Inhibition of platelet activation and thrombus formation by adenosine and inosine: studies on their relative contribution and molecular modeling.

Eduardo Fuentes,Marcelo Alarcón,Iván Palomo,Jaime Pereira,Diego Mezzano,Julio Caballero,James P Brody

doi:10.1371/journal.pone.0112741

Abstract

BackgroundThe inhibitory effect of adenosine on platelet aggregation is abrogated after the addition of adenosine-deaminase. Inosine is a naturally occurring nucleoside degraded from adenosine.ObjectivesThe mechanisms of antiplatelet action of adenosine and inosine in vitro and in vivo, and their differential biological effects by molecular modeling were investigated.ResultsAdenosine (0.5, 1 and 2 mmol/L) inhibited phosphatidylserine exposure from 52±4% in the control group to 44±4 (p<0.05), 29±2 (p<0.01) and 20±3% (p<0.001). P-selectin expression in the presence of adenosine 0.5, 1 and 2 mmol/L was inhibited from 32±4 to 27±2 (p<0.05), 14±3 (p<0.01) and 9±3% (p<0.001), respectively. At the concentrations tested, only inosine to 4 mmol/L had effect on platelet P-selectin expression (p<0.05). Adenosine and inosine inhibited platelet aggregation and ATP release stimulated by ADP and collagen. Adenosine and inosine reduced collagen-induced platelet adhesion and aggregate formation under flow. At the same concentrations adenosine inhibited platelet aggregation, decreased the levels of sCD40L and increased intraplatelet cAMP. In addition, SQ22536 (an adenylate cyclase inhibitor) and ZM241385 (a potent adenosine receptor A2A antagonist) attenuated the effect of adenosine on platelet aggregation induced by ADP and intraplatelet level of cAMP. Adenosine and inosine significantly inhibited thrombosis formation in vivo (62±2% occlusion at 60 min [n = 6, p<0.01] and 72±1.9% occlusion at 60 min, [n = 6, p<0.05], respectively) compared with the control (98±2% occlusion at 60 min, n = 6). A2A is the adenosine receptor present in platelets; it is known that inosine is not an A2A ligand. Docking of adenosine and inosine inside A2A showed that the main difference is the formation by adenosine of an additional hydrogen bond between the NH2 of the adenine group and the residues Asn253 in H6 and Glu169 in EL2 of the A2A receptor.ConclusionTherefore, adenosine and inosine may represent novel agents lowering the risk of arterial thrombosis.

Highlights

Cardiovascular diseases (CVD) have increased significantly in recent years [1,2]
Collagen/adenosine 59- diphosphate bis (ADP)-induced externalization of PS assessed by annexin-V binding in the presence of adenosine 0.5, 1 and 2 mmol/L was inhibited from 5264% in the control group to 4464% (p,0.05), 2962 (p,0.01), and 2063% (p,0.001), respectively
The influence of adenosine (0.5 to 2 mmol/L) and inosine (1 to 4 mmol/L) on Pselectin expression by human platelet after stimulation of ADP/ collagen in citrated whole blood was measured by flow cytometry

Summary

Introduction

Cardiovascular diseases (CVD) (i.e., acute myocardial infarction, cerebrovascular disease and peripheral arterial thrombosis) have increased significantly in recent years [1,2]. The major and independent risk factors for CVD are cigarette smoking, elevated blood pressure, elevated serum total cholesterol and diabetes, among others [3,4]. Platelet hyper-aggregability is associated with risk factors for CVD [5]. Platelets from patients with type 1 and type 2 diabetes exhibit enhanced platelet aggregation activity early [6,7]. Platelet accumulation at vascular injury sites is the primary event in arterial thrombosis and its activation is a critical component of atherothrombosis [8]. Patients with unstable complex lesions had a fivefold higher expression of platelet activation than patients with stable angina, indicating an intense thrombogenic potential [9]. The inhibitory effect of adenosine on platelet aggregation is abrogated after the addition of adenosinedeaminase.

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