Reciprocal cross-talk between P2Y1 and P2Y12 receptors at the level of calcium signaling in human platelets

Adenosine diphosphate (ADP), an important platelet agonist, acts through 2 G-protein-coupled receptors (GPCRs), P2Y(1) and P2Y(12), which signal through Gq and Gi, respectively. There is increasing evidence for cross-talk between signaling pathways downstream of GPCRs and here we demonstrate cross-talk between these 2 ADP receptors in human platelets. We show that P2Y(12) contributes to platelet signaling by potentiating the P2Y(1)-induced calcium response. This potentiation is mediated by 2 mechanisms: inhibition of adenylate cyclase and activation of phosphatidylinositol 3 (PI 3)-kinase. Furthermore, the Src family kinase inhibitor PP1 selectively potentiates the contribution to the calcium response by P2Y(12), although inhibition of adenylate cyclase by P2Y(12) is unaffected. Using PP1 in combination with the inhibitor of PI 3-kinase LY294002, we show that Src negatively regulates the PI 3-kinase-mediated component of the P2Y(12) calcium response. Finally, we were able to show that Src kinase is activated through P2Y(1) but not P2Y(12). Taken together, we present evidence for a complex signaling interplay between P2Y(1) and P2Y(12), where P2Y(12) is able to positively regulate P2Y(1) action and P2Y(1) negatively regulates this action of P2Y(12). It is likely that this interplay between receptors plays an important role in maintaining the delicate balance between platelet activation and inhibition during normal hemostasis.     

The P2 receptors in platelet function

After vessel wall injury, platelets adhere to the exposed subendothelium, are activated, and release mediators such as thromboxane A (2) (TXA (2)) and nucleotides stored at very high concentration in the so-called dense granules. Among other soluble agents, released nucleotides act in a positive feedback mechanism to cause further platelet activation and amplify platelet responses induced by agents such as thrombin or collagen. Adenine nucleotides act on platelets through three distinct P2 receptors: two are G protein-coupled adenosine diphosphate (ADP) receptors, namely the P2Y (1) and P2Y (12) receptor subtypes; the P2X (1) receptor ligand-gated cation channel is activated by adenosine triphosphate (ATP). The P2Y (1) receptor initiates platelet aggregation but is not sufficient for a full platelet aggregation in response to ADP, whereas the P2Y (12) receptor is responsible for completion of the aggregation to ADP. This receptor, the molecular target of the antithrombotic drug clopidogrel, is responsible for most of the potentiating effects of ADP when platelets are stimulated by agents such as thrombin, collagen, or immune complexes. The P2X (1) receptor is involved in platelet shape change and in activation by collagen under shear conditions. Each of these receptors is coupled to specific signal transduction pathways in response to ADP or ATP and is differentially involved in all of the sequential events involved in platelet function and hemostasis. As such, they represent potential targets for antithrombotic drugs.     

Succinate reverses in-vitro platelet inhibition by acetylsalicylic acid and P2Y receptor antagonists

High on-treatment platelet reactivity has been associated with adverse cardiovascular events in patients receiving anti-platelet agents, but the molecular mechanisms underlying this phenomenon remain incompletely understood. Succinate, a citric acid cycle intermediate, is released into the circulation under conditions of mitochondrial dysfunction due to hypoxic organ damage, including sepsis, stroke, and myocardial infarction. Because the G protein-coupled receptor (GPCR) for succinate, SUCNR1 (GPR91), is present on human platelets, we hypothesized that succinate-mediated platelet stimulation may counteract the pharmacological effects of cyclooxygenase-1 and ADP receptor antagonists. To test this hypothesis in a controlled in-vitro study, washed platelets from healthy donors were treated with acetylsalicylic acid (ASA) or small-molecule P2Y(1) or P2Y(12) inhibitors and subsequently analyzed by light transmittance aggregometry using arachidonic acid (AA), ADP and succinate as platelet agonists. Aggregation in response to succinate alone was highly variable with only 29% of donors showing a (mostly delayed) platelet response. In contrast, succinate reproducibly and concentration-dependently (10-1000?μM) enhanced platelet aggregation in response to low concentrations of exogenous ADP. Furthermore, while succinate alone had no effect in the presence of platelet inhibitors, responsiveness of platelets to ADP after pretreatment with P2Y(1) or P2Y(12) antagonists was fully restored, when platelets were co-stimulated with 100?μM succinate. Similarly, succinate completely (at 1000?μM) or partially (at 100?μM) reversed the inhibitory effect of ASA on AA-induced platelet aggregation. In contrast, succinate failed to restore platelet responsiveness in the presence of both ASA and the P2Y(12) antagonist, suggesting that concomitant signaling via different GPCRs was required. Essentially identical results were obtained, when flow cytometric analysis of surface CD62P expression was used as a different readout for platelet activation. In summary, extracellular succinate may have a co-stimulatory role in platelet aggregation and, by (partially) antagonizing the effects of platelet inhibitors, may contribute to the inter-individual variability frequently observed in platelet function testing.     

