Impaired platelet function in a patient with P2Y12 deficiency caused by a mutation in the translation initiation codon

In this study, we have identified a patient (OSP-1) with a congenital P2Y12 deficiency showing a mild bleeding tendency from her childhood and examined the role of P2Y12 in platelet function. At low concentrations of agonists OSP-1 platelets showed an impaired aggregation to several kinds of stimuli, whereas at high concentrations they showed a specifically impaired platelet aggregation to adenosine diphosphate (ADP). ADP normally induced platelet shape change and failed to inhibit PGE1-stimulated cAMP accumulation in OSP-1 platelets. Molecular genetic analysis revealed that OSP-1 was a homozygous for a mutation in the translation initiation codon (ATG to AGG) in the P2Y12 gene. Heterologous cell expression of wild-type or mutant P2Y12 confirmed that the mutation was responsible for the deficiency in P2Y12. OSP-1 platelets showed a markedly impaired platelet spreading onto immobilized fibrinogen. Real-time observations of thrombogenesis under a high shear rate (2000 s(-1)) revealed that thrombi over collagen were small and loosely packed and most of the aggregates were unable to resist against high shear stress in OSP-1. Our data suggest that secretion of endogenous ADP and subsequent P2Y12-mediated signaling are critical for platelet aggregation, platelet spreading, and as a consequence, for stabilization of thrombus.     

Platelet activation by ADP: the role of ADP antagonists

ADP plays a key role in haemostasis and thrombosis. Despite its early identification in 1961 as the first known aggregating agent, the molecular basis of ADP-induced platelet activation is only beginning to be understood. Two purinergic receptors contribute separately to the complex process of ADP-induced platelet aggregation: the P2Y1 metabotropic receptor responsible for mobilization of ionized calcium from internal stores, which initiates aggregation, and P2Y receptor coupled to adenylyl cyclase inhibition, which is essential for the full aggregation response to ADP and stabilization of platelet aggregates. The latter is the molecular target of the ADP-selective antiaggregating drugs ticlopidine and clopidogrel and the ATP analogues of the AR-C series. In addition, it is probably defective in patients with a bleeding diathesis characterized by selective impairment of platelet responses to ADP. Finally, the P2X1 ionotropic receptor is also present in platelets, but its role is not yet known. Studies with P2Y1 knock-out mice as well as the use of selective P2Y1 antagonists have shown that, in addition to the P2Y receptor, which is the target of clopidogrel, the P2Y1 receptor is an important potential target for new antithrombotic drugs.     

Nucleotide receptor signaling in platelets

Upon injury to a vessel wall the exposure of subendothelial collagen results in the activation of platelets. Platelet activation culminates in shape change, aggregation, release of granule contents and generation of lipid mediators. These secreted and generated mediators trigger a positive feedback mechanism potentiating the platelet activation induced by physiological agonists such as collagen and thrombin. Adenine nucleotides, adenosine diphosphate (ADP) and adenosine triphosphate (ATP), released from damaged cells and that are secreted from platelet-dense granules, contribute to the positive feedback mechanism by acting through nucleotide receptors on the platelet surface. ADP acts through two G protein-coupled receptors, the Gq-coupled P2Y1 receptor, and the Gi-coupled P2Y12 receptor. ATP, on the other hand, acts through the ligand-gated channel P2X1. Stimulation of platelets by ADP leads to shape change, aggregation and thromboxane A2 generation. ADP-induced dense granule release depends on generated thromboxane A2. Furthermore, costimulation of both P2Y1 and P2Y12 receptors is required for ADP-induced platelet aggregation. ATP stimulation of P2X1 is involved in platelet shape change and helps to amplify platelet responses mediated by agonists such as collagen. Activation of each of these nucleotide receptors results in unique signal transduction pathways that are important in the regulation of thrombosis and hemostasis.     

Transcriptional down-regulation of the platelet ADP receptor P2Y12 and clusterin in patients with systemic lupus erythematosus

