P2Y12 and EP3 antagonists promote the inhibitory effects of natural modulators of platelet aggregation that act via cAMP

Several antiplatelet drugs that are used or in development as antithrombotic agents, such as antagonists of P2Y₁₂ and EP3 receptors, act as antagonists at G_i-coupled receptors, thus preventing a reduction in intracellular cyclic adenosine monophosphate (cAMP) in platelets. Other antiplatelet agents, including vascular prostaglandins, inhibit platelet function by raising intracellular cAMP. Agents that act as antagonists at G_i-coupled receptors might be expected to promote the inhibitory effects of agents that raise cAMP. Here, we investigate the ability of the P2Y₁₂ antagonists cangrelor, ticagrelor and prasugrel active metabolite (PAM), and the EP3 antagonist DG-041 to promote the inhibitory effects of modulators of platelet aggregation that act via cAMP. Platelet aggregation was measured by platelet counting in whole blood in response to the TXA₂ mimetic U46619, thrombin receptor activating peptide and the combination of these. Vasodilator-stimulated phosphoprotein phosphorylation (VASP-P) was measured using a cytometric bead assay. Cangrelor always increased the potency of inhibitory agents that act by raising cAMP (PGI₂, iloprost, PGD₂, adenosine and forskolin). Ticagrelor and PAM acted similarly to cangrelor. DG-041 increased the potency of PGE₁ and PGE₂ as inhibitors of aggregation, and cangrelor and DG-041 together had more effect than either agent alone. Cangrelor and DG-041 were able to increase the ability of agents to raise cAMP in platelets as measured by increases in VASP-P. Thus, P2Y₁₂ antagonists and the EP3 antagonist DG-041 are able to promote inhibition of platelet aggregation brought about by natural and other agents that raise intracellular cAMP. This action is likely to contribute to the overall clinical effects of such antagonists after administration to man.     

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.     

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.     

血小板功能检测与P2Y12受体拮抗剂个体化抗血小板治疗的研究进展

P2Y12受体拮抗剂广泛应用于急性冠脉综合征（ACS）及经皮冠状动脉介入治疗（PCI）后血栓事件的预防。由于抗血小板治疗反应多样性的存在,经典的P2Y12受体拮抗剂氯吡格雷的治疗期间高血小板反应性（HTPR）被证实与患者不良心血管事件的发生密切相关。尽管新药普拉格雷和替格瑞洛的抗栓疗效优于氯吡格雷,但是由于治疗期间低血小板反应性（LTPR）的存在,出血风险明显增加。如何权衡血栓和出血风险,实现个体化抗血小板治疗,已经成为临床的重要挑战。研究证实,血小板反应性（PR）与缺血和出血事件的发生密切有关,基于血小板功能检测（PFT）的治疗窗将有助于个体化抗血小板治疗。本文就PFT与P2Y12受体拮抗剂个体化抗血小板治疗的研究进展作一综述。     

Evolving pattern of platelet P2Y12 inhibition in patients with acute coronary syndromes

Abstract

Dual antiplatelet therapy consisting of clopidogrel in addition to aspirin has previously been the standard of care for patients with acute coronary syndromes (ACS) but international guidelines have been evolving over the last 4 years with the introduction of prasugrel and ticagrelor. In October 2009, prasugrel was approved in the UK by the National Institute of Health and Clinical Excellence (NICE) for use in patients with ST-elevation myocardial infarction (STEMI) undergoing primary percutaneous coronary intervention (PCI), diabetic patients with non-ST-elevation (NSTE) ACS undergoing PCI and patients with stent thrombosis while other ACS patients were to continue receiving clopidogrel. Ticagrelor was approved in October 2011 by NICE for use in patients with moderate-to-high risk NSTE ACS and STEMI undergoing primary PCI and was recommended in preference to clopidogrel in European guidelines. These recommendations were adopted in our region, constituting a population of 1.8 million. We studied the effect of changing patterns of P2Y₁₂ inhibitor usage on levels of platelet inhibition during maintenance therapy. Patients admitted to Northern General Hospital, Sheffield, with NSTE ACS or STEMI managed with primary PCI were enrolled over two periods of time: May 2010 to November 2011 (T1); and October 2012 to February 2013 (T2). Venous blood samples were obtained at 1 month after the onset of ACS. Light transmittance aggregometry (LTA) was performed and maximum aggregation response to ADP 20?μM was determined. A total of 116 patients were enrolled in T1 of whom 82 were receiving clopidogrel and 34 were receiving prasugrel. Twenty-nine patients were enrolled in T2, all of whom were receiving ticagrelor. Mean LTA results according to treatment with clopidogrel, prasugrel and ticagrelor were 57?±?18%, 41?±?20%, and 31?±?12%, respectively. Prasugrel was associated with significantly lower platelet aggregation responses than clopidogrel (p?<?0.001) and ticagrelor was associated with significantly lower platelet aggregation responses than both prasugrel (p?=?0.015) and clopidogrel (p?<?0.001). We conclude that international guidelines and NICE approval have led to increasing levels of P2Y₁₂ inhibition in ACS patients in this UK centre between May 2010 and February 2013. Ticagrelor was associated with significantly greater P2Y₁₂ inhibition than both clopidogrel and prasugrel during maintenance therapy.     

