The platelet ATP and ADP receptors

Adenine nucleotides, ADP and ATP, are coreleased from dense granules during platelet activation, as well as from endothelial cells and damaged red blood cells following vascular injury. Through autocrine and paracrine mechanisms, these extracellular signaling molecules interact with the platelet P2 receptors to amplify ongoing platelet activation. Two receptors for ADP, the G(q)-protein-coupled P2Y1 and G(i)-protein-coupled P2Y12 and one receptor for ATP, the P2X1 ion channel, have been identified on platelets. Due to distinct pharmacological properties and differential regulation, the P2Y and P2X receptors essentially operate on different scales of time and distance and trigger selective intracellular signaling cascades. Recent advances in the understanding of the P2Y receptor physiology have reinforced the concept of these receptors as useful targets for antithrombotic therapy. The function of P2X1 in platelet activation only recently started to be unraveled. This review focuses on recent findings on the physiology of these platelet ADP and ATP receptors, their distinct downstream intracellular signaling pathways as well as on the available agonists, antagonists and inhibitors that allow their pharmacological discrimination.     

ADP Receptor Antagonism

ADP is one of the most important mediators of both physiologic hemostasis and thrombosis. Development and utilization of agents that block ADP receptors on the platelet membrane, namely thienopyridines, has represented a major advancement for treatment of patients undergoing percutaneous coronary interventions and those with acute coronary syndromes. Currently, clopidogrel, a second-generation thienopyridine that inhibits the ADP P2Y(12) receptor, represents the treatment of choice, in addition to aspirin, for the prevention of stent thrombosis. Further, long-term adjunctive use of this ADP P2Y(12) receptor antagonist is also associated with improved clinical outcomes in high-risk patients, and represents the standard of care for these patients. Despite the unambiguous clinical benefit associated with clopidogrel, accumulating experience with this drug has also led to identification of some of its drawbacks, which are related to inadequate platelet inhibition with standard dosage regimens as well as to its irreversible antiplatelet effects. This has led to the questioning of currently recommended clopidogrel dosage regimens as well as to the development of novel and more potent ADP P2Y(12) receptor antagonists, some of which are also reversible agents. Numerous studies are currently ongoing with the objective of demonstrating how more potent platelet inhibition using higher loading and maintenance dose regimens of clopidogrel or novel ADP P2Y(12) receptor antagonists - such as prasugrel, ticagrelor (AZD 6140) and cangrelor - will affect clinical outcomes. This article reviews the current knowledge of platelet ADP P2Y(12) receptor antagonism and the projected developments in this field.     

ADP receptor antagonists as antiplatelet therapeutics

With the cloning of the P2Y12 receptor, the molecular basis for ADP-induced platelet aggregation is seemingly complete. Two platelet-bound ADP receptors, P2Y1 and P2Y12, operate through unique pathways to induce and sustain platelet aggregation via the glycoprotein (GP)IIb-IIIa integrin. P2Y1 operates via a glycoprotein q (Gq) pathway, activates phospholipase C, induces platelet shape change and is responsible for intracellular calcium mobilisation. P2Y12 inhibits adenylyl cyclase through a glycoprotein i (Gi)-dependent pathway, and is the target of the clinically used thienopyridines, ticlopidine (Ticlid, F. Hoffman-La Roche) and clopidogrel (Plavix, Bristol-Myers Squibb/Sanofi-Synthelabo). In addition, the receptor is targeted by the ADP analogue AR-C66096, which is currently in Phase IIb clinical trials, as well as other non-nucleoside-based preclinical leads.     

Validation of a VerifyNow-P2Y12 cartridge for monitoring platelet inhibition with clopidogrel