Cardiovascular complications are common in systemic lupus erythematosus (SLE) and myocardial infarctions are the leading cause of increased mortality. The ADP receptor P2Y(12) plays a central role in platelet activation and the P2Y(12) blocker clopidogrel reduces the incidence of cardiovascular events. Clusterin, a complement inhibitory protein suggested to be involved in the pathogenesis of SLE, has been found recently in a microarray study to be expressed at very high levels in platelets. Using a new protocol for mRNA quantification in platelets we set out to study if gene expression is altered in SLE patients compared with a healthy control group. Quantitative assay based on real-time PCR was used to measure mRNA expression, Western blot for P2 receptor protein expression and PFA-100 for platelet aggregation. The P2Y(12) receptor expression was decreased in SLE compared to the controls (P < 0.05), while expression of P2Y(1) and P2X(1) were unaltered. These findings were consistent at the protein level. The clusterin mRNA expression was very high. However, SLE patients had significantly lower levels than controls (P < 0.05). Platelet aggregation was similar in both groups. It may be suggested that a decreased level of P2Y(12) receptors could represent a protective response in SLE against thrombotic complications. Lowered clusterin levels could be involved in the pathogenesis of SLE due to decreased protective effects. These findings could help to achieve a better understanding of the platelet function in SLE and serve as a guide for further research and drug use.     

(N)-methanocarba-2MeSADP (MRS2365) is a subtype-specific agonist that induces rapid desensitization of the P2Y1 receptor of human platelets

Adenosine diphosphate (ADP) initiates and maintains sustained aggregation of platelets through simultaneous activation of both the Gq-coupled P2Y1 receptor and the Gi-coupled P2Y12 receptor. We recently described the synthesis and P2Y1 receptor-specific agonist activity of (N)-methanocarba-2MeSADP (MRS2365). Consequences of selective activation of the P2Y1 receptor by MRS2365 have been further examined in human platelets. Whereas MRS2365 alone only induced shape change, addition of MRS2365 following epinephrine treatment, which activates the Gi/z-linked, alpha2A-adrenergic receptor, resulted in sustained aggregation that was indistinguishable from that observed with ADP. Conversely, the platelet shape change promoted by ADP in the presence of the GPIIb/IIIa antagonist eptifibatide was similar to that promoted by MRS2365. Preaddition of the high affinity P2Y1 receptor antagonist MRS2500 inhibited the effect of MRS2365, whereas addition of MRS2500 subsequent to MRS2365 reversed the MRS2365-induced shape change. Preactivation of the P2Y1 receptor with MRS2365 for 2 min resulted in marked loss of capacity of ADP to induce aggregation as evidenced by a greater than 20-fold rightward shift in the concentration effect curve of ADP. This inhibitory effect of P2Y1 receptor activation was dependent on the concentration of MRS2365 (EC50 = 34 nm). The inhibitory effect of preincubation with MRS2365 was circumvented by activation of the Gq-coupled 5-HT2A receptor suggesting that MRS2365 induces loss of the ADP response as a consequence of desensitization of the Gq-coupled P2Y1 receptor. The time course of MRS2365-induced loss of aggregation response to epinephrine was similar to that observed with ADP. These results further demonstrate the P2Y1 receptor selectivity of MRS2365 and illustrate the occurrence of agonist-induced desensitization of the P2Y1 receptor of human platelets studied in the absence of P2Y12 receptor activation .     

αIIbβ3-mediated outside-in signaling induced by the agonist peptide LSARLAF utilizes ADP and thromboxane A2 receptors to cause α-granule secretion by platelets

Summary.  The peptide LSARLAF (LSA) causes αIIbβ3-dependent platelet activation that results in α-granule secretion and aggregation. LSARLAF-induced, αIIbβ3-mediated outside-in signaling causing α-granule secretion and platelet aggregation was studied using washed mouse platelets. ADP receptor antagonists, enzyme inhibitors, normal platelets and platelets from mice that lack either Gαq or thromboxane (Tx) A2 receptors were used for this investigation. The results demonstrate that LSA-induced αIIbβ3-mediated signaling producing aggregation of washed platelets is mediated through the release of ADP and thromboxane, which cause α-granule release by mediating their effects though Gαq and/or Gi depending on the level of LSA used to activate the platelets. Specifically, αIIbβ3 elicited aggregation of washed platelets in response to a low level of LSA requires signaling through the ADP receptor P2Y1 and Gαq, and the ADP receptor P2Y12 and Gi as well as TxA2 receptors. However, this aggregation is independent of Gαq and TxA2 signaling in response to high LSA concentrations, but is dependent on ADP signaling through its receptor P2Y12, and therefore presumably Gi, regardless of the level of LSA used to activate the platelets. PKC function is required for ADP secretion and the subsequent signaling through P2Y12 regardless of the level of LSA used to activate the platelets. The end point of the LSA-induced αIIbβ3-mediated signaling characterized in this study is α-granule secretion, which provides the fibrinogen required for aggregation of washed platelets.     

