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.     

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.     

MRS2500 [2-iodo-N6-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphate], a potent, selective, and stable antagonist of the platelet P2Y1 receptor with strong antithrombotic activity in mice

The platelet P2Y(1) ADP receptor is an attractive target for new antiplatelet drugs. However, because of the lack of strong and stable antagonists, only a few studies have suggested that pharmacological inhibition of the P2Y(1) receptor could efficiently inhibit experimental thrombosis in vivo. Our aim was to determine whether the newly described potent and selective P2Y(1) receptor antagonist MRS2500 [2-iodo-N(6)-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphate] could inhibit platelet function ex vivo and experimental thrombosis in mice in vivo. MRS2500 was injected intravenously into mice, and its effect on ex vivo platelet aggregation and in several models of thrombosis in vivo was determined. MRS2500 displayed high potency and stable and selective P2Y(1) receptor inhibition ex vivo. Although MRS2500 injection resulted in only moderate prolongation of the bleeding time, it provided strong protection in systemic thromboembolism induced by infusion of a mixture of collagen and adrenaline. MRS2500 also potently inhibited localized arterial thrombosis in a model of laser-induced vessel wall injury with two degrees of severity. Moreover, combination of MRS2500 with clopidogrel, the irreversible inhibitor of the platelet P2Y(12) receptor for ADP, led to increased antithrombotic efficacy compared with each alone. These results add further evidence for a role of the P2Y(1) receptor in thrombosis and validate the concept that targeting the P2Y(1) receptor could be a relevant alternative or complement to current antiplatelet strategies.     

A clopidogrel-insensitive inducible pool of P2Y12 receptors contributes to thrombus formation: inhibition by elinogrel, a direct-acting, reversible P2Y12 antagonist

It is known that hepatic metabolism limits the antiaggregatory activity of clopidogrel and, as a consequence, its clinical benefits. In this study, we investigated whether other factors exist that could account for clopidogrel's suboptimal antithrombotic activity. Using an in vivo murine FeCl(3) thrombosis model coupled with intravital microscopy, we found that at equivalent, maximal levels of inhibition of ADP-induced platelet aggregation, clopidogrel (50 mg/kg p.o.) failed to reproduce the phenotype associated with P2Y(12) deficiency. However, elinogrel (60 mg/kg p.o.), a direct-acting reversible P2Y(12) antagonist, achieved maximal levels of inhibition in vivo, and its administration (1 mg/kg i.v.) abolished residual thrombosis associated with clopidogrel dosing. Because elinogrel is constantly present in the plasma, whereas the active metabolite of clopidogrel exists for ～2 h, we evaluated whether an intracellular pool of P2Y(12) exists that would be inaccessible to clopidogrel and contribute to its limited antithrombotic activity. Using saturation [(3)H]2-(methylthio)ADP ([(3)H]2MeSADP) binding studies, we first demonstrated that platelet stimulation with thrombin and convulxin (mouse) and thrombin receptor activating peptide (TRAP) (human) significantly increased surface expression of P2Y(12) relative to that of resting platelets. We next found that clopidogrel dose-dependently inhibited ADP-induced aggregation, signaling (cAMP), and surface P2Y(12) on resting mouse platelets, achieving complete inhibition at the highest dose (50 mg/kg), but failed to block this inducible pool. Thus, an inducible pool of P2Y(12) exists on platelets that can be exposed upon platelet activation by strong agonists. This inducible pool is not blocked completely by clopidogrel, contributes to thrombosis in vivo, and can be blocked by elinogrel.     

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.     

Evidence that pre-existent variability in platelet response to ADP accounts for ''clopidogrel resistance''

BACKGROUND: Clopidogrel is a widely used antithrombotic agent that inhibits the platelet P2Y(12) adenosine diphosphate (ADP) receptor. There is increasing interest in 'clopidogrel resistance'. OBJECTIVES: To determine whether 'clopidogrel resistance' is accounted for by a pre-existent variability in platelet response to ADP. METHODS: Platelet response to 20 microm ADP was analyzed by four independent whole blood flow cytometric assays: platelet surface activated GPIIb-IIIa, platelet surface P-selectin, monocyte-platelet aggregates and neutrophil-platelet aggregates. In 25 consecutive, non-aspirin-treated healthy subjects, we studied platelet response before and after clopidogrel administration. In addition, we studied the platelet response in 613 consecutive aspirinated patients with or without coronary artery disease (CAD, as determined by angiography) who had or had not been treated with clopidogrel. In these patients, we tested for homogeneity of variance across all durations of clopidogrel exposure and severity of CAD by estimating the 'goodness of fit' of two independent models. RESULTS: In the healthy subjects, pre-clopidogrel response to ADP predicted post-clopidogrel response to ADP. In the patients, clopidogrel, as expected, inhibited the platelet response to ADP. However, irrespective of the duration of clopidogrel administration, the severity of CAD, and the dose of aspirin, clopidogrel did not increase the variance in the platelet response to ADP in any of the four assays of platelet response. CONCLUSIONS: These studies provide evidence that 'clopidogrel resistance' is accounted for by a pre-existent variability in platelet response to ADP and this variability is not increased by clopidogrel administration.     