Roles and interactions among protease-activated receptors and P2ry12 in hemostasis and thrombosis

Toward understanding their redundancies and interactions in hemostasis and thrombosis, we examined the roles of thrombin receptors (protease-activated receptors, PARs) and the ADP receptor P2RY12 (purinergic receptor P2Y G protein-coupled 12) in human and mouse platelets ex vivo and in mouse models. Par3(-/-) and Par4(+/-) mouse platelets showed partially decreased responses to thrombin, resembling those in PAR1 antagonist-treated human platelets. P2ry12(+/-) mouse platelets showed partially decreased responses to ADP, resembling those in clopidogrel-treated human platelets. Par3(-/-) mice showed nearly complete protection against carotid artery thrombosis caused by low FeCl(3) injury. Par4(+/-) and P2ry12(+/-) mice showed partial protection. Increasing FeCl(3) injury abolished such protection; combining partial attenuation of thrombin and ADP signaling, as in Par3(-/-):P2ry12(+/-) mice, restored it. Par4(-/-) mice, which lack platelet thrombin responses, showed still better protection. Our data suggest that (i) the level of thrombin driving platelet activation and carotid thrombosis was low at low levels of arterial injury and increased along with the contribution of thrombin-independent pathways of platelet activation with increasing levels of injury; (ii) although P2ry12 acts downstream of PARs to amplify platelet responses to thrombin ex vivo, P2ry12 functioned in thrombin/PAR-independent pathways in our in vivo models; and (iii) P2ry12 signaling was more important than PAR signaling in hemostasis models; the converse was noted for arterial thrombosis models. These results make predictions being tested by ongoing human trials and suggest hypotheses for new antithrombotic strategies.     

Platelet receptors for adenine nucleotides and thromboxane A2

Adenosine diphosphate (ADP) and thromboxane A (2) (TXA (2)) are important physiological activators of platelets and exert their effects by acting on cell surface receptors. Platelet nucleotide receptors can be distinguished as three separate subtypes of the P2 receptor family. The P2X (1) receptor is a ligand-gated adenosine triphosphate (ATP) receptor that was originally mistaken for an ADP receptor. This calcium-influx-causing receptor mediates platelet shape change and plays an important role in thrombus formation in small arterioles. The P2Y (1) receptor, through activation of G (q) and phospholipase C, is required for ADP-induced platelet shape change, fibrinogen receptor activation, and TXA (2) generation. The G (i)-coupled P2Y (12) receptor plays an important role in platelet aggregation, potentiation of dense granule release, and TXA (2) generation. Both the P2Y receptors are crucial for in vivo thrombus formation. TXA (2) stimulates two subtypes of G protein-coupled TP receptor, TPalpha and TPbeta, but its effects in platelets are mediated predominantly through the alpha isoform. Although interference with the activation of G protein-coupled ADP or TP receptors results in increased bleeding times and protection from thromboembolism, TP receptor antagonists did not translate into effective antiplatelet drugs. Blockade of ADP receptor is a mode of newer classes of antithrombotic drugs in the coming era. This review focuses on the contribution of different nucleotide receptors and TP receptors to platelet function and their potential as antithrombotic agents.     

Evidence that the purinergic receptor P2Y12 potentiates platelet shape change by a Rho kinase-dependent mechanism

ADP activates human platelets through two G-protein coupled receptors, P2Y1 and P2Y12, to induce a range of functional responses. Here we have addressed the role and mechanism of P2Y12 in modulating ADP-induced platelet shape change. Although the response depended upon activation of P2Y1, it was potentiated by P2Y12 as the P2Y12-selective antagonists AR-C69931MX and 2MeSAMP partially inhibited shape change in the later phase of the response. This was paralleled by inhibition of pseudopod formation, platelet spheration, actin polymerisation and myosin light chain phosphorylation. P2Y12 is known to couple to activation of PI3 kinase and inhibition of adenylate cyclase, but we showed that neither of these signalling events couples to regulation of shape change by this receptor. However, by assessment of phosphorylation of its major substrate myosin light chain phosphatase, we provide direct evidence for activation of Rho kinase by ADP, and that although P2Y1 is required for activation of Rho kinase, P2Y12 is able to potentiate its activity. We conclude that P2Y12 plays a potentiatory role in ADP-induced shape change through regulation of the Rho kinase pathway, potentiating both myosin phosphorylation and actin polymerisation, and this forms part of an important signalling pathway additional to its well-established Gi-coupled pathways.     

