P2 Receptors and Platelet Activation

Adenosine diphosphate (ADP) plays a crucial role in hemostasis and thrombosis by activating platelets. In platelets, the classical P2T receptor is now resolved into three P2 receptor subtypes: the P2Y1, the P2Y12, and the P2X1 receptors. Both pharmacological and molecular biological approaches have confirmed the role of the P2Y1 and P2Y12 receptors in the ADP-induced platelet fibrinogen receptor activation. The P2Y1 and the P2X1 receptors independently contribute to platelet shape change. Whereas the P2Y12 receptor mediates the potentiation of dense granule release reaction, both the P2Y1 and P2Y12 receptors play an important role in the ADP-induced phospholipase A2 activation. The signaling events downstream of these receptors leading to the physiological effects remain elusive, and they are yet to be delineated

The P2Y12 receptor induces platelet aggregation through weak activation of the αIIbβ3 integrin – a phosphoinositide 3-kinase-dependent mechanism

AbstractHigh concentrations of adenosine-5′-diphosphate ADP are able to induce partial aggregation without shape change of P2Y1 receptor-deficient mouse platelets through activation of the P2Y12 receptor. In the present work we studied the transduction pathways selectively involved in this phenomenon. Flow cytometric analyses using R-phycoerythrin-conjugated JON/A antibody (JON/A-PE), an antibody which recognizes activated mouse αIIbβ3 integrin, revealed a low level activation of αIIbβ3 in P2Y1 receptor-deficient platelets in response to 100 μM ADP or 1 μM 2MeS-ADP. Adrenaline induced no such activation but strongly potentiated the effect of ADP in a dose-dependent manner. Global phosphorylation of 32P-labeled platelets showed that P2Y12-mediated aggregation was not accompanied by an increase in the phosphorylation of myosin light chain (P20) or pleckstrin (P47) and was not affected by the protein kinase C (PKC) inhibitor staurosporine. On the other hand, two unrelated phosphoinositide 3-kinase inhibitors, wortmannin and LY294002, inhibited this aggregation. Our results indicate that (i) the P2Y12 receptor is able to trigger a P2Y1 receptor-independent inside-out signal leading to αIIbβ3 integrin activation and platelet aggregation, (ii) ADP and adrenaline use different signaling pathways which synergize to activate the αIIbβ3 integrin, and (iii) the transduction pathway triggered by the P2Y12 receptor is independent of PKC but dependent on phosphoinositide 3-kinase

Agonist-Promoted Regulation of the P2Y1 Receptor: Quantification of Native and Recombinant Receptors with the Novel Radioligand [32P]MRS2500

