P2Y12 receptor in platelet activation

ADP plays an important role in hemostasis and thrombosis. The P2Y12 receptor, activated by ADP, plays a central role in platelet activation and thrombus formation. Thus, the P2Y12 receptor has been an effective target for antithrombotic drugs.     

P2Y12 receptor in platelet activation

ADP plays an important role in hemostasis and thrombosis. The P2Y12 receptor, activated by ADP, plays a central role in platelet activation and thrombus formation. Thus, the P2Y12 receptor has been an effective target for antithrombotic drugs.     

血小板ADP-P2Y12受体拮抗剂研究进展

血小板在动脉粥样硬化斑块破裂后血栓形成发病机制中发挥关键作用,因而针对血小板激活途径的药物成为抗栓治疗的主要手段。循证医学表明,抗血小板治疗可以有效地预防动脉粥样硬化疾病患者发生严重的心血管事件。已经成为防治急性冠脉综合征的主要方法。     

P2Y12 receptor: platelet thrombus formation and medical interventions

Platelets express a wide range of receptors and proteins that play essential roles in thrombus formation. Among these, the P2Y₁₂ receptor, a member of the G protein-coupled receptor family, has attracted a significant amount of attention. Stimulation of the P2Y₁₂ receptor by ADP results in activation of various signaling pathways involved in amplification of platelet activation and aggregation. There have been extensive attempts to design an ideal antithrombotic agent to block P2Y₁₂, which shows selective expression, as an intervention for cardiovascular disease. Current inhibitors of the P2Y₁₂ receptor include indirect inhibitor members of the thienopyridine family (ticlopidine, clopidogrel, and prasugrel), and direct P2Y₁₂ inhibitors (ticagrelor, cangrelor and elinogrel). Of these, clopidogrel is the most commonly prescribed P2Y₁₂ blocker; however, this product does not fulfill the ideal therapeutic requirements. The main limitations of clopidogrel administration include slow onset, prevention of recovery of platelet functions, and interindividual variability. Hence, advanced studies have been carried out to achieve more efficient and safer P2Y₁₂ blockade. In this review, we provide a brief but comprehensive report on P2Y₁₂, its role on platelet thrombus formation, and the targeting of this receptor as an intervention for cardiovascular disease, for the benefit of basic science and clinical researchers.     

The platelet P2Y12 receptor contributes to granule secretion through Ephrin A4 receptor

The main responses of P2Y₁ ligation are platelet shape change and transient aggregation while P2Y₁₂ ligation amplifies P2Y₁-induced aggregation and accelerates aggregation, secretion and thromboxane A₂ production induced by other agonist-receptor complexes. We searched for new targets of P2Y signalling using micro-arrays with 144 peptides representing known phosphosites of protein tyrosine kinases. ADP induced phosphorylation of peptides representing surface receptors, second messenger enzymes and cytoskeletal proteins. Strong phosphorylation was found in peptides representing Ephrin-receptor family members. Blockade of P2Y_1/12 inhibited phosphorylation of EphA4- and EphB1-peptides on micro-arrays. The EphA2/4 inhibitor 2,5-dimethylpyrrolyl benzoic acid derivative interfered with P2Y_1/12-induced EphA4 phosphorylation, left P2Y₁-induced aggregation unchanged but inhibited with P2Y₁₂-induced secretion, second phase aggregation and thrombus formation on collagen at 1600?s^?1. These results show that platelet EphA4 is an important intermediate in P2Y₁₂-induced granule secretion.     