Both the ADP receptors P2Y1 and P2Y12, play a role in controlling shape change in human platelets

Two types of ADP receptors, P2Y(1) and P2Y(12) are involved in platelet aggregation. The P2X(1) receptor is also present but its role, in terms of platelet function, is not yet defined. The aim of this study was to establish if the ADP receptors, P2Y(1,) P2Y(12) and P2X(1) play a role in controlling platelet shape change (PSC) in human platelets. PSC is an early phase of platelet activation that precedes aggregation. Using a high-resolution channelyzer, PSC was assessed by measuring the median platelet volume (MPV). The P2Y(1) receptor antagonist MRS 2179 (1.06 - 10.25 micro mol/l) blocked ADP-induced PSC (by 100%). The median IC(50) was 3.16 micro mol/l. MRS 2179 also significantly (P = 0.01) inhibited PSC induced by the combination of ADP + serotonin (5HT). The P2Y(12) receptor antagonist AR-C69931MX significantly inhibited (at 10s, P = 0.009; 15 s, P = 0.001 and 30 s, P = 0.015) ADP-induced PSC. The P2X(1) receptor antagonist TNP-ATP had no significant effect on ADP- or ADP + 5HT-induced PSC. We conclude that the IC(50) of a P2Y(1)-blocker can be derived because of the high-resolution and reproducibility of the channelyzer technique. In addition to the P2Y(1) purinoceptor, the P2Y(12)receptor appears to be involved in ADP-induced PSC since this process was significantly inhibited by AR-C69931MX. The channelyzer technique may be more reliable than optical aggregometry to assess PSC.     

Role of G protein-gated inwardly rectifying potassium channels in P2Y12 receptor-mediated platelet functional responses

The role of the G(i)-coupled platelet P2Y(12) receptor in platelet function has been well established. However, the functional effector or effectors contributing directly to alphaIIbbeta3 activation in human platelets has not been delineated. As the P2Y(12) receptor has been shown to activate G protein-gated, inwardly rectifying potassium (GIRK) channels, we investigated whether GIRK channels mediate any of the functional responses of the platelet P2Y(12) receptor. Western blot analysis revealed that platelets express GIRK1, GIRK2, and GIRK4. In aspirin-treated and washed human platelets, 2 structurally distinct GIRK inhibitors, SCH23390 (R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride) and U50488H (trans-(+/-)-3,4-dichloro-N-methyl-N-[2-(pyrrolidinyl)cyclohexyl] benzeneacetamide methanesulfonate), inhibited adenosine diphosphate (ADP)-, 2-methylthioADP (2-MeSADP)-, U46619-, and low-dose thrombin-mediated platelet aggregation. However, the GIRK channel inhibitors did not affect platelet aggregation induced by high concentrations of thrombin, AYPGKF, or convulxin. Furthermore, the GIRK channel inhibitors reversed SFLLRN-induced platelet aggregation, inhibited the P2Y(12)-mediated potentiation of dense granule secretion and Akt phosphorylation, and did not affect the agonist-induced G(q)-mediated platelet shape change and intracellular calcium mobilization. Unlike AR-C 69931MX, a P2Y(12) receptor-selective antagonist, the GIRK channel blockers did not affect the ADP-induced adenlylyl cyclase inhibition, indicating that they do not directly antagonize the P2Y(12) receptor. We conclude that GIRK channels are important functional effectors of the P2Y(12) receptor in human platelets.     

Identification of P2Y12-dependent and -independent mechanisms of glycoprotein VI-mediated Rap1 activation in platelets

Glycoprotein (GP) VI is a critical platelet collagen receptor, yet the steps involved in GPVI-mediated platelet activation remain incompletely understood. Because activation of Rap1, an abundant small guanosine triphosphatase (GTPase) in platelets, contributes to integrin alpha(IIb)beta(3) activation, we asked whether and how GPVI signaling activates Rap1 in platelets. Here we show that platelet Rap1 is robustly activated upon addition of convulxin, a GPVI-specific agonist. Using a reconstituted system in RBL-2H3 cells, we found that GPVI-mediated Rap1 activation is dependent on FcRgamma but independent of another platelet collagen receptor, alpha(2)beta(1). Interestingly, GPVI-mediated Rap1 activation in human platelets is largely dependent on adenosine diphosphate (ADP) signaling through the P2Y(12) and not the P2Y(1) receptor. However, experiments with specific ADP receptor antagonists and platelets from knockout mice deficient in P2Y(1) or the P2Y(12)-associated G-protein, Galphai(2), indicate that human and murine platelets also have a significant P2Y(12)-independent component of GPVI-mediated Rap1 activation. The P2Y(12)-independent component is dependent on phosphatidylinositol 3-kinase and is augmented by epinephrine-mediated signaling. P2Y(12)-dependent and -independent components are also observed in GPVI-mediated platelet aggregation, further supporting a role for Rap1 in aggregation. These results define mechanisms of GPVI-mediated platelet activation and implicate Rap1 as a key signaling protein in GPVI-induced platelet signaling.     