Cangrelor inhibits the binding of the active metabolites of clopidogrel and prasugrel to P2Y12 receptors in vitro

Cangrelor is a rapid-acting, direct-binding, and reversible P2Y₁₂ antagonist which has been studied for use during percutaneous coronary intervention (PCI) in patients with or without pretreatment with an oral P2Y₁₂ antagonist. As cangrelor is administered intravenously, it is necessary to switch to an oral P2Y₁₂ antagonist following PCI, such as the thienopyridines clopidogrel, and prasugrel or the non-pyridine ticagrelor. Previous studies have suggested a negative pharmacodynamic interaction between cangrelor and thienopyridines. This in vitro study evaluated the receptor-level interaction between cangrelor and the active metabolite (AM) of clopidogrel or prasugrel by assessing functional P2Y₁₂ receptor number using a ³³P-2MeSADP binding assay. All P2Y₁₂ antagonists studied resulted in strong P2Y₁₂ receptor blockade (cangrelor: 93.6%; clopidogrel AM: 93.0%; prasugrel AM: 97.9%). Adding a thienopyridine AM in the presence of cangrelor strongly reduces P2Y₁₂ receptor blockade by the AM (clopidogrel AM: 7%, prasugrel AM: 3.2%). The thienopyridine AMs had limited ability to compete with cangrelor for binding to P2Y₁₂ (% P2Y₁₂ receptor blockade after co-incubation with cangrelor 1000?nmol/L: 11.7% for clopidogrel AM 3?µmol/L; 34.1% for prasugrel AM 3?µmol/L). In conclusion, in vitro cangrelor strongly inhibits the binding of clopidogrel and prasugrel AMs to the P2Y₁₂ receptor, consistent with the previous observation of a negative pharmacodynamic interaction. Care may need to be taken to not overlap exposure to thienopyridine AMs and cangrelor in order to reduce the risk of thrombotic complications following PCI.     

The effect of P2Y12 inhibition on platelet activation assessed with aggregation- and flow cytometry-based assays

Patients on P2Y₁₂ inhibitors may still develop thrombosis or bleeding complications. Tailored antiplatelet therapy, based on platelet reactivity testing, might reduce these complications. Several tests have been used, but failed to show a benefit of tailored antiplatelet therapy. This could be due to the narrowness of current platelet reactivity tests, which are limited to analysis of platelet aggregation after stimulation of the adenosine diphosphate (ADP)-pathway. However, the response to ADP does not necessarily reflect the effect of P2Y₁₂ inhibition on platelet function in vivo. Therefore, we investigated whether measuring platelet reactivity toward other physiologically relevant agonists could provide more insight in the efficacy of P2Y₁₂ inhibitors.

The effect of in vitro and in vivo P2Y₁₂ inhibition on αIIbβ3-activation, P-selectin and CD63-expression, aggregate formation, release of alpha, and dense granules content was assessed after stimulation of different platelet activation pathways. Platelet reactivity measured with flow cytometry in 72 patients on P2Y₁₂ inhibitors was compared to VerifyNow results.