Platelet P2 receptors: old and new targets for antithrombotic drugs

Platelets possess three P2 receptors for adenine nucleotides: P2Y1 and P2Y12, which interact with ADP, and P2X1, which interacts with ATP. The interaction of adenine nucleotides with their platelet receptors plays an important role in thrombogenesis. The thienopyridine ticlopidine, an antagonist of the platelet P2Y12 ADP receptor, reduces the incidence of vascular events in patients at risk, but it also has some important drawbacks: a relatively high incidence of toxic effects; delayed onset of action; high inter-individual variability in response. Another thienopyridine, clopidogrel, has superseded ticlopidine, because it is an efficacious antithrombotic drug and is less toxic than ticlopidine. However, the high inter-patient variability in response still remains an important issue. These drawbacks justify the continuing 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 P2Y12 antagonists, cangrelor and AZD6140, have 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 P2Y12 antagonists, inhibitors of the other platelet receptor for ADP, P2Y1, and of the receptor for ATP, P2X1, are under development and may prove to be effective antithrombotic agents.     

(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 .     

Novel molecular defect in the platelet ADP receptor P2Y12 of a patient with haemorrhagic diathesis.

BACKGROUND: The platelet adenosine 5'-diphosphate (ADP) receptor P2Y(12) plays a crucial role in haemostasis. Only a few patients with haemorrhagic diathesis due to molecular defects in the P2Y(12) receptor have been described so far. We report a novel molecular defect in the gene coding for P2Y(12) in a patient with a history of epistaxis, easy bruising and excessive posttraumatic blood loss. METHODS: Platelet aggregation studies, perfusion studies, in which patient blood was perfused over collagen surfaces at arterial shear rates, and PCR and sequencing were used. RESULTS: Platelet aggregation studies showed impaired ADP and collagen-induced aggregation for patient G.S. Perfusion of patient blood over collagen surfaces showed small thrombi consisting of spread platelets overlayered with non-spread platelets. These thrombi were identical to control thrombi formed in the presence of a P2Y(12) antagonist. DNA analysis of the P2Y(12) gene revealed a novel heterozygous base pair C-->A substitution in exon 3, changing codon 258 from proline to threonine in the third extracellular loop of the P2Y(12) receptor. CONCLUSIONS: We conclude that perfusion studies with patient blood are of added value in the diagnostic process, which resulted in identification of a novel molecular defect in the P2Y(12) gene of a patient with haemorrhagic diathesis.     

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.     

Inhibition of the platelet P2Y12 receptor for adenosine diphosphate potentiates the antiplatelet effect of prostacyclin

BACKGROUND: Activation of two receptors for adenosine diphosphate (ADP), P2Y(1) and P2Y(12), is necessary for ADP-induced platelet aggregation (PA). It is generally believed that the antithrombotic effects of drugs inhibiting P2Y(12), such as clopidogrel, are uniquely mediated by inhibition of P2Y(12)-dependent PA. However, as P2Y(12) is negatively coupled to adenylyl cyclase (AC), its inhibition may also exert antithrombotic effects through the potentiation of prostacyclin (PGI(2)), which inhibit PA by stimulating AC. OBJECTIVES: To test whether inhibition of P2Y(12) potentiates the antiplatelet effects of PGI(2). METHODS: We measured the effects of PGI(2) (0.01-10 microm) on PA of washed human platelets induced by thrombin (0.5 U mL(-1)) in the presence or absence of ARC69931MX (anti-P2Y(12)) or MRS2500 (anti-P2Y(1)). Results: PGI(2) inhibited PA in the presence of anti-P2Y(12), but not in the presence of anti-P2Y(1) or in the absence of inhibitors. In contrast, dibutyryl-cyclicAMP inhibited PA both in the presence and absence of anti-P2Y(1) or anti-P2Y(12). PGI(2) increased platelet cyclicAMP levels only in the absence of thrombin or in the presence of thrombin plus anti-P2Y(12). CONCLUSIONS: PGI(2) did not inhibit PA induced by thrombin, because its effect on AC was prevented by released ADP interacting with P2Y(12). Anti-P2Y(12) drugs, by rescuing AC activity, potentiate the antiplatelet effect of PGI(2), which may contribute to their antithrombotic effect.     

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.     

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.     