P2Y12 regulates platelet adhesion/activation,thrombus growth,and thrombus stability in injured arteries

The critical role for ADP in arterial thrombogenesis was established by the clinical success of P2Y12 antagonists, currently used at doses that block 40-50% of the P2Y12 on platelets. This study was designed to determine the role of P2Y12 in platelet thrombosis and how its complete absence affects the thrombotic process. P2Y12-null mice were generated by a gene-targeting strategy. Using an in vivo mesenteric artery injury model and real-time continuous analysis of the thrombotic process, we observed that the time for appearance of first thrombus was delayed and that only small, unstable thrombi formed in P2Y12-/- mice without reaching occlusive size, in the absence of aspirin. Platelet adhesion to vWF was impaired in P2Y12-/- platelets. While adhesion to fibrinogen and collagen appeared normal, the platelets in thrombi from P2Y12-/- mice on collagen were less dense and less activated than their WT counterparts. P2Y12-/- platelet activation was also reduced in response to ADP or a PAR-4-activating peptide. Thus, P2Y12 is involved in several key steps of thrombosis: platelet adhesion/activation, thrombus growth, and stability. The data suggest that more aggressive strategies of P2Y12 antagonism will be antithrombotic without the requirement of aspirin cotherapy and may provide benefits even to the aspirin-nonresponder population.     

The P2Y1 receptor plays an essential role in the platelet shape change induced by collagen when TxA2 formation is prevented.

ADP and TxA2 are secondary agonists which play an important role as cofactors when platelets are activated by agonists such as collagen or thrombin. The aim of the present study was to characterize the role of the ADP receptor P2Y(1) in collagen-induced activation of washed platelets. Inhibition of P2Y(1) alone with the selective antagonist MRS2179 prolonged the lag phase preceding aggregation in response to low or high concentrations of fibrillar collagen, without affecting the maximum amplitude of aggregation or secretion. A combination of MRS2179 and aspirin resulted in complete inhibition of platelet shape change at low and high collagen concentrations, together with a profound decrease in aggregation and secretion. Scanning electron microscopy showed that these platelets had conserved the discoid morphology typical of the resting state. A lack of shape change was also observed in aspirin-treated P2Y(1)- and G(alphaq)-deficient mouse platelets and in delta-storage pool-deficient platelets from Fawn Hooded rats. In contrast, when the second ADP receptor P2Y(12) was inhibited with AR-C69931MX, aspirin-treated platelets were still able to change shape and displayed only a moderate decrease in aggregation and secretion. In conclusion, this study provides evidence that collagen requires not only the TxA2 receptor Tpalpha, but also P2Y(1), to induce platelet shape change.     

Molecular identification and characterization of the platelet ADP receptor targeted by thienopyridine antithrombotic drugs

ADP plays a critical role in modulating thrombosis and hemostasis. ADP initiates platelet aggregation by simultaneous activation of two G protein-coupled receptors, P2Y1 and P2Y12. Activation of P2Y1 activates phospholipase C and triggers shape change, while P2Y12 couples to Gi to reduce adenylyl cyclase activity. P2Y12 has been shown to be the target of the thienopyridine drugs, ticlopidine and clopidogrel. Recently, we cloned a human orphan receptor, SP1999, highly expressed in brain and platelets, which responded to ADP and had a pharmacological profile similar to that of P2Y12. To determine whether SP1999 is P2Y12, we generated SP1999-null mice. These mice appear normal, but they exhibit highly prolonged bleeding times, and their platelets aggregate poorly in responses to ADP and display a reduced sensitivity to thrombin and collagen. These platelets retain normal shape change and calcium flux in response to ADP but fail to inhibit adenylyl cyclase. In addition, oral clopidogrel does not inhibit aggregation responses to ADP in these mice. These results demonstrate that SP1999 is indeed the elusive receptor, P2Y12. Identification of the target receptor of the thienopyridine drugs affords us a better understanding of platelet function and provides tools that may lead to the discovery of more effective antithrombotic therapies.     