P2Y₁₂ inhibitors caused strongly attenuated platelet fibrinogen binding after stimulation with peptide agonists for protease activated receptor (PAR)-1 and -4, or glycoprotein VI ligand crosslinked collagen-related peptide (CRP-xl), while aggregation was normal at high agonist concentration. P2Y₁₂ inhibitors decreased PAR-agonist and CRP-induced dense granule secretion, but not alpha granule secretion. A proportion of P2Y₁₂-inhibitor responsive patients according to VerifyNow, displayed normal fibrinogen binding assessed with flow cytometry after stimulation with PAR-agonists or CRP despite full inhibition of the response to ADP, indicating suboptimal platelet inhibition.

Concluding, measurement of platelet fibrinogen binding with flow cytometry after stimulation of thrombin- or collagen receptors in addition to ADP response identifies different patients as nonresponders to P2Y₁₂ inhibitors, compared to only ADP-induced aggregation-based assays. Future studies should investigate the value of both assays for monitoring on-treatment platelet reactivity.     

Acyl derivatives of coenzyme A inhibit platelet function via antagonism at P2Y1 and P2Y12 receptors: A new finding that may influence the design of anti-thrombotic agents

We have performed a detailed investigation of the effects on platelet function of coenzyme A (CoA) and several acyl-CoAs. Platelet aggregation was measured by turbidimetry and by platelet counting; platelet shape change was measured using light scattering; P-selectin, Ca²⁺ mobilization and vasodilator-stimulated phosphoprotein (VASP) phosphorylation were measured by flow cytometry. The compounds investigated inhibited ADP-induced platelet aggregation; those with saturated acyl groups containing 16-18 carbons were most effective. The effects of palmitoyl-CoA (16:0) were studied in depth. It inhibited platelet shape change and Ca²⁺ mobilization brought about by ADP (but not other agonists) indicating antagonism at P2Y₁ receptors, and also inhibited ADP-induced P-selectin expression. Effects of palmitoyl-CoA on the platelet aggregation and Ca²⁺ mobilization induced by several different agonists and agonist combinations were compared with those of MRS 2179 (a P2Y₁ antagonist) and AR-C69931 (a P2Y₁₂ antagonist), and were consistent with palmitoyl-CoA acting mainly at P2Y₁ but also with partial antagonism at P2Y₁₂ receptors. Antagonism at P2Y₁₂ receptors was confirmed in studies of VASP-phosphorylation. Palmitoyl-CoA did not act as an antagonist at P2X₁ receptors. The results are discussed in relation to the possibility that acyl-CoAs may contribute as endogenous modulators of platelet function and might serve as lead compounds for the design of novel antithrombotics.     

新型P2Y12受体拮抗剂替格瑞洛广谱抗血小板作用机制的研究进展

双联抗血小板是急性冠状动脉综合征患者抗栓治疗的基石,临床上常用的抗血小板药物主要包括阿司匹林与P2Y12受体拮抗剂。目前,新型P2Y12受体拮抗剂,尤其替格瑞洛单药治疗是抗血小板优化治疗领域正在深入研究的课题。研究发现,P2Y12受体拮抗剂可以减少血小板中的血栓素A2受体表达及血栓素A2生成。替格瑞洛具有广谱的抗血小板效应机制。本文针对新型P2Y12受体拮抗剂特别是替格瑞洛抗血小板机制的相关研究归纳总结,对优化抗血小板治疗提出新思路。     

PGE2 reverses Gs-mediated inhibition of platelet aggregation by interaction with EP3 receptors,but adds to non-Gs-mediated inhibition of platelet aggregation by interaction with EP4 receptors