Monitoring of clopidogrel action: comparison of methods

BACKGROUND: Clopidogrel is a potent drug for prevention of adverse effects during and after coronary intervention. Increasing experience indicates that a significant proportion of patients do not respond adequately to clopidogrel. Because failure of antiplatelet therapy can have severe consequences, there is need for a reliable assay to quantify the effectiveness of clopidogrel treatment. METHODS: Of 24 healthy volunteers admitted to the study, 18 were treated for 1 week with clopidogrel (300-mg loading dose and 75-mg maintenance dose), and 6 with placebo. Platelet function was monitored by 2 assays, based on flow cytometry and enzyme immunoassay, that measure the phosphorylation status of vasodilator-stimulated phosphoprotein (VASP) and by aggregometry, flow cytometry of P-selectin, and the platelet function analyzer at baseline, on days 1-5, and on day 9 of treatment. RESULTS: Aggregometry and VASP phosphorylation revealed a loss of platelet response to ADP within 12 h after clopidogrel intake. The phosphorylation status of VASP correlated with the inhibition of platelet aggregation. In contrast, neither P-selectin expression nor PFA-100 closure time was a clear indicator of clopidogrel effects on platelets. CONCLUSIONS: VASP phosphorylation assays are reliable for quantifying clopidogrel effects. Because the VASP assay directly measures the function of the clopidogrel target, the P2Y12 receptor, the assay is selective for clopidogrel effects rather than effects of other platelet inhibitors commonly in use.     

The future of platelet function testing to guide therapy in clopidogrel low and enhanced responders

Dual oral antiplatelet therapy with aspirin and clopidogrel is the therapy of choice in patients with acute coronary syndromes and in patients undergoing coronary stent placement to lower the risk of thrombotic events. Responsiveness to aspirin and especially to clopidogrel is not uniform and is subject to considerable interindividual variability. Furthermore, there is a broad consensus that clopidogrel low response or so-called high on-treatment platelet reactivity is linked to the occurrence of ischemic events. On the other hand, evidence is accumulating that enhanced clopidogrel responders are at increased risk of bleeding. Newer antiplatelet drugs, such as prasugrel and ticagrelor, are more potent and produce more consistent inhibition of platelet aggregation via the P2Y(12) ADP platelet receptor. A variety of methods of platelet function testing are available for evaluating platelet inhibition in percutaneous coronary intervention-treated patients in order to help determine the individual risk for ischemic and bleeding complications. Although not yet routinely undertaken, platelet function testing offers the potential to tailor antiplatelet therapy for individual patients. Whether alteration of therapy based on platelet function testing improves patients' outcomes remains unclear and is currently under investigation. This article reviews the impact of antiplatelet drug responsiveness on clinical outcomes with a focus on P2Y(12) receptor inhibition as well as on current and future concepts for personalized antiplatelet strategies.     

The greater in vivo antiplatelet effects of prasugrel as compared to clopidogrel reflect more efficient generation of its active metabolite with similar antiplatelet activity to that of clopidogrel''s active metabolite

BACKGROUND AND METHODS: Prasugrel is a novel orally active thienopyridine prodrug with potent and long-lasting antiplatelet effects. Platelet inhibition reflects inhibition of P2Y(12) receptors by its active metabolite (AM). Previous studies have shown that the antiplatelet potency of prasugrel is at least 10 times higher than that of clopidogrel in rats and humans, but the mechanism of its higher potency has not yet been fully elucidated. RESULTS: Oral administration of prasugrel to rats resulted in dose-related and time-related inhibition of ex vivo platelet aggregation, and its effect was about 10 times more potent than that of clopidogrel. The plasma concentration of prasugrel AM was higher than that of clopidogrel AM despite tenfold higher doses of clopidogrel, indicating more efficient in vivo production of prasugrel AM than of clopidogrel AM. In rat platelets, prasugrel AM inhibited in vitro platelet aggregation induced by adenosine 5'-diphosphate (ADP) (10 microm) with an IC(50) value of 1.8 microm. Clopidogrel AM similarly inhibited platelet aggregation with an IC(50) value of 2.4 microm. Similar results were also observed for ADP-induced (10 microm) decreases in prostaglandin E(1)-stimulated rat platelet cAMP levels. These results indicate that both AMs have similar in vitro antiplatelet activities. CONCLUSIONS: The greater in vivo antiplatelet potency of prasugrel as compared to clopidogrel reflects more efficient in vivo generation of its AM, which demonstrates similar in vitro activity to clopidogrel AM.     

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.     

Adrenomedullin Does Not Inhibit Human Platelet Aggregation

BACKGROUND: Adrenomedullin (ADM) is a hypotensive peptide isolated from human pheochromocytoma extracts discovered in 1993 using an assay system designed to monitor its ability to increase rat platelet adenosine 3',5'-cyclic monophosphate (cAMP) levels. Physiological mediators that elevate cAMP levels, such as prostaglandin (PG)E(1) and PGI(2), have also been shown to inhibit platelet aggregation. Therefore, we have chosen to investigate the effect of ADM, a peptide shown to increase platelet cAMP levels, on human platelet aggregation. METHODS AND RESULTS: Platelet-rich plasma prepared from blood donors was incubated with ADM (10(-9)-10(-6) M) for 1 min at 37 degrees C before the addition of a submaximal dose of adenosine 5'-diphosphate (ADP). ADM did not alter the platelet aggregatory response to ADP. PGE(1), a substance known to inhibit ADP-induced platelet aggregation (10(-6) M), however, inhibited ADP-induced platelet aggregation. In addition, the ADM induced a dose-dependent relation in rings of human chorionic arteries. CONCLUSIONS: These data may be interpreted to suggest that human platelets do not possess a functional ADM receptor couple with adenylate cyclase.