The platelet P2 receptors as molecular targets for old and new antiplatelet drugs

Platelet activation by ADP and ATP plays a crucial role in haemostasis and thrombosis, and their so-called P2 receptors are potential targets for antithrombotic drugs. The ATP-gated channel P2X1 and the 2 G protein-coupled P2Y1 and P2Y12 ADP receptors selectively contribute to platelet aggregation. The P2Y1 receptor is responsible for ADP-induced shape change and weak and transient aggregation, while the P2Y12 receptor is responsible for the completion and amplification of the response to ADP and to all platelet agonists, including thromboxane A2 (TXA2), thrombin, and collagen. The P2X1 receptor is involved in platelet shape change and in activation by collagen under shear conditions. Due to its central role in the formation and stabilization of a thrombus, the P2Y12 receptor is a well-established target of antithrombotic drugs like ticlopidine or clopidogrel, which have proved efficacy in many clinical trials and experimental models of thrombosis. Competitive P2Y12 antagonists have also been shown to be effective in experimental thrombosis as well as in several clinical trials. Studies in P2Y1 and P2X1 knockout mice and experimental thrombosis models using selective P2Y1 and P2X1 antagonists have shown that, depending on the conditions, these receptors could also be potential targets for new antithrombotic drugs.     

An inherited bleeding disorder linked to a defective interaction between ADP and its receptor on platelets. Its influence on glycoprotein IIb-IIIa complex function.

Much discussion has concerned the central role of ADP in platelet aggregation. We now describe a patient (M.L.) with an inherited bleeding disorder whose specific feature is that ADP induces a limited and rapidly reversible platelet aggregation even at high doses. Platelet shape change and other hemostatic parameters were unmodified. A receptor defect was indicated, for, while epinephrine normally lowered cAMP levels of PGE1-treated (M.L.) platelets, ADP was without effect. The binding of [3H]2-methylthio-ADP decreased from 836 +/- 126 molecules/platelet for normals to 30 +/- 17 molecules/platelet for the patient. Flow cytometry confirmed that ADP induced a much lower fibrinogen binding to (M.L.) platelets. Nonetheless, the binding in whole blood of activation-dependent monoclonal antibodies showed that some activation of GP IIb-IIIa complexes by ADP was occurring. Platelets of a patient with type I Glanzmann's thrombasthenia bound [3H]2-methylthio-ADP and responded normally to ADP in the presence of PGE1. Electron microscopy showed that ADP-induced aggregates of (M. L.) platelets were composed of loosely bound shape-changed platelets with few contact points. Thus this receptor defect has a direct influence on the capacity of platelets to bind to each other in response to ADP.     

Defective platelet aggregation and increased resistance to thrombosis in purinergic P2Y(1) receptor-null mice

ADP is a key agonist in hemostasis and thrombosis. ADP-induced platelet activation involves the purinergic P2Y(1) receptor, which is responsible for shape change through intracellular calcium mobilization. This process also depends on an unidentified P2 receptor (P2cyc) that leads to adenylyl cyclase inhibition and promotes the completion and amplification of the platelet response. P2Y(1)-null mice were generated to define the role of the P2Y(1) receptor and to determine whether the unidentified P2cyc receptor is distinct from P2Y(1). These mice are viable with no apparent abnormalities affecting their development, survival, reproduction, or the morphology of their platelets, and the platelet count in these animals is identical to that of wild-type mice. However, platelets from P2Y(1)-deficient mice are unable to aggregate in response to usual concentrations of ADP and display impaired aggregation to other agonists, while high concentrations of ADP induce platelet aggregation without shape change. In addition, ADP-induced inhibition of adenylyl cyclase still occurs, demonstrating the existence of an ADP receptor distinct from P2Y(1). P2Y(1)-null mice have no spontaneous bleeding tendency but are resistant to thromboembolism induced by intravenous injection of ADP or collagen and adrenaline. Hence, the P2Y(1) receptor plays an essential role in thrombotic states and represents a potential target for antithrombotic drugs.     

Impaired activation of murine platelets lacking G alpha(i2)

The intracellular signaling pathways by which G protein-coupled receptors on the platelet surface initiate aggregation, a critical process for hemostasis and thrombosis, are not well understood. In particular, the contribution of the G(i) pathway has not been directly addressed. We have investigated the activation of platelets from mice in which the gene for the predominant platelet G alpha(i) subtype, G alpha(i2), has been disrupted. In intact platelets from G alpha(i2)-deficient mice, the inhibition of adenylyl cyclase by ADP was found to be partially impaired compared with wild-type platelets. Moreover, both ADP-dependent platelet aggregation and the activation of the integrin alpha IIb beta 3 (GPIIb-IIIa) were strongly reduced in platelets from G alpha(i2)-deficient mice. In addition, G alpha(i2)-deficient platelets displayed impaired activation at low thrombin concentrations. This defect was mimicked by blocking the adenylyl cyclase--coupled platelet ADP receptor (P2Y(12)) on wild-type platelets with a selective antagonist. These observations suggest that G alpha(i2) is involved in the inhibition of platelet adenylyl cyclase in vivo and is a critical component of the signaling pathway for integrin activation by ADP, resulting in platelet aggregation. In addition, thrombin-dependent activation of mouse platelets is mediated, at least in part, by secreted ADP acting on the G alpha(i2)-linked ADP receptor.     