Prostaglandin E₂ (PGE₂) has intriguing effects on platelet function in the presence of agents that raise cyclic adenosine 3′5′-monophosphate (cAMP). PGE₂ reverses inhibition of platelet aggregation by agents that stimulate cAMP production via a G_s-linked receptor, but adds to the inhibition of platelet function brought about by agents that raise cAMP through other mechanisms. Here, we used the EP receptor antagonists DG-041 (which acts at the EP3 receptor) and ONO-AE3-208 (which acts at the EP4 receptor) to investigate the role of these receptors in mediating these effects of PGE₂. Platelet aggregation was measured in platelet-rich plasma obtained from healthy volunteers in response to adenosine diphosphate (ADP) using single platelet counting. The effects of a range of concentrations of PGE₂ were determined in the presence of (1) the prostacyclin mimetic iloprost, which operates through G_s-linked IP receptors, (2) the cAMP PDE inhibitor DN9693 and (3) the direct-acting adenylate cyclase stimulator forskolin. Vasodilator-stimulated phosphoprotein (VASP) phosphorylation was also determined as a measure of cAMP. PGE₂ reversed the inhibition of aggregation brought about by iloprost; this was prevented in the presence of the EP3 antagonist DG-041, indicating that this effect of PGE₂ is mediated via the EP3 receptor. In contrast, PGE₂ added to the inhibition of aggregation brought about by DN9693 or forskolin; this was reversed by the EP4 antagonist ONO-AE3-208, indicating that this effect of PGE₂ is mediated via the EP4 receptor. Effects on aggregation were accompanied by corresponding changes in VASP phosphorylation. The dominant role of EP3 receptors circumstances where cAMP is increased through a Gs-linked mechanism may be relevant to the situation in vivo where platelets are maintained in an inactive state through constant exposure to prostacyclin, and thus the main effect of PGE₂ may be prothrombotic. If so, the results described here further support the potential use of an EP3 receptor antagonist in the control of atherothrombosis.     

Evaluation of the INNOVANCE PFA P2Y test cartridge: Sensitivity to P2Y12 blockade and influence of anticoagulant

Monitoring of platelet ADP receptor P2Y₁₂ inhibition may be performed by a variety of platelet function assays. Given the lack of sensitivity of the existing PFA-100® cartridge formulations to detect P2Y₁₂ inhibition, a new cartridge for the PFA-100 (INNOVANCE® PFA P2Y) has recently been developed. The performance of the new PFA-100 test cartridge was compared with standard collagen/ADP (CADP) and collagen/epinephrine (CEPI) cartridges, light transmission aggregometry, vasodilator-stimulated phosphoprotein, the VerifyNow® P2Y₁₂ assay and multiple electrode aggregometry. In this study, 20 normal blood samples anticoagulated with either citrate or hirudin were spiked with two different clinically relevant concentrations (1 and 10?µM final concentration) of the prasugrel active metabolite (R-138727, Lilly/Daiichi Sankyo) for 30?min at 37°C. Comparison of the platelet function tests demonstrated that all tests (except CADP and CEPI) were substantially inhibited by 10?µM R-138727. Intermediate results were typically obtained with 1?µM R-138727 in citrated blood. However, both MEA ADP and ADPHS tests were highly sensitive to 1?µM R-138727 in hirudin anticoagulated blood. Further comparison of citrate or hirudin blood samples (N?=?5) revealed that all platelet tests (except CEPI) became more sensitive to 1?µM R-138727 in hirudinized blood. The INNOVANCE PFA P2Y cartridge proved to be sensitive to P2Y₁₂ inhibition and was comparable to other currently available platelet function tests. The sensitivity of all platelet function tests for detecting in vitro inhibition of P2Y₁₂ is markedly different depending on the anticoagulant used.     

Potentiation of TRAP-6-induced platelet dense granule release by blockade of P2Y12 signaling with MRS2395

The release of ADP from platelet dense granules and its binding to platelet P2Y₁₂ receptors is key to amplifying the initial hemostatic response and propagating thrombus formation. P2Y₁₂ has thus emerged as a therapeutic target to safely and effectively prevent secondary thrombotic events in patients with acute coronary syndrome or a history of myocardial infarction. Pharmacological inhibition of P2Y₁₂ receptors represents a useful approach to better understand the signaling mediated by these receptors and to elucidate the role of these receptors in a multitude of platelet hemostatic and thrombotic responses. The present work examined and compared the effects of four different P2Y₁₂ inhibitors (MRS2395, ticagrelor, PSB 0739, and AR-C 66096) on platelet function in a series of in vitro studies of platelet dense granule secretion and trafficking, calcium generation, and protein phosphorylation. Our results show that in platelets activated with the PAR-1 agonist TRAP-6 (thrombin receptor-activating peptide), inhibition of P2Y₁₂ with the antagonist MRS2395, but not ticagrelor, PSB 0739 or AR-C 66096, potentiated human platelet dense granule trafficking to the plasma membrane and release into the extracellular space, cytosolic Ca²⁺ influx, and phosphorylation of GSK3β-Ser9 through a PKC-dependent pathway. These results suggest that inhibition of P2Y₁₂ with MRS2395 may act in concert with PAR-1 signaling and result in the aberrant release of ADP by platelet dense granules, thus reducing or counteracting the anticipated anti-platelet efficacy of this inhibitor.     