P2Y12, a new platelet ADP receptor, target of clopidogrel

Clopidogrel is a potent antithrombotic drug that inhibits ADP-induced platelet aggregation. The results of large clinical trials have demonstrated an overall benefit of clopidogrel over aspirin in the prevention of vascular ischemic events (myocardial infarction, stroke, vascular death) in patients with a history of symptomatic atherosclerotic disease. The antiaggregating effect of clopidogrel is attributed to an irreversible inhibition of ADP binding to a purinergic receptor present at the platelet surface. Clopidogrel is not active in vitro and can be considered a precursor of an active metabolite formed in the liver. The chemical structure of this active metabolite and its biological activity have been described recently. Several purinergic receptors have been described on platelets; P2X (1), a calcium channel, and P2Y1 a Gq-coupled seven-transmembrane domain receptor, have been found not to be antagonized by clopidogrel. Another Gi (2)-coupled receptor (named P2Y12) has been recently cloned and stably expressed in CHO cells. These cells displayed a strong affinity for (33)P-2MeS-ADP, a stable analogue of ADP, the binding characteristics of which corresponded in all points to those observed on platelets. The binding of (33)P-2MeS-ADP to these cells was strongly inhibited by the active metabolite of clopidogrel with a potency that was consistent with that observed for this compound on platelets. In these transfected CHO cells, as in platelets, ADP and 2MeS-ADP induced adenylyl cyclase downregulation, an effect that was inhibited by the active metabolite of clopidogrel. These results demonstrate that this receptor corresponds to the previously called "P2t" platelet receptor and show that the active metabolite of clopidogrel binds in a covalent manner to this receptor, thus explaining how it blocks the aggregating effect of ADP on platelets.     

P2Y12 platelet inhibition in clinical practice

Platelet adhesion, activation and aggregation play a pivotal role in atherothrombosis. Intracoronary atherothrombosis is the most common cause of the development of acute coronary syndrome (ACS), and plays a central role in complications occurring around percutaneous coronary intervention (PCI) including recurrent ACS, procedure-related myocardial infarction or stent thrombosis. Inhibition of platelet aggregation by medical treatment impairs formation and progression of thrombotic processes and is therefore of great importance in the prevention of complications after an ACS or around PCI. An essential part in the platelet activation process is the interaction of adenosine diphosphate (ADP) with the platelet P2Y12 receptor. The P2Y12 receptor is the predominant receptor involved in the ADP-stimulated activation of the glycoprotein IIb/IIIa receptor. Activation of the glycoprotein IIb/IIIa receptor results in enhanced platelet degranulation and thromboxane production, and prolonged platelet aggregation. The objectives of this review are to discuss the pharmacological limitations of the P2Y12 inhibitor clopidogrel, and describe the novel alternative P2Y12 inhibitors prasugrel and ticagrelor and the clinical implications of the introduction of these new medicines.     

PCI相关血小板P2Y12受体阻滞剂的研究现况及进展

抗血小板治疗是心血管领域永恒的话题,全世界学者都努力在出血和血栓之间寻找平衡,对于新型抗血小板药物的研究亦从未停止。目前,阿司匹林与氯吡格雷的双重抗血小板治疗是急性冠状动脉综合征(ACS)患者经皮冠状动脉介入（PCI）治疗围手术期的标准化治疗方案,近年来新型P2Y12受体阻滞剂的出现对这一标准方案提出挑战。本文通过综述各种P2Y12受体阻滞剂抗血小板作用机制及临床特点的异同,为临床上新型P2Y12受体阻滞剂的应用及进一步研究提供理论依据。     

The active metabolite of prasugrel effectively blocks the platelet P2Y12 receptor and inhibits procoagulant and pro-inflammatory platelet responses

The aim of these studies was to investigate the extent of platelet P2Y(12) receptor inhibition by the thienopyridine active metabolite of prasugrel, R-138727. Blood was taken from healthy volunteers and pre-incubated with R-138727 or cangrelor (AR-C66931MX). Platelet aggregation was assessed in platelet rich plasma (PRP) and whole blood (WB). Vasodilator stimulated phosphoprotein (VASP) phosphorylation, platelet procoagulant activity (annexin V binding and microparticle formation) and calcium mobilisation were measured by flow cytometry. Platelet-leukocyte co-aggregate formation and sCD40L release, both pro-inflammatory responses of platelets, were measured by flow cytometry and ELISA, respectively. P2Y(12) receptor antagonism was determined using a radioligand binding assay ((33)P 2-MeSADP) in resting and stimulated platelets and the effects of clopidogrel administration were also assessed. R-138727 yielded concentration-dependent inhibition of platelet aggregation, VASP phosphorylation inhibition, procoagulant activity and pro-inflammatory responses. In the presence of R-138727 or cangrelor there was increased calcium reuptake following agonist stimulation. R-138727 30 micromol/L and cangrelor 1 micromol/L completely inhibited (33)P 2-MeSADP binding, compared to partial inhibition following clopidogrel administration. Platelet activation and granule secretion did not expose an additional pool of P2Y(12) receptors. Prasugrel's active metabolite effectively blocks the P2Y(12) receptor with the highest concentrations tested yielding complete inhibition of P2Y(12)-mediated amplification of several important platelet responses.     

