amide (P4pal10), were evaluated in two rat models of myocardial I/R injury. P4pal10 (10 mu g/kg) treatment before ischemia significantly decreased infarct size (IS) by 31, 21, and 19% when given before, during, and after ischemia in the in vivo model. tc-Y-NH2 (5 mu M) treatment before ischemia decreased IS by 51% in the in vitro model and increased recovery of ventricular function by 26%. To assess whether the cardioprotective effects of PAR4 blockade were due to endogenous adenosine, isolated hearts were treated with a nonselective adenosine receptor blocker, 8-sulfaphenyltheophylline (8-SPT), and tc-Y-NH2 before ischemia. 8-SPT abolished the protective effects of tc-Y-NH2 but did not affect IS when given alone. Adenosine-mediated survival pathways were then explored. The cardioprotective effects of tc-Y-NH2 were abolished by inhibition of Akt (wortmannin), extracellular signal-regulated kinase 1/2 [PD98059 (2 '-amino-3 '-methoxyflavone)], nitric-oxide synthase [N-G-monomethyl-L-arginine (L-NMA)], and K-ATP channels (glibenclamide). PD98059, L-NMA, and glibenclamide alone had no effect on cardioprotection in vitro. Furthermore, inhibition of mitochondrial K-ATP channels [5-hydroxydecanoic acid (5-HD)] and sarcolemmal K-ATP channels ( sodium (5-(2-(5-chloro-2-methoxybenzamido)ethyl)-2-methoxyphenylsulfonyl)(methylcarbamothioyl) amide; HMR 1098) abolished P4pal10- induced cardioprotection in vivo. Thrombin receptor blockade by PAR4 inhibition provides protection against injury from myocardial I/R by unmasking adenosine receptor signaling and supports the hypothesis of a coupling between thrombin receptors and adenosine receptors.