Platelet CD36 mediates interactions with endothelial cell-derived microparticles and contributes to thrombosis in mice

CD36 is a scavenger receptor that binds multiple ligands, including phosphatidyl serine (PS). Although CD36(-) mice do not have a bleeding diathesis, we show here that they do have significantly prolonged thrombotic occlusion times in response to FeCl(3)-induced vascular injury. Because cell-derived microparticles (MPs) are generated in response to vascular injury and circulate in patients with prothrombotic diseases, we hypothesized that PS exposed on their surfaces could be an endogenous CD36 ligand that transmits an activating signal to platelets. We found that MPs prepared from human ECs, monocytes, or platelets or isolated from blood of normal subjects bound to platelets. Binding was not observed with platelets from CD36(-) donors and was inhibited by an anti-CD36 antibody or by blockade of exposed PS by annexin V or anti-PS IgM. Preincubation of platelets with MPs led to CD36-dependent augmentation of platelet activation in response to low doses of ADP, as assessed by measuring alpha(2b)beta(3) activation, P-selectin expression, and aggregation. Immunofluorescence confocal microscopy of murine carotid thrombi from CD36(-) mice showed a significant decrement in endothelial antigen accumulation, which suggests that CD36 plays a role in MP recruitment into thrombi. These results provide what we believe to be a novel role for CD36 in thrombosis.     

A differential role of the platelet ADP receptors P2Y1 and P2Y12 in Rac activation

The dynamics of the actin cytoskeleton, largely controlled by the Rho family of small GTPases (Rho, Rac and Cdc42), is critical for the regulation of platelet responses such as shape change, adhesion, spreading and aggregation. Here, we investigated the role of adenosine diphosphate (ADP), a major co-activator of platelets, on the activation of Rac. ADP rapidly activated Rac in a dose-dependent manner and independently of GPIIb/IIIa and phosphoinositide 3-kinase. ADP alone, used as a primary agonist, activated Rac and its effector PAK via its P2Y1 receptor, through a G(q)-dependent pathway and independently of P2Y12. The P2Y12 receptor appeared unable to activate the GTPase per se as also observed for the adenosine triphosphate receptor P2X1. Conversely, secreted ADP strongly potentiated Rac activation induced by FcgammaRIIa clustering or TRAP via its P2Y12 receptor, the target of antithrombotic thienopyridines. Stimulation of the alpha(2A)-adrenergic receptor/G(z) pathway by epinephrine was able to replace the P2Y12/G(i)-mediated pathway to amplify Rac activation by FcgammaRIIa or by the thrombin receptor PAR-1. This co-activation appeared necessary to reach a full stimulation of Rac as well as PAK activation and actin polymerization and was blocked by a G-protein betagamma subunits scavenger peptide.     

Plasma ectonucleotidases prevent desensitization of purinergic receptors in stored platelets: importance for platelet activity during thrombus formation

BACKGROUND: Platelets (PLTs) contain purinergic receptors for ATP (P2X1) and ADP (P2Y1 and P2Y12) that rapidly desensitize upon stimulation with these nucleotides. In vivo, this is antagonized by ectonucleotidases on the surface of endothelial cells and white blood cells (WBCs). The receptor desensitization of ATP- and ADP-induced responses of PLTs stored in plasma without WBCs was investigated. STUDY DESIGN AND METHODS: ATP- and ADP-induced PLT shape change (shear-induced) aggregation and Ca2+ signaling were measured in the presence or absence of plasma. Degradation of nucleotides in plasma was quantified by high-performance liquid chromatography. RESULTS: Washed PLTs became refractory for ATP and ADP in shape change, aggregation, and Ca2+ responses during a 90-minute incubation at 37 degrees C. The PLT responses mediated by P2X1, P2Y1, and P2Y12 receptors gradually reduced or disappeared. When plasma was present, however, the PLTs persistently showed high responses to ATP and ADP. Heat treatment of plasma abolished this effect. Also under conditions of flow and high shear, PLTs in plasma kept high P2X1 activity, mediating aggregate formation. In isolated plasma, not containing WBCs, nucleotides were degraded in the order of ADP/UDP>ATP/UTP. Degradation of ATP was partly inhibited by blocking the ecto-NTPDase CD39, whereas degradation of both ATP and ADP was inhibited by blocking ectopyrophosphatase/phosphodiesterase activity. Part of the nucleotide-degrading activities appeared to be membrane-bound. CONCLUSION: Ectonucleotidases in plasma preserve the functionality of P2X1 and P2Y receptors. Upon PLT storage, these plasma activities are essential to ensure adequate (shear-dependent) formation of aggregates and thrombi.     