The active metabolite of prasugrel effectively blocks the platelet P2Y12 receptor and inhibits procoagulant and pro-inflammatory platelet responses

The aim of these studies was to investigate the extent of platelet P2Y₁₂ receptor inhibition by the thienopyridine active metabolite of prasugrel, R-138727. Blood was taken from healthy volunteers and pre-incubated with R-138727 or cangrelor (AR-C66931MX). Platelet aggregation was assessed in platelet rich plasma (PRP) and whole blood (WB). Vasodilator stimulated phosphoprotein (VASP) phosphorylation, platelet procoagulant activity (annexin V binding and microparticle formation) and calcium mobilisation were measured by flow cytometry. Platelet-leukocyte co-aggregate formation and sCD40L release, both pro-inflammatory responses of platelets, were measured by flow cytometry and ELISA, respectively. P2Y₁₂ receptor antagonism was determined using a radioligand binding assay (³³P 2-MeSADP) in resting and stimulated platelets and the effects of clopidogrel administration were also assessed. R-138727 yielded concentration-dependent inhibition of platelet aggregation, VASP phosphorylation inhibition, procoagulant activity and pro-inflammatory responses. In the presence of R-138727 or cangrelor there was increased calcium reuptake following agonist stimulation. R-138727 30 µmol/L and cangrelor 1 µmol/L completely inhibited ³³P 2-MeSADP binding, compared to partial inhibition following clopidogrel administration. Platelet activation and granule secretion did not expose an additional pool of P2Y₁₂ receptors. Prasugrel's active metabolite effectively blocks the P2Y₁₂ receptor with the highest concentrations tested yielding complete inhibition of P2Y₁₂-mediated amplification of several important platelet responses.     

Comparison of four methods to assess high-on platelet reactivity under P2Y12 receptor inhibitor

P2Y12 receptor inhibitors are antiplatelet agents commonly prescribed in the treatment of coronary artery disease. Their efficacy can be limited by high on-treatment platelet reactivity (HPR), which can be evaluated by different biological assays. Most commonly, HPR is evaluated by flow cytometric vasodilator-stimulated phosphoprotein-phosphorylation (VASP-P) assay, which can be time consuming. To evaluate the potential interest of novel technologies, we compared four different assays.

Ninety patients receiving P2Y12 inhibitors were included. Four technologies were evaluated: the current standard test measuring VASP-P by flow cytometry, the historical reference test based on light transmittance aggregation (LTA), and two relatively novel techniques: whole blood multiple electrode aggregometry (MEA) and platelet function analyzer (PFA), which are less time consuming.

The three latter tests were compared with the VASP-P assay as a reference using receiver operating characteristics (ROC) analysis: LTA has an excellent comparability with the VASP test (ROC AUC > 0.9); the other two tests (multiplate and PFA) have only satisfactory comparability (ROC AUC around 0.7) and therefore may not replace the VASP “gold standard” test, if importance is attached to a quantitative assessment of the substitution parameter of VASP. Nevertheless, if a binary approach of the anti-aggregation result is sought, then one can conclude that the three tests are equivalent since Cohen’s kappa coefficients are very close for the three tests (k = 0.548 for LTA; k = 0.554 for MEA; k = 0.570 for PFA/P2Y), and a similar proportion of patients are misclassified (15% for LTA, 14% for MEA, and 13.6% for PFA). Discriminant factor analysis using all the parameters provided by each test did not improve the diagnostic performance of MEA or PFA.