Despite common use of clopidogrel in patients with vascular disease, monitoring of platelet inhibition is still not conventional in clinical practice. Considering substantial response variability, when some patients may experience inadequate protection, and/or increased risk of bleeding, simple and reliable methods to control adequate antiplatelet regimen is mandatory. We validated a new VerifyNow-P2Y12 assay to measure inhibition of the P2Y12 platelet receptors by clopidogrel by evaluating its receptor specificity, precision, and potential interference with platelet count, hematocrit, age, cholesterol, triglycerides, and other antiplatelet agents. Platelet aggregation induced by ADP or ADP + prostaglandin E1 (ADP + PGE1) in the presence of specific P2Y12 inhibitor 2-methylthio-AMP (2MeSAMP) for the assessment of assay specificity was performed in 10 volunteers. Seventeen medications were used for the VerifyNow-P2Y12 interference testing, and assay interplay with blood constituents was evaluated in a clinical setting in 131 patients with coronary artery disease. In the presence of 2MeSAMP, the average residual aggregation level across the 10 donors was 27% for ADP and 5% for ADP + PGE1. There also was a strong agreement between ADP + PGE1 aggregometry and VerifyNow-P2Y12 assay (93% vs. 95% average inhibition across all donors). The coefficient of variation for the test precision was less than 8%. The VerifyNow-P2Y12 readings were not influenced by age, platelet count, hematocrit, fibrinogen, cholesterol, or triglycerides level. There was an interference with abciximab before P2Y12 inhibition; however, after platelet suppression with cilostazol, the interference with all tested substances was minimal. VerifyNow-P2Y12 is a reliable, simple, and sensitive device suitable for monitoring of P2Y12 platelet receptor inhibitors in the clinical arena.     

Role of purinergic receptors in platelet-nanoparticle interactions

Primary objective. Elevation of the thrombotic responses mediated by a variety of carbon-derived nanoparticles was recently reported in the literature. In this paper our objective was to investigate whether metal nanoparticles (iron, copper, gold or cadmium sulfide [CdS]) impart such prothrombotic effects on human platelets. Secondly, we wanted to examine whether such effects were mediated through any specific platelet receptor. Experimental design. The size distributions and zeta potentials of characterized gold, copper, iron and CdS (rod & sphere) nanoparticles were measured using photon correlation spectroscopy and laser Doppler velocimetry. The effect of two classes of agonists, adenosine diphosphate (ADP) and epinephrine were studied. To study the effect of ADP, a suboptimal concentration was chosen below a critical concentration. Above the critical concentration, the aggregation assumed its standard hyperbolic shape (and de-aggregation disappeared). Pro-aggregatory action of a given agent can be understood with better sensitivity using a transition from deaggregation to aggregation at this suboptimal agonist level. For epinephrine at low concentration this criticality was absent, however the aggregatory profile showed a delayed response. Two classes of human subjects (a) normal and (b) individuals with acute coronary syndrome, who were under a therapeutic regime of clopidogrel were chosen, as clopidogrel is a specific inhibitor of the low affinity ADP receptor P2Y12. This enabled us to understand the pro-aggregatory effects of nanoparticles with only P2Y1 (high affinity ADP receptor) active. In another set of aggregation experiments, the inhibitor MRS2179 was used to specifically block the high affinity ADP receptor P2Y1. Methods. The threshold ADP concentration was determined using an ADP titration. Nanoparticle rich platelet suspensions were exposed to a previously determined sub-optimal ADP concentration. The experiment was repeated with iron, copper, gold and CdS nanoparticles (later with two different morphologies, rod and sphere). Results. The primary result was that the nanoparticles, composed of various materials and shape features, are likely to impart a pro-aggregatory response in platelets. That the pro-aggregatory effect is not solely a physical self-assembly process and has ADP dependence, is evident from the reversal of the said response by apyrase. The fact that the response was absent in the case of P2Y12 blocked subjects (CdS nanoparticles being an exception) suggests that the low-affinity P2Y12 receptor may be an important target for the nanoparticles. If on the other hand P2Y1 (the high affinity receptor) was blocked by the specific inhibitor MRS2179, nanoparticles could still induce higher aggregation in normal subjects. No significant nanoparticle induced proaggregatory effect was observed for epinephrine. Inference. It is inferred, that the said platelet effect is mediated through ADP receptors, the probable target being the low affinity purinergic receptor P2Y12. The indication is that P2Y12 is a potential target for a wide class of nanoparticles. However the extent of the induced pro-aggregatory effect may be dependent upon the constitutive material and/or the shape of the nanoparticles. This may have important implications in the use of nano-materials in human drug delivery systems. The fact that clopidogrel prevents this nanoparticle mediated prothrombotic effect (with CdS as an exception) may help making nanodrug administration safer.     

