Evidence from our laboratory has revealed that brief episodes of antecedent 'preconditioning' ischemia, in addition to limiting infarct size: (1) improve coronary artery patency in a canine model of primary hemostasis mimicking unstable angina; and (2) enhance the speed and efficacy of thrombolysis in a canine model of coagulation and persistent thrombotic occlusion. Our results have further implicated release of adenosine from ischemic/reperfused myocardium, and subsequent stimulation of platelet adenosine A2 receptors, as playing a pivotal role in this phenomenon. However, the specific 'distal' molecular mechanisms responsible for these improvements in coronary patency are unknown. Platelet adhesion, activation -- and, ultimately, aggregation via the binding of fibrinogen to glycoprotein (GP) llb/lll receptors on adjacent platelets -- represents the underlying 'pathophysiology' of recurrent ischemia in the setting of unstable angina. Thus, our first hypothesis is that the improved coronary patency seen with brief antecedent ischemia/platelet A2 receptor stimulation is achieved by a down-regulation in the expression of adhesion molecules and/or ligands involved in the formation and stabilization of platelet aggregates -- specifically, GP IIb/IIIa, P-selectin, von Willebrand Factor, PECAM and/or GP Ib. The evolution from an unstable platelet 'plug' to a persistent, fibrin-containing clot involves the thrombin-catalyzed conversion of fibrinogen (the molecular 'bridge' in platelet aggregates) to fibrin, thrombin-mediated activation of Factor XIII and subsequent Factor XIII-catalyzed fibrin cross-linking. Accordingly, our second hypothesis is that brief antecedent ischemia/A2 receptor stimulation elicits -- as a consequence of changes in platelet activation/aggregation and adhesion molecule expression -- modifications in coagulation and thus fibrin polymerization during clot development that facilitate penetration and binding of the lytic agent and thus accelerate thrombolysis. These concepts will be tested via an integrated analysis of both in vivo and in vitro endpoints. First, we will employ in viva canine models of unstable angina and persistent thrombosis to evaluate the effects of brief antecedent ischemia and selective; pharmacologic A2 receptor stimulation on: coronary patency; platelet adhesion molecule expression (by flow cytometry; platelet aggregometry); and Factor XlIl-catalyzed fibrin polymerization (by quantitative light microscopy) in the evolving coronary thrombus. Second, in concurrent studies, we will assess the effects of exogenous A2 receptor stimulation on in vitro, surrogate indices of vessel patency (in vitro platelet aggregation; in vitro thrombosis/thrombolysis). Finally, to address our third hypothesis -- that the favorable effects of A2-mediated signaling observed in our experimental models have potential clinical relevance -- we will conduct an identical in vitro analysis of platelet aggregation and thrombosis/thrombolysis on blood samples obtained from human volunteers. These hypotheses, if confirmed, may ultimately aid in the design of novel therapeutic strategies aimed at enhancing vessel patency in clinical instances of unstable angina and reperfusion for the treatment of acute myocardial infarction.