The P2Y family of G-protein coupled receptors are activated by adenine and uridine di- and triphosphate nucleotides and nucleotide sugars and are implicated in a wide range of important human physiologies. Difficulty studying these receptors and in their successful manipulation as therapeutic targets has historically derived from a lack of available pharmacological tools that discriminate among members of the P2Y receptor family. The studies described here focus on the P2Y1 receptor, a key mediator of ADP-induced platelet aggregation. Based on the structure of the recently synthesized, high-affinity P2Y1 receptorselective antagonist, 2-iodo-N6-methyl-(N)-methanocarba-2 -deoxyadenosine-3´,5´- bisphosphate (MRS2500), we undertook the development of a high-specific radioactivity radioligand for the P2Y1 receptor, suitable for the study of endogenous receptors in mammalian tissues and cell lines. Using an enzymatic phosphorylation reaction, we successfully generated [32P]MRS2500 with a specific activity of 9120 Ci/mmol. The selectivity and affinity of [32P]MRS2500 for the P2Y1 receptor were confirmed in radioligand binding assays with Sf9 insect cell membranes overexpressing recombinant P2Y1 receptors. The utility of [32P]MRS2500 for the study of endogenous P2Y1 receptors was examined using washed human platelets and membranes prepared from various tissues of the adult rat. We applied this high-specific radioactivity radioligand to observe surface expression of P2Y1 receptor binding sites in human platelets and MDCK(II) epithelial cells following incubation with P2Y1 receptor agonists. In human platelets, the rapid, agonist-promoted desensitization of the P2Y1 receptor observed after incubation with the selective agonist (N)- methanocarba-2-methylthioadenosine-diphosphate (MRS2365) also occurs for the Gqcoupled 5-HT2A receptor after incubation of platelets with 5-hydroxytryptamine (5-HT). The rapid, agonist-promoted desensitization of the P2Y1 receptor of platelets was accompanied by a modest decrease (< 20%) in the number of surface [32P]MRS2500 binding sites and only a partial recovery of P2Y1 receptor responsiveness after removal of the selective agonist MRS2365. Platelets, therefore, appear to employ a unique mechanism for prolonged termination of P2Y1 receptor signaling in which desensitized receptors are maintained at the cell surface, unable to respond to subsequent agonist stimulation. In intact MDCK(II) cells overexpressing recombinant P2Y1 receptors, incubation with 2MeSADP was followed by a 50% loss of surface [32P]MRS2500 binding sites that was agonist-concentration dependent and required the formation of clathrin-coated pits. Mutagenesis studies indicated that this rapid, agonist-promoted loss of surface binding sites requires two serine residues, Ser352 and Ser354, in the receptor carboxyl terminus. The findings presented here indicate that P2Y1 receptor cell surface expression is regulated in an agonist-dependent manner that differs in at least two cell types and suggests an important role for phosphorylation in agonist-dependent desensitization and internalization of the P2Y1 receptor

Pharmacological evaluation of NF279 as a P2 receptor antagonist : structure-activity relationship studies of analogues of the P2 receptor antagonists Suramin and NF023 at native P2 receptor subtypes and ecto-nucleotidases

Zu Beginn der 70er Jahre wurde für Adenosin-5'-triphosphat (ATP), das bereits lange als innerhalb der Zelle vorkommender Energieträger bekannt war, eine extrazelluläre Wirkung als Neurotransmitter postuliert. Es dauerte allerdings noch eine Reihe von Jahren, bis die von der Arbeitsgruppe um G. Burnstock aufgestellte Hypothese der "purinergen Nerven" und die damit verbundene Existenz von sogenannten P2-Rezeptoren (früher purinerge Rezeptoren) allgemein anerkannt wurde. Heute besteht kein Zweifel mehr, dass ATP alle der unten genannten Anforderungen an eine als endogener Überträgerstoff innerhalb des vegetativen und zentralen Nervensystems fungierende Substanz erfüllt: • Synthese und Speicherung des entsprechenden Überträgerstoffes, • Freisetzung einer definierten Menge an Transmitter bei einem einlaufenden Aktionspotential, • Rezeptoren für die Reaktion mit dem Transmitter sowie • die rasche Inaktivierung des freigesetzten Neurotransmitters. ...More than 70 years ago, the effects of extracellular adenosine 5'-triphosphate (ATP), a newly identified and purified biomolecule at that time (Fiske and Subbarow, 1925; Lohmann, 1929) were observed by Drury and Szent-Györgyi (1929). Since then, many pharmacological studies were carried out with extracellular adenine nucleotides in various intact organ systems, isolated tissues, and purified cell preparations. Yet it was not until 1972 that Burnstock introduced the concept of "purinergic nerves" and suggested that ATP might fulfil the criteria generally regarded as necessary for establishing a substance as a neurotransmitter, summarised by Eccles (1964): • synthesis and storage of transmitter in nerve terminals Strips of guinea-pig taenia coli (GPTC) were shown to take up large amounts of tritium-labelled adenosine when incubated with tritium-labelled adenosine, adenosine 5'-monophosphate (AMP), adenosine 5'-diphosphate (ADP) and ATP. The nucleoside was rapidly converted into and retained largely as [ 3 H]-ATP (Su et al., 1971). • release of transmitter during nerve stimulation Spontaneous relaxation of GPTC as well as relaxations induced by nerve stimulation or nicotine, respectively, in the presence of compounds which block adrenergic and cholinergic responses were accompanied by a remarkable increase in release of tritium-labelled material from taenia coli incubated in [ 3 H]-adenosine (Su et al., 1971). • postjunctional responses to exogenous transmitters that mimic responses to nerve stimulation Burnstock et al. (1966) characterised ATP and ADP as the most potent inhibitory purine compounds in the gut and observed that the effects of ATP mimic more closely the inhibitory response of the taenia to non-adrenergic nerve-stimulation than to adrenergic nerve stimulation (Burnstock et al., 1970). enzymes that inactivate the transmitter and/or uptake systems for the transmitter or its breakdown products When ATP was added to a perfusion fluid recycled through the vasculature of the stomach, very little ATP remained, but the perfusate contained substantially increased amounts of adenosine and inosine, as well as some ADP and AMP (Burnstock et al., 1970). • drugs that can produce parallel blocking of potentiating effects on the responses of both exogenous transmitter and nerve stimulation Tachyphylaxis to ATP produced in the rabbit ileum resulted in a consistent depression of responses to non-adrenergic inhibitory nerve stimulation, whereas responses to adrenergic nerve stimulation remained unaffected (Burnstock et al., 1970). Lower concentrations of quinidine reduced and finally abolished relaxation of GPTC induced by noradrenaline (NA) and by adrenergic nerve stimulation. Using higher concentrations of the compound, relaxant responses of GPTC to ATP as well as to non-adrenergic inhibitory nerve stimulation were abolished (Burnstock et al., 1970). ..