ADP receptors--targets for developing antithrombotic agents

Platelet P2 receptors--P2Y1, P2Y12, and P2X1--constitute the means by which adenine nucleotides can activate platelets. Coactivation of the Galphaq-coupled P2Y1 and Galphai2-coupled P2Y12 receptors is necessary for ADP-mediated platelet activation, which forms the basis of using P2 antagonists as antithrombotic drugs. P2Y1 receptor antagonists inhibit platelet activation, while P2Y1 knockout mice show longer bleeding times than normal mice but few other problems; however, its ubiquitous expression in other tissues renders P2Y1 questionable as an antithrombotic target. The P2Y12 receptor is expressed nearly exclusively in platelets and brain, making it an attractive antithrombotic target. Antagonists for the P2Y12 receptor have been developed that either require metabolic activation to covalently inhibit P2Y12 and are irreversible, or simply are competitive in nature and thus reversible. Ticlopidine and clopidogrel are irreversible P2Y12 antagonists and have been repeatedly proven as clinical antithrombotic agents. In addition, a recently reported P2Y12 antagonist, CS-747, shows promise as a future antithrombotic drug. The AR-C series of compounds represent reversible P2Y12 antagonists and have been used extensively to characterize the function of P2Y12 in platelets. Clinical studies show that AR-C69931MX is as effective as clopidogrel; furthermore, the combination of AR-C69931MX (cangrelor) and clopidogrel confers greater antagonism of P2Y12 than either antagonist alone. The P2X1 receptor is a calcium channel that functions to potentiate agonist-induced platelet shape change, and its inhibition or loss has little if any effect on hemostasis. A combination of P2Y1 and P2Y12 antagonists may represent an additional course of antithrombotic treatment.     

Platelet purinergic receptors

Activation of P2Y(1) and P2Y(12) receptors, through secreted ADP that is stimulated by agonists such as thrombin, thromboxane and collagen, is a major mechanism of platelet activation. P2X(1) receptors also participate in platelet shape change and potentiation of calcium mobilization. The cloning of the P2Y(12) receptor and its subsequent knockout in mice promises further understanding of its downstream signaling events.     

P2Y receptor antagonists in thrombosis

The dual role of P2Y1 and P2Y12 receptors in platelet aggregation by ADP has been firmly established, based on the action of selective inhibitors, gene targeting in mice and human genetic evidence. Both of these receptor subtypes constitute targets for antithrombotic agents, and compounds with a dual action might also be of interest. However, the agents currently on the market (ticlopidine and clopidogrel), or known to be in development (cangrelor, AZD-6140 and prasugrel), all target the P2Y12 receptor. The thienopyridines (ticlopidine, clopidogrel and prasugrel) irreversibly inactivate the P2Y12 receptor via the covalent binding of an active metabolite generated in the liver, while the other compounds are competitive antagonists. Cangrelor, an ATP derivative, is suitable for intravenous perfusion, whereas AZD-6140 is in clinical development as an orally active agent.     

Pharmacokinetic, pharmacodynamic and pharmacogenetic profile of the oral antiplatelet agent ticagrelor

Acute coronary syndromes (ACS) remain life-threatening disorders associated with high morbidity and mortality, despite advances in treatment over the last decade. Adenosine diphosphate-induced platelet activation via P2Y(12) receptors plays a pivotal role in the pathophysiology of ACS. The current standard of treatment involves dual antiplatelet therapy with aspirin (acetylsalicylic acid) and the thienopyridine clopidogrel. Numerous studies and wide use in clinical practice have established the value of this approach in the treatment of ACS. However, clopidogrel treatment has a number of limitations, including a delayed onset of action due to the need for metabolic activation, variable and reduced antiplatelet effects in patients with certain genotypes, and prolonged recovery of platelet function due to irreversible P2Y(12) receptor binding. Prasugrel, a new thienopyridine, has demonstrated more consistent inhibition of platelet aggregation (IPA) than clopidogrel, although this thienopyridine also requires metabolic activation and treatment is associated with a significantly increased risk of life-threatening and fatal bleeding. The recently approved oral antiplatelet agent ticagrelor has the potential to overcome some of the limitations of current therapy due to its unique pharmacokinetic and pharmacodynamic profiles. It is a member of a new chemical class, the cyclopentyltriazolopyrimidines, and is a potent P2Y(12) receptor antagonist. Ticagrelor is rapidly absorbed, with a median time to maximum concentration of 1.3-2.0 hours. Ticagrelor does not require metabolic activation to an active form and binds rapidly and reversibly to the P2Y(12) receptor. As well as exerting effects via platelet P2Y(12) receptors, ticagrelor may confer additional benefits via inhibition of non-platelet P2Y(12) receptors. The pharmacokinetic profile of ticagrelor is not significantly affected by age, gender or administration with food, nor by prior treatment with, or responsiveness to, clopidogrel. Ticagrelor is primarily metabolized via the cytochrome P450 (CYP) 3A4 enzyme, rapidly produces plasma concentration-dependent IPA that is greater and more consistent than that observed with clopidogrel, and can also enhance platelet inhibition and overcome non-responsiveness in patients previously treated with clopidogrel. Importantly, the pharmacodynamic characteristics of ticagrelor are not influenced by CYP2C19 and ABCB1 genotypes. This article summarizes our current knowledge regarding the pharmacokinetic, pharmacodynamic and pharmacogenetic profile of ticagrelor.     

