Adenosine Receptor Identification for Controlling Platelet Aggregation

Introduction : Adenosine, an autacoid and metabolite of ATP, has been known to have anti-platelet properties. Of the 4 adenosine receptors, both A 2A and/or A 2B have been implicated in adenosine-mediated anti-platelet properties, while the roles of A 1 and A 3 have not been clearly defined in humans. In addition, previous studies show that A 2A /A 2B on platelets are G-Protein Coupled Receptors and are coupled to a stimulatory G-protein that activate adenylyl cyclase and subsequently increase intracellular cAMP. An elevation of cAMP in platelets inhibits aggregation. In this study, we set out to determine which adenosine receptor subtype leads to inhibition of platelet aggregation, and change in intracellular cAMP. Materials and Methods : Platelet-rich plasma (PRP) was isolated from whole blood of human volunteers, and centrifuged at 200g for 10min. Light transmission aggregometry was performed by 100uM ADP with or without NECA (non-specific AR agonist), CCPA (A 1 AR agonist), CGS 21680 (A 2A AR agonist), BAY 60-6583 (A 2B AR agonist), DPCPX (A 1 AR antagonist), Sch 58261 (A 2A AR antagonist), GS 6201 (A 2B AR antagonist), and MRS 1220 (A 3 AR antagonist). Cyclic AMP was extracted from 100ul of PRP after adding 1ml of EtOH, 10mM ammonium formate, with 10ug/ml cGMP-Br as an internal control, and measured by liquid chromatography/ Tandem Mass Spectroscopy (Quantiva, ThrermoFisher). Results : ADP-induced platelet aggregation was inhibited in a dose dependent manner by the non-specific adenosine agonist, NECA (Figure 1). This inhibition of platelet aggregation was likely mediated by A 2A receptor as the specific A 2A receptor agonist had a similar effect (Figure 2). Furthermore, A 2A antagonist blocked the effects of NECA (Figure 5). Stimulation of A 1 receptor had no effect on the ADP-induced platelet aggregation, except at the highest concentration (250 µM), and is likely due to its non-specific effect on A 2A AR (Figures 3 and 4). Blockade of A 1 enhanced the effects of NECA (Figure 5). This suggest that A 2A and A 1 may have opposing roles for control of platelet aggregation. Stimulation of A 2B receptor, had no effect on ADP-induced platelet aggregation, except at the highest concentration (250 µM), which was likely due to the non-specific vehicle effects (2.5% DMSO, Figure 6). Blockade of A 2B receptor had no effect on NECA, while A 3 blockade showed slight inhibition on NECA9s anti-platelet effect (data not shown). NECA inhibition of platelet aggregation was likely due to elevation of intracellular cAMP as incubation for 5min with NECA stimulated intracellular cAMP (Figure 7). This effect was blocked by A 2A , not by A 1 antagonist. Conclusion : Our results support previous findings that adenosine receptor A 2A mediates adenosine-induced anti-platelet properties in human platelets. Adenosine and its analogs inhibit platelet aggregation to the natural stimulus, ADP. The mechanism appears to be due to elevation in intracellular cAMP. We did not find evidence that A 2B played a significant role in platelet aggregation. A 1 and A 3 . however, demonstrated modulatory effects that has not been previously described. Disclosures No relevant conflicts of interest to declare.