Évaluation pré-clinique et clinique de deux nouvelles stratégies dans le traitement des maladies cardiovasculaires : activité anti-thrombotique de l'inhibition du récepteur P2Y1 et activité anti-inflammatoire de l'inhibition du récepteur PAR-1

Alors que la plaquette a un rôle prépondérant dans le maintient de l'hémostase, son implication dans la formation de la thrombose est également incontestée. Il existe à présent des traitements antiplaquettaires mais ceux-ci présentent quelques failles. Le but de ma maîtrise a donc été de caractériser de nouvelles voies d'inhibition de l'activité plaquettaire. Les deux études qui ont été réalisées concernent l'inhibition de récepteurs couplés aux protéines G, soit le récepteur à l'ADP P2Y1 et le récepteur à la thrombine PAR-1. Dans un premier temps, l'étude du récepteur P2Y1 suggère que l'inhibition de celui-ci seul ou en combinaison avec l'inhibition du récepteur P2Y12 présente un potentiel thérapeutique chez des patients coronariens stables. Dans un deuxième temps, l'étude d'un nouvel antiplaquettaire qui est présentement en phase III de son développement clinique, soit le SCH 530348, a été effectuée. Étant un antagoniste du récepteur PAR-1, le SCH 530348 a des effets qui se répercutent à la fois chez la plaquette et les leucocytes qui possèdent eux aussi ce récepteur. Cette deuxième étude suggère que complémentairement à son effet chez la plaquette, cet inhibiteur diminue les marqueurs de l'inflammation systémique.Platelets have a central role in haemostasis as well as in the pathology of atherothrombotic disease. Antiplatelet therapy is the standard treatment for patients with coronary heart disease. However, the current antiplatelet agents show some disadvantages. The aim of my studies was then to describe the effect of inhibition of platelet activation pathways that are not yet the target of clinically used therapies. The two studies that were performed are regarding the inhibition of two G coupledprotein receptors, which are the ADP P2Y1 receptor and the thrombin PAR-1 receptor. First, the study of P2Y1 receptor revealed that its inhibition alone or in combination with the inhibition of P2Y12 receptor show a therapeutic potential in patients with stable coronary disease. Second, the study of a novel antiplatelet agent, SCH 530348, which is in phase III of its clinical development, has been carried out. SCH 530348, which is a PAR-1 receptor antagonist, can therefore act on platelets as well as on leukocytes which also express this receptor. This second study showed that additionally to its effect on platelet activation inhibition, SCH 530348 also reduces the cell-surface markers of systemic inflammation