In conclusion, only LTA shows a good comparability to the VASP assay using ROC curve analysis, probably because misclassified patients have results close to the cutoff values. All three tests have moderate agreement regarding the classification of patients as responders to P2Y12 inhibition.     

Effects of P2Y 1 and P2Y 12 receptor antagonists on platelet aggregation induced by different agonists in human whole blood

ADP plays a major role in the amplification of platelet aggregation induced by other platelet agonists. ADP initiates platelet activation via the P2Y 1 receptor and amplifies platelet activation via the P2Y 12 receptor. Using the selective P2Y 1 receptor antagonist A2P5P and the selective P2Y 12 receptor antagonist AR-C69931MX, we assessed the relative contributions of P2Y 1 receptor and P2Y 12 receptor activation to platelet aggregation in hirudin-anticoagulated whole blood induced by PAF, 5HT, epinephrine, TRAP, streptokinase, U46619 and collagen. The effects of aspirin were assessed concurrently. A2P5P and AR-C69931MX variably inhibited aggregation induced by most of the agonists studied, whereas aspirin only inhibited aggregation induced by streptokinase and collagen. In some experiments, A2P5P and AR-C69931MX yielded additive inhibition of aggregation. All three antagonists interacted synergistically to inhibit collagen-induced aggregation. These studies demonstrate that P2Y 1 receptor activation plays a significant role in amplifying aggregation induced by agonists other than ADP, in addition to the established roles of P2Y 12 receptor activation and thromboxane A 2 synthesis.     

Intensified P2Y12 inhibition for high-on treatment platelet reactivity

High on treatment platelet reactivity (HPR) during treatment with clopidogrel has been consistently found to be strong risk factor for recurrent ischemic events after percutaneous coronary intervention (PCI). Insufficient P2Y12 receptor inhibition contributes to HPR measured by the VerifyNow (VN) assay. Prasugrel and ticagrelor are more potent P2Y12 inhibitors than clopidogrel and commonly substituted for clopidogrel when HPR is documented, however benefit of VN guided intensified antiplatelet therapy is uncertain. We identified patients who had undergone platelet reactivity testing after PCI with VN after pretreatment with clopidogrel (n?=?252) in a single center observational analysis. Patients who had HPR defined as PRU?>?208 were switched to alternate P2Y12 inhibitors. Primary clinical endpoint was 1-year post PCI combined cardiovascular death, myocardial infarction (MI), and stent thrombosis. One hundred and eight (43%) subjects had HPR and were switched to prasugrel (n?=?60) and ticagrelor (n?=?48). Risk of recurrent 1-year primary endpoint remained higher for HPR patients switched to either ticagrelor or prasugrel as compared to subjects who had low on treatment platelet reactivity (n?=?144) (LPR) on clopidogrel [Hazard Ratio: 3.5 (95% CI 1.1–11.1); p?=?0.036)]. Propensity score matched analysis demonstrated higher event rates in patients with HPR on alternate P2Y12 inhibitor as compared to patients with LPR (log-rank: p?=?0.044). The increased risk of recurrent events associated with HPR measured by VN is not completely attenuated by switching to more potent P2Y12 inhibitors. Non-P2Y12 mediated pathways likely contribute to increased incidence of thrombotic events after PCI in subjects with HPR.

     

Scientific and therapeutic insights into the role of the platelet P2Y12 receptor in thrombosis

