Adenosine Receptor Agonists Exhibit Anti-Platelet Effects and the Potential to Overcome Resistance to P2Y12 Receptor Antagonists

Abstract

Large inter-individual variation in platelet response to endogenous agonists and pharmacological agents, including resistance to antiplatelet therapy, prompts a search for novel platelet inhibitors and development new antithrombotic strategies. The present in vitro study evaluates the beneficial effects of three adenosine receptor (AR) agonists (regadenoson, LUF 5835 and NECA), different in terms of their selectivity for platelet adenosine receptors, when used alone and in combination with P2Y 12 inhibitors, such as cangrelor or prasugrel metabolite. The anti-platelet effects of AR agonists were evaluated in healthy subjects (in the whole group and after stratification of individuals into high-and low-responders to P2Y 12 inhibitors), using whole blood techniques, under flow (thrombus formation) and static conditions (study of platelet activation and aggregation). Compared to P2Y 12 antagonists, AR agonists were much less or not effective under static conditions, but demonstrated similar antiplatelet activity in flow. In most cases, AR agonists significantly enhanced the anti-platelet effect of P2Y 12 antagonists, despite possessing different selectivity profiles and antiplatelet activities. Importantly, their inhibitory effects in combination with P2Y 12 antagonists were similar in high-and low-responders to P2Y 12 inhibitors. In conclusion, a combination of anti-platelet agents acting via the P1 and P2 purinergic receptors represents a promising alternative to existing antithrombotic therapy.Molecules 2020, 25, 130 2 of 17 ADP is one of the key mediators of both physiological haemostasis and thrombosis, being not only a direct agonist of platelets, but also an important factor released from platelet intracellular structures, enhancing the platelet response initially induced by other activators. Platelets have two ADP receptors on their surface: the P2Y 1 receptor initiates platelet aggregation, while the P2Y 12 receptor enhances this process, eventually leading to the formation of a clot. Due to this fact, the P2Y 12 receptor is the main therapeutic target in anti-platelet therapy targeted at the ADP-dependent activation pathway [5]. Generally, the most commonly used clinically approved P2Y 12 inhibitors include the thienopyridine-class inhibitors (ticlopidine, clopidogrel and prasugrel), the ATP analogue cangrelor, and the cyclo-pentyl-triazolo-pyrimidine derivative ticagrelor [3,5]. Thienopyridines are prodrugs: their short-lived active metabolites irreversibly inactivate the receptor and consequently inhibit ADP-induced platelet activation. Cangrelor is the first (recently approved) intravenous P2Y 12 receptor inhibitor that reversibly and non-competitively blocks ADP signalling [6].Adenosine is an important purine metabolite, serving not only as a component of nucleic acids and ATP, the most important energy carrier in the cell, but also as a signalling molecule regulating many cell processes [7,8]. Adenosine receptors (AR) are a subfamily of highly conserved G protein-coupled receptors located in the membr...

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

“…AR agonists were used in a combination with two types of P2Y 12 receptor antagonists: cangrelor (C) or prasugrel metabolite R-138727 (PM), the experimental set up was one AR agonist + one P2Y 12 antagonist. Each compound was used at its aggregation IC 50 value [ 19 , 21 ]: PSB0777 23 µM, CGS21680 1 µM, MRE0094 26 µM, 2-chloroadenosine 5 µM, CV1808 25 µM, HE-NECA 0.2 µM, NECA 0.5 µM, regadenoson 1.2 µM, and UK423,097 1 µM, cangrelor 17 nM, and PM 1.3 µM, unless otherwise specified.…”

“…In the literature, some encouraging results have been published for the application of AR agonists in arrhythmias, cardiac and cerebral ischaemias, neurodegenerative diseases, inflammation, sleep disorders, pain, diabetes, cancer, renal failure as well as glaucoma [ 26 ]. Interestingly, agonists of AR receptors expressed on blood platelets (A 2A and A 2B ) were previously reported to have remarkable anti-platelet properties [ 11 , 13 , 19 , 21 ]. Hypothetically, the agonists of the A 2A and A 2B adenosine receptors could be a beneficial supplement to current antithrombotic therapy, especially in the light of frequently observed, high inter-individual variability in response to platelet inhibitors.…”

“…Our previous findings demonstrated the anti-aggregatory activity of some of the AR agonists presented here (UK 432097, MRE 0094, PSB 0777) examined in the absence or presence of the P2Y 12 antagonists (cangrelor or prasugrel metabolite) [ 19 ], and that the use of combination of a P2Y 12 antagonist and an AR agonist (regadenoson, NECA, and LUF5835) leads to increased inhibition of platelet function than the P2Y 12 antagonist alone, and that their antiplatelet effect was much more pronounced in individuals with poor response to P2Y 12 inhibitors [ 21 ]. This study supports our recent in vitro work, extending further analyses of the influence of AR agonists, on the effects of the P2Y 12 antagonists by introducing measurements of a panel of platelet activation hallmarks.…”

“…Prasugrel is a pro-drug and requires metabolization to its active components, therefore its most abundant stable active metabolite R-138727 was used [ 28 ]. P2Y 12 antagonists, as well as AR agonists, were used at IC 50 values [ 19 , 21 ]. The relatively high dosage (aggregation IC 50 = 1.3 µM) required to achieve the effective platelet inhibition most likely stems from the fact that only one of the prasugrel active metabolites is used, whereas in vivo prasugrel metabolization results in a number of active metabolites, all of which may exert an antiplatelet effect of their own.…”

“…In summary, this work provides the comprehensive evidence of the antiplatelet potential of AR agonists, demonstrated on multiple levels of platelet activation process, from calcium flux inhibition, and cAMP formation increase to restriction of surface markers of platelet activation such as P-selectin expression and GPIIb-IIIa activation together with fibrinogen binding. All the AR agonists studied were able to strengthen the effect of at least one P2Y 12 receptor inhibitor; therefore, a dual experimental therapy involving combination of P2Y 12 inhibitors and AR agonists appears to be a feasible solution to overcoming problems of drug resistance leading to dosage increase and resulting in severe side effects, and a way of combating inter-individual variation [ 21 ]. The approach combining the blocking of P2Y 12 receptor and the activation of AR receptors using novel agonists of AR receptors may prove to be a favourable strategy of preventing thrombotic events, and should therefore be further investigated (including in vivo studies in animal models).…”