Role of the P2Y1 receptor in platelet activation

Understanding and manipulating thrombosis and blood hemostasis is critical for the effective treatment of patients at risk for heart attack and stroke. ADP is an important modulator of platelet function and vascular tone that acts by binding to and activating the P2Y1 and P2Y12 receptors. Deletion or inhibition of either receptor results in nearly a total loss of ADP-promoted aggregation. Interestingly, the P2Y1 receptor desensitizes extremely rapidly, with a half-life of approximately 18 sec, whereas the P2Y12 receptor continuously signals minutes after initial activation. We hypothesized that the rapid desensitization of the P2Y1 receptor in platelets prevents excess thrombosis and unwanted aggregation at lower concentrations of ADP. We have used ex vivo platelet activation experiments to demonstrate that the observed desensitization is specific to the P2Y1 receptor compared to other Gq-coupled receptors in platelets and is recapitulated in mouse platelets. We focused on serine and threonine residues on the C-terminus because of the involvement of various Ser and Thr phosphorylation on the regulation of the P2Y1 receptor in cell culture systems. Using an optimized viral transduction model to introduce variants of the P2Y1 receptor into bone marrow, we show preliminary data suggesting that mutating multiple serine and threonine residues in the C-terminus of the P2Y1 receptor (“340-0P”) results in prolonged activation of platelets in the absence of the P2Y12 receptor pathway, thus eliminating the observed desensitization in the wild type P2Y1 receptor. Furthermore, creation of a knock-in mouse for the 340-0P variant of the P2Y1 receptor revealed a loss of desensitization in platelets upon ADP stimulation in 340-0P heterozygous mice. These data suggest that the loss of Ser and Thr residues on the C-terminus of the P2Y1 receptor nullifies the observed desensitization and provides insights regarding the physiological relevance of this process in platelets.Doctor of Philosoph

Charakterisierung von Thrombozytenfunktion und Thrombozyten-Leukozyten Interaktionen im Rahmen von Koronarinterventionen unter einer Therapie mit Clopidogrel