ADP receptors: inhibitory strategies for antiplatelet therapy

The interaction of adenosine-5'-diphosphate (ADP) with its platelet receptors (P2Y(1) and P2Y(12)) plays a very important role in thrombogenesis. The thienopyridine ticlopidine was the first specific antagonist of the platelet P2Y(12) ADP receptor to be tested in randomized clinical trials for the prevention of arterial thrombotic events. Although ticlopidine reduces the incidence of vascular events in patients at risk, it also unfortunately has some significant drawbacks: a relatively high incidence of toxic effects, which may be fatal in some cases; delayed onset of action; and a high interindividual variability in response. A second thienopyridine, clopidogrel, has superseded ticlopidine, because it is also an efficacious antithrombotic drug and is less toxic than ticlopidine. However, clopidogrel is not completely free from faults: severe toxic effects, albeit occurring much less frequently than with ticlopidine, may still complicate its administration to patients; the onset of pharmacologic action can be accelerated by the use of large loading doses, but may still not be optimal; the high interpatient variability in response remains an important issue. These concerns justify the continued search for agents that can further improve the clinical outcome of patients with atherosclerosis through greater efficacy and/or safety. A new thienopyridyl compound, prasugrel, which is characterized by higher potency and faster onset of action compared with clopidogrel, is currently under clinical evaluation. Two direct and reversible P2Y(12) antagonists, cangrelor and AZD6140, feature very rapid onset and reversal of platelet inhibition, which make them attractive alternatives to thienopyridines, especially when rapid inhibition of platelet aggregation or its quick reversal are required. Along with new the P2Y(12) antagonists, inhibitors of the other platelet receptor for ADP, the antagonists P2Y(1), are under development and may prove to be effective antithrombotic agents.     

Identification of a potent inverse agonist at a constitutively active mutant of human P2Y12 receptor

Human platelets express two P2Y receptors: G(q)-coupled P2Y(1), and G(i)-coupled P2Y(12). Both P2Y(1) and P2Y(12) are ADP receptors on human platelets and are essential for ADP-induced platelet aggregation that plays pivotal roles in thrombosis and hemostasis. Numerous constitutively active G protein-coupled receptors have been described in natural or recombinant systems, but in the P2Y receptors, to date, no constitutive activity has been reported. In our effort to identify G protein coupling domains of the human platelet ADP receptor, we constructed a chimeric hemagglutinin-tagged human P2Y(12) receptor with its C terminus replaced by the corresponding part of human P2Y(1) receptor and stably expressed it in Chinese hamster ovary-K1 cells. It is interesting that the chimeric P2Y(12) mutant exhibited a high level of constitutive activity, as evidenced by decreased cAMP levels in the absence of agonists. The constitutive activation of the chimeric P2Y(12) mutant was dramatically inhibited by pertussis toxin, a G(i) inhibitor. The constitutively active P2Y(12) mutant retained normal responses to 2-methylthio-ADP, with an EC(50) of 0.15 +/- 0.04 nM. The constitutively active P2Y(12) mutant caused Akt phosphorylation that was abolished by the addition of pertussis toxin. Pharmacological evaluation of several P2Y(12) antagonists revealed (E)-N-[1-[7-(hexylamino)-5-(propylthio)-3H-1,2,3-triazolo-[4,5-d]-pyrimidin-3-yl]-1,5,6-trideoxy-beta-d-ribo-hept-5-enofuranuronoyl]-l-aspartic acid (AR-C78511) as a potent P2Y(12) inverse agonist and 5'-adenylic acid, N-[2-(methylthio)ethyl]-2-[(3,3,3-trifluoropropyl)thio]-, monoanhydride with (dichloromethylene)bis[phosphonic acid] (AR-C69931MX) as a neutral antagonist. In conclusion, this is the first report of a cell line stably expressing a constitutively active mutant of human platelet P2Y(12) receptor and the identification of potent inverse agonist.     