Platelets are important mediators of thrombosis in both healthy and diseased vessels. When platelets become activated by various soluble agonists or by adhesion to subendothelium under high shear, they release adenosine-5'-diphosphate that acts in a positive feedback mechanism on two different G-protein coupled receptors (P2Y(12), P2Y(1)) on platelets. This released adenosine-5'-diphosphate, acting through P2Y(12), is critical for sustained aggregation and stabilization of thrombi. P2Y(12) is the target of antithrombotic drugs (ticlopidine, clopidogrel), whereas the role of P2Y(1) in thrombosis remains to be fully established. Recent studies using either inhibitors of key components of signaling pathways or genetically engineered mice have contributed to our understanding of the signaling mechanisms in platelets mediated by adenosine-5'-diphosphate through the P2Y(12) receptor. Studies of patients with defective adenosine-5'-diphosphate mediated aggregation, as well as P2Y(12)-null mice, have revealed the importance of this receptor in mediating platelet activation and aggregation. Recent clinical trials using approved P2Y(12) blockers have extended the use of these drugs to additional patient populations. Recent data demonstrating the role of P2Y(12) in mediating platelet adhesion to thrombogenic surfaces (collagen, von Willebrand factor) provide further rationale as to the clinical efficacy of P2Y(12) blockers. P2Y(12) antagonists in combination with anticoagulants (thrombin inhibitors, factor Xa inhibitors) act synergistically in inhibiting thrombus formation (similar to aspirin) ex vivo. These findings suggest the potential for combination therapies (P2Y(12) antagonists with inhibitors of GPIIb-IIIa, thrombin or Factor Xa, etc.) to provide additional clinical benefit to patients with various cardiovascular diseases, especially those who may be aspirin-resistant.     

Blockade of the purinergic P2Y12 receptor greatly increases the platelet inhibitory actions of nitric oxide

Circulating platelets are constantly exposed to nitric oxide (NO) released from the vascular endothelium. This NO acts to reduce platelet reactivity, and in so doing blunts platelet aggregation and thrombus formation. For successful hemostasis, platelet activation and aggregation must occur at sites of vascular injury despite the constant presence of NO. As platelets aggregate, they release secondary mediators that drive further aggregation. Particularly significant among these secondary mediators is ADP, which, acting through platelet P2Y₁₂ receptors, strongly amplifies aggregation. Platelet P2Y₁₂ receptors are the targets of very widely used antithrombotic drugs such as clopidogrel, prasugrel, and ticagrelor. Here we show that blockade of platelet P2Y₁₂ receptors dramatically enhances the antiplatelet potency of NO, causing a 1,000- to 100,000-fold increase in inhibitory activity against platelet aggregation and release reactions in response to activation of receptors for either thrombin or collagen. This powerful synergism is explained by blockade of a P2Y₁₂ receptor-dependent, NO/cGMP-insensitive phosphatidylinositol 3-kinase pathway of platelet activation. These studies demonstrate that activation of the platelet ADP receptor, P2Y₁₂, severely blunts the inhibitory effects of NO. The powerful antithrombotic effects of P2Y₁₂ receptor blockers may, in part, be mediated by profound potentiation of the effects of endogenous NO.     

P2Y12-ADP receptor antagonists: Days of future and past

Antiplatelet therapy is the cornerstone of the therapeutic arsenal in coronary artery disease.Thanks to a better understanding in physiology,pharmacology and pharmacogenomics huge progress were made in the field of platelet reactivity inhibition thus allowing theexpansion of percutaneous coronary intervention.Stent implantation requires the combination of two antiplatelet agents acting in a synergistic way.Asprin inhibit the cyclo-oxygenase pathway of platelet activation while clopidogrel is a P2Y12 adenosine diphosphate(ADP)-receptor antagonist.This dual antiplatelet therapy has dramatically improved the prognosis of stented patients.However,due to pharmacological limitations of clopidogrel(interindividual variability in its biological efficacy,slow onset of action,mild platelet reactivity inhibition)ischemic recurrences remained high following stent implantation especially in acute coronary syndrome patients.Thus,more potent P2Y12-ADP receptor inhibitors were developped including prasugrel,ticagrelor and more recently cangrelor to overcome these pitfalls.These new agents reduced the rate of thrombotic events in acute coronary syndrome patients at the cost of an increased bleeding risk.The abundance in antiplatelet agents allow us to tailor our strategy based on the thrombotic/bleeding profile of each patient.Recently,the ACCOAST trial cast a doubt on the benefit of pre treatment in non-ST segment elevation acute coronary syndrome.The aim of the present review is to summarize the results of the main studies dealing with antiplatelet therapy in stented/acute coronary syndromes patients.