Central role of the P2Y12 receptor in platelet activation

Platelet activation occurs in response to vessel injury and is important for the arrest of bleeding. Platelet activation during disease states leads to vascular occlusion and ischemic damage. The P2Y(12) receptor, activated by ADP, plays a central role in platelet activation and is the target of P2Y(12) receptor antagonists that have proven therapeutic value.     

Limited usefulness of the PFA-100 for the monitoring of ADP receptor antagonists--in vitro experience.

We have evaluated the usefulness of the PFA-100 system (collagen/ADP and collagen/epinephrine cartridges) to assess the in vitro effects of a few platelet function inhibitors: Aspisol (60 microg/ml), 4-[4-[4-(aminoiminomethyl]-1-piperazinyl]-1-piperidineactetic acid, hydrochloride trihydrate (GR144053F, fibrinogen receptor antagonist, 100 nM), adenosine-3',5'-diphosphate (A3P5P, P2Y1 ADP receptor antagonist, 500 microM) and Bis[(adenosine-5'-O-phosphorodithioyl)methylene]-phosphinic acid (APTMPA, P2Y12 ADP receptor antagonist, 500 microM) on platelet function, as compared with the other commonly used diagnostic technique, a whole blood electrical aggregometry (20 microM ADP or 0.5 mM arachidonic acid). The in vitro studies were carried out on a group of 38 subjects. Whereas all the examined platelet antagonists and inhibitors almost completely blocked the 20 microM ADP- or 0.5 mM arachidonic acid-induced (in the case of acetylsalicylic acid) whole blood aggregation, only two inhibitors (Aspisol and GR144053F) remained effective in a significant prolongation of the PFA-100 occlusion time. Otherwise, using the PFA-100 system we were not able to detect the inhibitory actions of ADP receptor antagonists- P2Y1 and P2Y12. Our findings point to a limited usefulness of the PFA-100 system for the monitoring of the effectiveness of ADP receptor antagonists. The outcomes of this study show that platelet aggregometry in whole blood is characterised by the highest sensitivity in the monitoring of the investigated blood platelet inhibitors.     

Adenine triphosphate nucleotides are antagonists at the P2Y receptor.

The aim of the present study was to characterize the pharmacological profile of the P2Y(12) receptor for several adenine triphosphate nucleotides in view of their possible roles as partial agonists or true antagonists. Two distinct cellular systems were used: P2Y(1) receptor deficient mouse platelets ( platelets) previously shown to express a native and functional P2Y(12) receptor and 1321 N1 astrocytoma cells stably expressing the human P2Y(12) receptor (1321 N1 P2Y(12)). ADP and its structural analogues inhibited cAMP accumulation in a dose-dependent manner in both platelets and 1321 N1 P2Y(12) cells with a similar rank order of potency, 2 methylthio-ADP (2MeSADP) >ADP - Adenosine 5'-(betathio) diphosphate (AlphaDPbetaS). Commercial ATP, 2 chloro; ATP (2ClATP) and 2 methylthio-ATP (2MeSATP) also inhibited cAMP accumulation in both cell systems. In contrast, after creatine phosphate (CP)/creatine phosphokinase (CPK) regeneration, adenine triphosphate nucleotides lost their agonistic effect on platelets and behaved as antagonists of ADP (0.5 microm)-induced adenylyl cyclase inhibition with IC(50) of 13.5 +/- 4.8, 838 +/- 610, 1280 +/- 1246 microm for 2MeSATP, ATP and 2ClATP, respectively. In 1321 N1 P2Y(12) cells, CP/CPK regenerated ATP and 2ClATP lost their agonistic effect only when CP/CPK was maintained during the cAMP assay. The stable ATP analogue ATPgammaS antagonized ADPbetaS-induced inhibition of cAMP accumulation in both platelets and 1321 N1 P2Y(12) cells. Thus, ATP and its triphosphate analogues are not agonists but rather antagonists at the P2Y(12) receptor expressed in platelets or transfected cells, provided care is taken to remove diphosphate contaminants and to prevent the generation of diphosphate nucleotide derivatives by cell ectonucleotidases.