Regulation of death and survival in astrocytes by ADP activating P2Y1 and P2Y12 receptors

ADP is the endogenous agonist for both P2Y(1) and P2Y(12) receptors, which are important therapeutic targets. It was previously demonstrated that ADP and a synthetic agonist, 2-methylthioadenosine 5'-diphosphate (2MeSADP), can induce apoptosis by activating the human P2Y(1) receptor heterologously expressed in astrocytoma cells. However, it was not known whether the P2Y(12) receptor behaved similarly. We demonstrated here that, unlike with the G(q)-coupled P2Y(1) receptor, activation of the G(i)-coupled P2Y(12) receptor does not induce apoptosis. Furthermore, activation of the P2Y(12) receptor by either ADP or 2MeSADP significantly attenuates the tumor necrosis factor alpha (TNFalpha)-induced apoptosis in 1321N1 human astrocytoma cells. This protective effect was blocked by the P2Y(12) receptor antagonist 2-methylthioAMP and by inhibitors of phospholipase C (U73122) and protein kinase C (chelerythrin), but not by the P2Y(1) receptor antagonist MRS2179. Toward a greater mechanistic understanding, we showed that hP2Y(12) receptor activation by 10nM 2MeSADP, activates Erk1/2, Akt, and JNK by phosphorylation. However, at a lower protective concentration of 100pM 2MeSADP, activation of the hP2Y(12) receptor involves only phosphorylated Erk1/2, but not Akt or JNK. This activation is hypothesized as the major mechanism for the protective effect induced by P2Y(12) receptor activation. Apyrase did not affect the ability of TNFalpha to induce apoptosis in hP2Y(12)-1321N1 cells, suggesting that the endogenous nucleotides are not involved. These results may have important implications for understanding the signaling cascades that follow activation of P2Y(1) and P2Y(12) receptors and their opposing effects on cell death pathways.     

Platelet aggregometry and receptor binding to predict the magnitude of antithrombotic and bleeding time effects of clopidogrel in rabbits

Target levels of ex vivo inhibition of platelet aggregation (IPA) induced by adenosine diphosphate (ADP) that produce clinically relevant effects of clopidogrel, a P2Y12 antagonist, are unclear. We examined standard and modified IPA and P2Y12 receptor occupancy as predictors of antithrombotic (% thrombus weight reduction) and bleeding time (BT, fold-increase over control) effects of clopidogrel in rabbit models of carotid artery thrombosis and cuticle bleeding, respectively. Standard and modified IPA with 20 microM ADP were measured in the absence and presence of partial P2Y1 blockade, respectively. Clopidogrel maximally produced standard IPA of 57% +/- 5%, antithrombotic effect of 85% +/- 1%, BT increase of 6.0 +/- 0.4-fold and P2Y12 receptor occupancy of 87% +/- 5%. Surprisingly, a clopidogrel dose that produced a low standard IPA of 17% +/- 4% and P2Y12 receptor occupancy of 39% +/- 5% achieved a significant antithrombotic activity of 55% +/- 2% with a moderate increase in BT of 2.0 +/- 0.1-fold. This underestimation of clopidogrel efficacy by standard IPA was improved by measuring either modified IPA or P2Y12 receptor occupancy. These results suggest that in clopidogrel-treated rabbits, low standard IPA is associated with significant antithrombotic effects. Moreover, modified IPA and P2Y12 receptor occupancy appear to better predict the magnitude of clopidogrel's efficacy compared with standard IPA, which may be a better predictor of BT.     

An elusive receptor is finally caught: P2Y(12'), an important drug target in platelets

Despite intensive research, the nucleotide P2 receptor that is involved in the aggregation and activation of platelets by ADP has remained elusive. However, now two research groups have independently identified a new platelet receptor of unexpected structure, P2Y(12), that acts with the P2Y(1) receptor to form the site of ADP activation and explains the multiple transduction mechanisms observed in response to ADP in platelets. Recent evidence also suggests that a third component, ATP action on the P2X(1) receptor ion channel, contributes to platelet activation.     