Evaluation of the pharmacokinetics and pharmacodynamics of ticagrelor co-administered with aspirin in healthy volunteers

The results of two independent, randomized, two-period crossover, single-center studies, conducted to assess the pharmacokinetics of ticagrelor?±?aspirin, inhibition of platelet aggregation (IPA) with ticagrelor/aspirin vs. clopidogrel/aspirin, and safety, tolerability, and bleeding times are reported here. In Study A (open-label), 16 volunteers received ticagrelor (50?mg bid Days 1–5; 200?mg bid Days 6–9; one 200?mg dose on Day 10)?±?300?mg qd aspirin (Days 1–10). In Study B (double-blind, double-dummy), 16 volunteers received aspirin (300?mg loading dose/75?mg qd Days 2–9) with either ticagrelor (200?mg bid Days 4–8, one 200?mg dose on Day 9) or clopidogrel (300?mg loading dose Day 4, 75?mg qd Days 5–9). At steady-state ticagrelor (50?mg bid, or 200?mg bid), concomitant aspirin (300?mg qd) had no effect on mean maximum plasma concentration (C_max), median time to C_max (t_max), or mean area under the plasma concentration-time curve for the dosing interval (AUC_0–τ) for ticagrelor and its primary metabolite, AR-C124910XX. Following 200?mg bid ticagrelor, mean C_max and AUC_0–τ for both parent and metabolite were comparable with co-administration of aspirin at 75?mg and 300?mg qd. Aspirin (300?mg qd) had no effect on IPA (ADP-induced) by ticagrelor. However, aspirin and ticagrelor had an additive effect on IPA (collagen-induced). Ticagrelor/aspirin increased bleeding times vs. baseline. Ticagrelor/aspirin co-administration was well tolerated at all dose combinations evaluated. In summary, the findings of this study demonstrate that co-administration of aspirin (300?mg qd) with ticagrelor (50?mg bid, or 200?mg bid) had no effect on ticagrelor pharmacokinetics or IPA (ADP-induced) by ticagrelor.     

Dyspnea in patients treated with P2Y12 receptor antagonists: insights from the GReek AntiPlatElet (GRAPE) registry

In ‘real life’ acute coronary syndrome (ACS) patients undergoing percutaneous coronary intervention (PCI) and receiving contemporary antiplatelet treatment, data on dyspnea occurrence and impact on persistence with treatment are scarce. In a prospective, multicenter, cohort study, ACS patients undergoing PCI were recruited into the GReekAntiPlatElet (GRAPE) registry. During 1-year follow up, overall, 249/1989 (12.5%) patients reported dyspnea, more frequently at 1-month and decreasing thereafter. Multivariate analysis showed that ticagrelor administration (n = 738) at discharge was associated with the occurrence of dyspnea: Odds ratio 2.46 (95% confidence interval, CI, 1.87–3.25), p < 0.001. Older age, lower hematocrit, and prior bleeding event were also associated with dyspnea reports. Persistence, switching, and cessation rates were 68.3%, 20.9%, and 10.8% vs 76.7%, 12.5%, and 10.9% among patients reporting dyspnea compared with those who did not, p for trend = 0.002. In conclusion, in ACS patients undergoing PCI and treated with a P2Y₁₂ receptor antagonist, dyspnea occurs commonly, particularly when ticagrelor is administered. Non-persistence with antiplatelet agents at discharge is more frequently observed among dyspnea-reporters.     

