Vascular Pharmacology 113 (2019) 47–56 Contents lists available at ScienceDirect Vascular Pharmacology journal homepage: www.elsevier.com/locate/vph Adenosine receptor agonists deepen the inhibition of platelet aggregation by T P2Y12 antagonists ⁎ Magdalena Boncler ,1, Joanna Wzorek1, Nina Wolska, Dawid Polak, Cezary Watala, Marcin Rozalski Department of Haemostasis and Haemostatic Disorders, Medical University of Lodz, Lodz, Poland ARTICLE INFO ABSTRACT Keywords: Several adenosine receptor (AR) agonists have been shown in the past to possess anti-platelet potential; however, Platelet the adjunctive role of AR agonists in anti-platelet therapy with the use of P2Y12 receptor inhibitors has not been Adenosine receptor elucidated so far. P2Y12 antagonist This in vitro aggregation-based study investigates whether the inhibition of platelet function mediated by Dual anti-platelet therapy cangrelor or prasugrel metabolite can be potentiated by AR agonists. It evaluates the effect of non-selective (2- Aggregation chloroadenosine), A2A-selective (UK 432097, MRE 0094, PSB 0777) and A2B-selective AR agonists (BAY 60- 6583) on platelet function in relation to their toxicity, specificity towards adenosine receptor subtypes, structure and solubility. UK 432097, 2-chloroadenosine, MRE 0094 and PSB 0777 were found to be more or less potent inhibitors of ADP-induced platelet aggregation when acting alone, and that they remained non-cytotoxic to the cells. These AR agonists were also effective in the potentiation of the effects exerted12 byP2Y antagonists. Considering the estimated IC50 value, UK 432097, showing a relatively high binding affinity to the2A A adenosine receptor, has been identified as the most potent anti-aggregatory agent. This compound diminished platelet aggregation at nanomolar concentrations and further augmented platelet inhibition by P2Y12 antagonists by approx. 60% (P < .01). Our results indicate the importance of adenosine receptors as therapeutic targets and point out challenges and potential benefits of therapeutic use of a combined therapy12 ofP2Y antagonist and AR agonist in cardiopro- tection. Our comparative analysis of the effects of AR agonists on platelet response in plasma and whole blood may indirectly suggest that other blood morphology elements contribute little to the inhibition of platelet function by AR agonists. 1. Introduction or use combined therapy based on currently-available agents. Adenosine diphosphate (ADP) is a crucial mediator of both phy- Activation of blood platelets plays a crucial role in the initiation and siological haemostasis and thrombosis. Although ADP is regarded as a development of arterial thrombotic diseases, which are the leading weak agonist of blood platelets, it is nonetheless an important factor cause of morbidity and mortality in Western countries. Therefore, an- promoting platelet activation induced by other activators (thrombin, tiplatelet therapy is the ‘first choice’ in the management and treatment collagen); these activators themselves promote ADP release from in- of arterial thrombotic disorders [1]. Despite the fact that many anti- traplatelet storage pools, such as dense granules, where it is present in platelet agents are currently available and some are widely applied as molar concentrations. It results in a positive feedback that enhances drugs, the clinical problem of the effective treatment of arterial platelet aggregation and platelet plug formation. Additionally, ADP acts thrombosis has not been fully solved [2–4]. Therefore, there is still a synergistically to other platelet agonists, even the weak ones, such as need to develop novel platelet inhibitors with better efficacy and safety serotonin, adrenaline or chemokines [5]. ⁎ Corresponding author at: Department of Haemostasis and Haemostatic Disorders, Chair of Biomedical Sciences, Medical University of Lodz, ul. Mazowiecka 6/8, 92-215 Lodz, Poland. E-mail address: [email protected] (M. Boncler). 1 Both authors contributed equally to this manuscript. https://doi.org/10.1016/j.vph.2018.11.005 Received 24 July 2018; Received in revised form 1 October 2018; Accepted 18 November 2018 Available online 22 November 2018 1537-1891/ © 2018 Elsevier Inc. All rights reserved. M. Boncler et al. Vascular Pharmacology 113 (2019) 47–56 Platelets express two receptors for ADP: the P2Y1 receptor, which 2. Materials and methods initiates platelet aggregation, and the P2Y12 receptor, which enhances this process, leading to thrombus formation. P2Y12 has become a more 2.1. Chemicals attractive therapeutic target than P2Y1 for preventing arterial throm- botic disorders [6]. The major clinically approved P2Y12 inhibitors in- Adenosine receptor agonists were purchased from Tocris Bioscience clude the