Gegenstand der vorliegenden Arbeit war die Charakterisierung der Thrombozytenfunktion und der Thrombozyten-Leukozyten Adhäsion (TLA) im Rahmen von Koronarinterventionen unter einer Therapie mit dem Thienopyridin Clopidogrel. Untersucht wurden die Expression aktivierungsab-hängiger membranständiger Thrombozytenrezeptoren, die Thrombozytenadhäsion an Leukozyten und die Aggregation bei 40 Patienten mit koronarer Herzerkrankung, die vor geplanter Koronar-intervention mit Clopidogrel (300mg Aufsättigungsdosis, 75mg/d Erhaltungsdosis) behandelt wurden. Bereits 6 Stunden nach Therapiebeginn war eine signifikante Hemmung der durch Adenosin-diphosphat (ADP) induzierten Expression der Membranrezeptoren P-Selektin und Glykoprotein (GP) 53 zu beobachten, deren Ausmaß unter der Erhaltungsdosis bestehen blieb. Auch die durch das Thrombin Rezeptor aktivierende Peptid (TRAP) induzierte Rezeptorexpression war unter Clopidogrel signifikant vermindert. Daneben wurde eine vermehrte Hemmung der Internalisierung des GP Ib Rezeptors und eine deutliche Hemmung der thrombozytären Fibrinogenbindung beobachtet. Auch wurde die Rekrutierung von GP IIb/IIIa Rezeptoren auf der Zelloberfläche und die Expression aktivierter GP IIb/IIIa Rezeptoren signifikant gehemmt. Neben der verminderten Thrombozyten-rezeptorexpression war die Thrombozytenadhäsion an Neutrophile und an Monozyten unter Clopidogrel deutlich vermindert. Auch hier konnte bereits nach 6 Stunden eine signifikante Hemmung der ADP-induzierten TLA-Bildung verzeichnet werden, die im weiteren Verlauf erhalten blieb. Das Ausmaß der TLA-Bildung vor Therapiebeginn und unter Clopidogrel korrelierte signifikant mit der Expression des thrombozytären P-Selektin. Die Thrombozytenaggregation wurde ebenfalls rasch und signifikant durch Clopidogrel gehemmt. Neben der Hemmung der ADP-induzierten Aggregation war auch die TRAP-induzierte Aggregation leicht vermindert. Unter der Clopidogrel-Therapie wurde postinterventionell kein signifikanter Anstieg der Expression thrombozytärer Membranrezeptoren, der TLA-Bildung oder der Thrombozytenaggregation beobachtet. Clopidogrel führt als Aufsättigungsdosis zur raschen Hemmung der Thrombozytenfunktion, welche die agonisten-induzierte Expression der intrazellulär gespeicherter Membranrezeptoren P-Selektin und GP 53, die Internalisierung des GP Ib, die Rekrutierung und Expression aktivierter GP IIb/IIIa Rezeptoren sowie die Fibrinogenbindung und die Thrombozytenaggregation beinhaltet. Darüber hinaus wird die Thrombozytenadhäsion an zirkulierende Leukozyten signifikant gehemmt. Das Ausmaß der Funktionshemmung bleibt unter der Erhaltungsdosis bestehen. Durch die Therapie mit Clopidogrel scheint eine Passivierung der Thrombozytenfunktion ohne signifikanten reaktiven Anstieg der Expression membranständiger Rezeptoren oder der Aggregation nach elektiver Koronar-intervention induziert zu werden

Pharmacological agents that distinguish between P2X receptor subtypes.

The activity of novel pharmacological agents at recombinant P2X receptors was studied to find agents that distinguish between P2X receptor subtypes, particularly P2X1 and P2X3. Adenine nucleotide derivatives and diadenosine polyphosphates (ApnA, n = 2-6) were investigated as P2X receptor agonists. PAPET and HT-AMP were agonists, to varying degrees, at P2X1-4 receptors. PAPET displayed higher affinity but lower efficacy than ATP at P2X1 and P2X3 receptors. HT-AMP showed higher affinity than ATP at P2X3 receptors yet acted as a partial agonist at P2X1-4 receptors. Diadenosine polyphosphates also showed selectivity in their actions at P2X1-4 receptors. Ap2A was inactive and Ap3-6A showed varying affinities and efficacies as agonists at P2X1-4. Ap3A was most effective at distinguishing between P2X1 and P2X3 receptors with over 100 fold difference between their respective EC50 values A series of PPADS derivatives, involving chemical manipulation of the phenylazo moiety and/or the pyridoxal phosphate moiety, showed nanomolar activity at P2X1 and P2X3 receptors with variable degrees of selectivity between these receptor subtypes. The most potent compounds were studied in detail and shown to be nonsurmountable antagonists. A comparison of like data for recombinant P2X1 receptors and native P2X1-like receptors in vas deferens revealed a number of pharmacological anomalies. Co-expression of P2X1 and P2X2 revealed a novel pH-sensitive phenotype although this heteromeric receptor is unlikely to account for the difference between rP2X1 and the native P2X subtype(s) in this tissue. A step forward has been made in the search of pharmacological agents that distinguish between P2X1 and P2X3 receptors. Antagonist-resistant ATP responses in the vas deferens lend weight for other contraction-mediating P2 receptors in this preparation. Greater diversity of purinergic signalling was revealed through co-expression of P2X receptors and underlines the need for further novel pharmacological tools