Antagonism of P2Y12 reduces physiological thromboxane levels

Antiplatelet therapy for the management of patients with cardiovascular risks often includes a combination therapy of aspirin and clopidogrel, acting through inhibition of thromboxane generation and blockade of G_i-coupled P2Y₁₂ receptor, respectively. We hypothesized that ADP acting through P2Y₁₂ regulates physiological thromboxane levels. The serum thromboxane levels in mice (n?=?3) dosed with clopidogrel and prasugrel were decreased by 83.1?±?5.3% and 94.26?±?1.75% respectively compared to untreated mice. Pre-treatment of human blood (n?=?3) ex vivo with active metabolites of clopidogrel or prasugrel led to a reduction in thromboxane levels to 16.3?±?3.2% and 4.9?±?0.8% respectively, compared to untreated human serum. We also evaluated serum thromboxane levels in P2Y receptor null mice (n?=?4). Whereas serum thromboxane levels in P2Y₁ null mice were similar to those in wild type littermates, those in the P2Y₁₂ null mice were inhibited by 83.15?±?3.8%. Finally, in a pilot study, serum thromboxane levels were reduced by 76.05?±?8.41% in healthy human volunteers (n?=?6) upon dosing with clopidogrel, compared to the levels before dosing. In conclusion, P2Y₁₂ antagonism alone can decrease physiological thromboxane levels. Thus, this study could pave way the for newer/modified treatment regimens for the management of patients with thrombotic complications who are allergic or non-responsive to aspirin.     

Advances in the monitoring of anti-P2Y12 therapy

Although there are many new and effective anti-P2Y₁₂ drugs available, clopidogrel in its original or generic forms will probably remain the most widely used and cheaper option. As clopidogrel is a pro-drug, there is marked heterogeneity in drug responsiveness between individuals and lack of responsiveness is associated with poorer clinical outcomes. Various platelet function and genetic tests are now available for potentially measuring whether clopidogrel effectively inhibits platelet function. Monitoring of P2Y₁₂ inhibition and/or identification of loss of function cytochrome P-450 genotypes could, therefore, offer the potential of tailoring therapy by identifying poor responders to clopidogrel and optimizing the levels of platelet inhibition using, for example, alternative drugs such as prasugrel or ticagrelor. The question remains whether any of these tests have prognostic utility with a defined therapeutic window to reliably identify hypo or hyper-responsive patients who may have an increased risk of thrombosis or bleeding, respectively? Once such patients are identified, can the tests then be subsequently used to demonstrate a change or improvement in platelet reactivity by using alternative therapies and equate this with improved clinical outcome? In this review, we describe an overview of the current platelet and genetic tests available and discuss whether these tests will ever become used routinely.     

Rapid and reversible modulation of platelet function in man by a novel P2Y12 ADP-receptor antagonist,INS50589

P2Y₁₂ receptors participate in ADP-induced activation and aggregation of human platelets. INS50589, a selective P2Y₁₂ receptor antagonist, is being developed for use where controlled, reversible modulation of the platelet hemostatic function is needed. The tolerability, pharmacokinetics, and pharmacodynamics of INS50589 were tested in healthy human volunteers. Thirty-six subjects received intravenous infusions of placebo or INS50589 at 0.1–3?mg/kg/h for four hours. Platelet function, clotting parameters, bleeding time, safety assessments, and plasma concentrations of INS50589 and its major metabolite were monitored for 24 hours. Near-steady state plasma concentrations of INS50589 and effects on platelet function were achieved rapidly. The average maximal plasma concentration of INS50589 was linearly related to the dose administered. Intravenous INS50589 produced dose-dependent inhibition of platelet activation and aggregation in response to ADP in vitro until nearly full inhibition was achieved at the higher doses. Bleeding time was correspondingly increased, without any effect on activated clotting time, prothrombin time, or activated partial thromboplastin time. Platelet response to ADP had returned to at least 75% of the baseline value within 0.25–4?h after cessation of the intravenous infusion of INS50589, depending upon the dose and ADP challenge concentration. Infusions were well tolerated up to the highest dose tested. There was no evidence that the principal metabolite (INS51088) contributed to these effects. INS50589 is a well-tolerated, reversible, competitive antagonist of ADP at the P2Y₁₂ human platelet receptor, and its potential therapeutic utility in various cardiovascular settings is discussed.