thienopyridines (ticlopidine, clopidogrel and prasugrel), the (Bristol, UK) (BAY 60-6583, PSB 0777 and 2-chloroadenosine), Toronto ATP analogue cangrelor and the cyclopentyl-triazolo-pyrimidine Research Chemicals (Toronto, Canada) (MRE 0094) or Axon Medchem (CPTP) derivative-ticagrelor [3,6]. Thienopyridines are prodrugs that (Reston, VA, USA) (UK 432097). Cangrelor (AR-C69931MX) was from are converted to short-living active metabolites that irreversibly in- Cayman Chemical (Ann Arbor, MI, USA). Prasugrel metabolite (R- activate the receptor and consequently inhibit ADP-induced platelet 138727) was obtained from BOC Sciences (Shirley, NY, USA). Calcein activation. Cangrelor is the first intravenous 12P2Y receptor inhibitor, AM was obtained from Molecular Probes (Eugene, OR, USA). which reversibly blocks ADP signaling in a non-competitive manner. Monoclonal antibodies anti-human CD61/PerCP, CD62/PE, PAC-1/ Ticagrelor is an allosteric antagonist of P2Y12, acting directly via re- FITC, mouse IgG1/PE isotype control, mouse IgG1/FITC isotype con- versible binding to the P2Y12 receptor, resulting in the non-competitive trol, Cellfix, buffered sodium citrate was obtained from Becton- inhibition of ADP-induced P2Y12 activation; it is therefore used in the Dickinson (San Diego, CA, USA). Phosphate buffered saline (PBS) was prevention of thromboembolic events in patients with acute coronary obtained from Biomed Lublin (Lublin, Poland). Dimethyl sulfoxide syndromes [3,6,7]. Clopidogrel, prasugrel, and ticagrelor are the most (DMSO), adenosine diphosphate (ADP) were obtained from Sigma (St. frequently-used oral platelet P2Y12 inhibitors in current clinical prac- Louis, MO, USA). All other chemicals, unless otherwise stated, were tice. Although all three agents have indications for use in acute cor- purchased from POCH (Gliwice, Poland). onary syndromes, current guidelines favour prasugrel and ticagrelor over clopidogrel because of their superior clinical benefits [8]. In turn, 2.2. Subjects cangrelor, being the first intravenously-administered 12P2Y inhibitor to be approved, seems to be the most promising option for percutaneous Human blood was obtained from healthy donors, who gave their coronary interventions [9]. written informed consent (35 men and 65 women; mean age Adenosine serves not only as a component of nucleic acids and the 29 ± 10 years). All individuals confirmed not having taken medica- most important energy carrier in the cell (ATP), but also as a signaling tions known to influence platelet function (e. g. non-steroidal anti-in- molecule which regulates tissue function [10,11]. Adenosine receptors flammatory drugs, NSAIDs) for at least two weeks prior to the study. (AR) are a subfamily of G-protein-coupled receptors, found in mem- The study was approved by the Human Studies Committee of the branes of various human cells and playing various physiological func- Medical University of Lodz (Poland) and was conducted in accordance tions. Of four known adenosine receptor subtypes (A1,A2A,A2B and with the guidelines established by the Declaration of Helsinki. A3), platelets express only A2A and A2B. Of the platelet AR receptors, A2A is regarded as a high affinity receptor, and platelets have a sig- 2.3. Collection and processing of blood samples nificantly higher density2A ofA than A2B [10,12,13]. Agonization of platelet AR results in an enhanced intracellular cAMP level and con- Whole blood was collected into the polypropylene tubes with a sequently leads to the inhibition of platelet activation and aggregation blood to citrate ratio of 9:1 (vol:vol). Platelet-rich plasma (PRP) was [11,14]. Therefore, the adenosine receptors A2A and A2B could act as prepared by 12-min of 190 ×g centrifugation of a withdrawn blood at targets for antiplatelet therapy, especially under circumstances when 37 °C. Platelet-poor plasma (PPP) was obtained by centrifuging PRP classical therapy of antagonizing the other purinergic receptor P2Y12 is samples at 2000 x g for 15 min at 37 °C. Platelets in plasma were insufficient or problematic. Apart from the natural agonist, adenosine, counted using photometry [38] and their count was adjusted to 8 a group of synthetic, longer-lasting agonists of A2A and A2B receptors 2 × 10 cells/ml. To ensure that the washing procedure did not arte- also exists [11–13,15]. This group includes agonists with good se- factually activate the platelets, the expression of P-selectin and active lectivity for A2A or A2B receptors, as well as non-selective compounds form of GPIIbIIIa in PRP was randomly monitored with the use of flow that activate more than one type of adenosine receptor. Several A2A cytometry (CD62P/PE-positive and PAC-1/FITC objects within CD61/ and A2B agonists have been tested clinically, and two