Differential effects of P2Y1 and P2Y12 nucleotide receptors on ERK1/ERK2 and phosphatidylinositol 3-kinase signalling and cell proliferation in serum-deprived and nonstarved glioma C6 cells

We have previously shown that, in glioma C6 cells, two nucleotide ADP-sensitive receptors coexist: P2Y1, coupled to PLC and responsible for Ca2+ release, and P2Y12, negatively coupled to adenylate cyclase. In the present study, we examined the effects of the stimulation of these two receptors on ERK1/2 and PI3-K activation, and cell proliferation in either serum-deprived or nonstarved C6 cells. In response to ADP and its analogues, in serum-starved cells, both p44 ERK1 and p42 ERK2 were activated in a time-dependent manner, as monitored by Western blot analysis using an antiphospho-p42/p44 MAPK antibody. The phosphorylation was reduced both by removal of the extracellular Ca2+ and partially or almost completely by MRS2179 or AR-C69931MX, specific antagonists of the P2Y1 and P2Y12 receptors, respectively. The inhibitory effect of antagonists was additive. These data indicate the involvement of both receptors, P2Y1 and P2Y12, in the ERK1/2 activation, but the P2Y12 receptor contribution predominates. ERK1/2 activity was positively correlated with cell proliferation of cultured glioma C6 cells. In nonstarved cells, ADP markedly decreased the PI3-K activity. In contrast, in serum-starved cells, ADP evoked an increase in the PI3-K activity. Blocking of the P2Y1 receptor by MRS2179 additionally increased this ADP response. These results suggest that the P2Y1 receptor has an inhibitory and the P2Y12 receptor a stimulatory effect on PI3-K signalling pathway. RT-PCR analysis revealed different mRNA expression of both receptors in starved and nonstarved cells. In nonstarved cells, the P2Y1 receptor mRNA predominates, whereas in serum-deprived cells the expression of P2Y12 mRNA becomes more pronounced.British Journal of Pharmacology (2004) 141, 497-507. doi:10.1038/sj.bjp.0705639     

A study of P2X1 receptor function in murine megakaryocytes and human platelets reveals synergy with P2Y receptors

We have examined the role of ATP-dependent P2X(1) receptors in megakaryocytes (MKs) and platelets using receptor-deficient mice and selective agonists. Alpha,beta-meATP- and ATP- evoked ionotropic inward currents were absent in whole-cell recordings from MKs of P2X(1)(-/-) mice, demonstrating that the P2X receptor phenotype in MKs, and by inference, platelets, is due to expression of homomeric P2X(1) receptors. P2X(1) receptor deficiency had no effect on MK (CD 41) numbers or size distribution, showing that it is not essential for normal MK development. P2Y receptor-stimulated [Ca(2+)](i) responses were unaffected in MKs from P2X(1)(-/-) mice, however the inward cation current associated with Ca(2+) release was reduced by approximately 50%, suggesting an interaction between the membrane conductances activated by P2X(1) and P2Y receptors. Interaction between P2X(1) and P2Y receptors in human platelets was also examined using [Ca(2+)](i) recordings from cell suspensions. Alpha,beta-meATP (10 microM) evoked a rapid transient P2X(1) receptor-mediated increase in [Ca(2+)](i), whereas ADP-(10 microM) evoked P2Y receptor responses were slower, peaked at a higher level and remained elevated for longer periods. Co-application of alpha, beta-meATP and ADP resulted in marked acceleration and amplification of the peak [Ca(2+)](i) response. We conclude that ionotropic P2X(1) receptors may play a priming role in the subsequent activation of metabotropic P2Y receptors during platelet stimulation.     

Distinct roles for protein kinase C isoforms in regulating platelet purinergic receptor function

