Adenosine Receptor Agonists Increase the Inhibition of Platelet Function by P2Y12 Antagonists in a cAMP- and Calcium-Dependent Manner.

Nina Wolska,Marcin Rozalski,Cezary Watala,Hassan Kassassir,Boguslawa Luzak

doi:10.3390/ph13080177

Abstract

We have shown previously that platelet activity can be lowered through the simultaneous inhibition of P2Y12 receptor and activation of adenosine receptors (AR). This work explores this concept by testing the antiplatelet potential of nine AR agonists in combination with P2Y12 receptor antagonists—cangrelor and prasugrel metabolite. A panel of in vitro methods was used to assess platelet viability, P-selectin expression, GPIIb-IIIa activation, fibrinogen binding, calcium ion mobilization, VASP-P level, and cAMP formation, utilizing whole blood or isolated platelets from healthy volunteers. The AR agonists demonstrated anti-platelet effects, but stimulated signaling pathways to varying degrees. AR agonists and P2Y12 antagonists reduced expression of both P-selectin and the activated form of GPIIb-IIIa on platelets; however, the combined systems (AR agonist + P2Y12 antagonist) demonstrated stronger effects. The antiplatelet effects of AR when combined with P2Y12 were more pronounced with regard to exogenous fibrinogen binding and calcium mobilization. The cAMP levels in both resting and ADPactivated platelets were increased by AR agonist treatment, and more so when combined with P2Y12 inhibitor. In conclusion, as AR agonists are fast-acting compounds, the methods detecting early activation events are more suitable for assessing their antiplatelet action. The exogenous fibrinogen binding, calcium mobilisation and cAMP level turned out to be sensitive markers for detecting the inhibition caused by AR agonists alone or in combination with P2Y12 receptor antagonists.

Highlights

The leading cause of death in Western countries, according to current World Health Organisation data, is cardiovascular disease that results primarily from arterial thrombosis dependent on blood platelet hyperactivity
adenosine receptors (AR) agonists were used in a combination with two types of P2Y12 receptor antagonists: cangrelor (C) or prasugrel metabolite R-138727 (PM), the experimental set up was one AR agonist + one P2Y12 antagonist
We found that a whole set of markers of platelet activation, such as P-selectin expression, GPIIb-IIIa activation together with fibrinogen binding are reduced by AR agonists alone, and, what is noteworthy, the effects of P2Y12 inhibitors on those markers are strengthened by the addition of AR agonists

Summary

Introduction

The leading cause of death in Western countries, according to current World Health Organisation data, is cardiovascular disease that results primarily from arterial thrombosis dependent on blood platelet hyperactivity. Currently available therapeutic strategies often demonstrate unsatisfactory safety and efficiency, with one of the key issues being drug resistance stemming from high inter-individual variation among patients [2,3]. Novel platelet inhibitors and/or new therapeutic strategies are needed to provide safe and efficient treatments. Its inhibition blocks the ADP-dependent platelet activation pathway [4]—the enhancement of platelet aggregation initiated by another ADP receptor (P2Y1), rendering the clot formation process ineffective. The inhibition of the third platelet receptor from the P2 class, ATP-gated ion channel receptor P2X1, which activation does not directly induce the platelet aggregation but causes fast calcium mobilization and platelet shape change, was suggested as a potential way of reducing thrombotic events [5]. The most commonly used clinically-approved P2Y12 inhibitors are thienopyridines (ticlopidine, clopidogrel, and prasugrel—prodrugs whose short-lived active metabolites are irreversible P2Y12 inhibitors), the ATP analogue cangrelor (the first intravenous, reversible, non-competitive P2Y12 inhibitor) and the cyclopentyltriazolopyrimidine derivative ticagrelor [2,4]

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