ADP is a critical regulator of platelet activation, mediating its actions through two G protein-coupled receptors (GPCRs), P2Y1 and P2Y12. We have shown previously that the receptors are functionally desensitized, in a homologous manner, by distinct kinase-dependent mechanisms in which P2Y1 is regulated by protein kinase C (PKC) and P2Y12 by G protein-coupled receptor kinases. In this study, we addressed whether different PKC isoforms play different roles in regulating the trafficking and activity of these two GPCRs. Expression of PKCalpha and PKCdelta dominant-negative mutants in 1321N1 cells revealed that both isoforms regulated P2Y1 receptor signaling and trafficking, although only PKCdelta was capable of regulating P2Y12, in experiments in which PKC was directly activated by the phorbol ester phorbol 12-myristate 13-acetate (PMA). These results were paralleled in human platelets, in which PMA reduced subsequent ADP-induced P2Y1 and P2Y12 receptor signaling. PKC isoform-selective inhibitors revealed that novel, but not conventional, isoforms of PKC regulate P2Y12 function, whereas both novel and classic isoforms regulate P2Y1 activity. It is also noteworthy that we studied receptor internalization in platelets by a radioligand binding approach showing that both receptors internalize rapidly in these cells. ADP-induced P2Y1 receptor internalization is attenuated by PKC inhibitors, whereas that of the P2Y12 receptor is unaffected. Both P2Y1 and P2Y12 receptors can also undergo PMA-stimulated internalization, and here again, novel but not classic PKCs regulate P2Y12, whereas both novel and classic isoforms regulate P2Y1 internalization. This study therefore is the first to reveal distinct roles for PKC isoforms in the regulation of platelet P2Y receptor function and trafficking.     

ADP is not an agonist at P2X(1) receptors: evidence for separate receptors stimulated by ATP and ADP on human platelets

ADP, an important agonist in thrombosis and haemostasis, has been reported to activate platelets via three receptors, P2X(1), P2Y(1) and P2T(AC). Given the low potency of ADP at P2X(1) receptors and recognized contamination of commercial samples of adenosine nucleotides, we have re-examined the activation of P2X(1) receptors by ADP following HPLC and enzymatic purification. Native P2X(1) receptor currents in megakaryocytes were activated by alpha, beta-meATP (10 microM) and commercial samples of ADP (10 microM), but not by purified ADP (10 - 100 microM). Purified ADP (up to 1 mM) was also inactive at recombinant human P2X(1) receptors expressed in XENOPUS: oocytes. Purification did not modify the ability of ADP to activate P2Y receptors coupled to Ca(2+) mobilization in rat megakaryocytes. In human platelets, P2X(1) and P2Y receptor-mediated [Ca(2+)](i) responses were distinguished by their different kinetics at 13 degrees C. In 1 mM Ca(2+) saline, alpha,beta-meATP (10 microM) and commercial ADP (40 microM) activated a rapid [Ca(2+)](i) increase (lag time < or =0.5 s) through the activation of P2X(1) receptors. Hexokinase treatment of ADP shifted the lag time by approximately 2 s, indicating loss of the P2X(1) receptor-mediated response. A revised scheme is proposed for physiological activation of P2 receptors in human platelets. ATP stimulates P2X(1) receptors, whereas ADP is a selective agonist at metabotropic (P2Y(1) and P2T(AC)) receptors.     

The role of the cytochrome P450 polymorphisms in clopidogrel efficacy and clinical utility

Clopidogrel, an antiplatelet agent, prevents platelet aggregation by inhibiting the adenosine disphosphate (ADP) P2Y12 receptor, which is located on the platelet surface. Although dual antiplatelet therapy appears to be efficient, a considerable number of patients continue to experience adverse cardiovascular events, such as stent thrombosis. The percentage of low response to antiplatelet therapy varies from 4% to 30% of patients depending on the cut-off values. In addition, several factors such as poor absorption, drug-to-drug interactions, inadequate dosing, elevated body mass index, insulin resistance and the nature of acute coronary syndromes have been implicated in low clopidogrel response. Recently, studies have focused on the role of genetic polymorphisms encoding enzymes that participate in clopidogrel hepatic metabolism or receptors involved in intestinal absorption and ADP induced platelet aggregation, which may affect the percentage of platelet inhibition after clopidogrel administration. The management of clopidogrel resistance remains a controversial issue and additional studies are required to evaluate the safety and efficacy of increased loading of clopidogrel or replacement with other new antiplatelet agents such as prasugrel.     

AZD6140

Oral antiplatelet therapy with P2Y(12) receptor blockers (especially clopidogrel) is the current choice of treatment during acute coronary syndromes and percutaneous interventions. To address the various limitations of thienopyridine therapy (including response variability and non-responsiveness) a novel drug, AZD6140, is under clinical development. AZD6140 is an oral and reversible P2Y(12) receptor blocker that does not require hepatic conversion to an active metabolite and produces an overall superior ADP-induced platelet inhibition with less response variability than clopidogrel. It has fast onset and offset actions that may be advantageous in patients who may have to undergo immediate surgery.