Immunolocalization of P2Y1 and TPalpha receptors in platelets showed a major pool associated with the membranes of alpha -granules and the open canalicular system

P2Y(1) and thromboxane-prostanoid-alpha (TPalpha) receptors on platelets belong to the G-protein-coupled 7-transmembrane domain family. They transmit signals for shape change, mobilization of calcium, and platelet aggregation. Immunogold labeling with a monoclonal antibody (MoAb) to the amino-terminal domain of P2Y(1) and a polyclonal antibody to the C-terminal domain of TPalpha revealed that while present at the platelet surface, both receptors were abundantly represented inside the platelet. Specifically, receptors were found in membranes of alpha-granules and elements of the open-canalicular system. A similar organization was found in mature megakaryocytes. Activation of platelets by adenosine diphosphate (ADP) and the thromboxane A(2) (TXA(2)) analog, I-BOP [1S-(1 alpha,2 beta(5Z),3 alpha-(1E,3S)4 alpha)-7-(3-(3- hydroxy-4-(p-iodophenoxy)-1-butenyl)-7-oxabicyclo(2.2.1)hept-2-yl)-5-heptenoic acid], increased the labeling of both P2Y(1) and TPalpha at the surface and in intracellular pools, suggesting that activation resulted in greater antibody accessibility to the receptor. A return to a platelet discoid shape and to basal values of labeling accompanied receptor desensitization. Platelets lacking the P2Y(12) ADP receptor normally expressed P2Y(1) and TPalpha, both before and after activation. Studies with the anti-ligand-induced binding site (anti-LIBS) MoAb, AP-6, confirmed that stored fibrinogen associated with internal pools of alpha(IIb)beta(3) at the start of secretion in a microenvironment containing agonist receptors. Pharmacologic antagonism of ADP or TXA(2) receptors in antithrombotic therapy may need to take into account blockade of internal receptor pools.     

Inactivation of the human P2Y12 receptor by thiol reagents requires interaction with both extracellular cysteine residues,Cys17 and Cys270

Human platelets express 2 G protein-coupled nucleotide receptors: the platelet adenosine diphosphate (ADP) receptor coupled to stimulation of phospholipase C (P2Y(1)) via heterotrimeric guanosine 5-triphosphate (GTP)-binding protein G(q), and the platelet ADP receptor coupled to inhibition of adenylyl cyclase (P2Y(12)) via heterotrimeric GTP-binding protein G(i). Although these 2 receptors are encoded on the same chromosome and have similar pharmacologic profiles, they have different reactivities toward thiol reagents. The thiol agent p-chloromercuribenzene sulfonic acid (pCMBS) and the active metabolites from antiplatelet drugs, clopidogrel and CS-747, inactivate the P2Y(12) receptor and are predicted to interact with the extracellular cysteine residues on the P2Y(12) receptor. In this study we identified the reactive cysteine residues on the human P2Y(12) receptor by site-directed mutagenesis using pCMBS as the thiol reagent. Cys97Ser and Cys175Ser mutants of the P2Y(12) receptor did not express when transfected into Chinese hamster ovary (CHO-K1) cells, indicating the essential nature of a disulfide bridge between these residues. The Cys17Ser, Cys270Ser, and Cys17Ser/Cys270Ser double mutants had similar median effective concentration (EC(50)) values for ADP and 2-methylthio-ADP (2-MeSADP) when compared with the wild-type P2Y(12). Similarly, the median inhibitory concentration (IC(50)) values for BzATP (2',3'-O-(4- benzoylbenzoyl) adenosine 5'-triphosphate), an antagonist of the P2Y(12) receptor, also did not differ dramatically among these mutants and the wild-type P2Y(12) receptor. pCMBS inactivated the wild-type P2Y(12) receptor in a concentration-dependent manner, whereas it had no effect on the P2Y(1) receptor. Finally, pCMBS partially affected the G(i) coupling of Cys17Ser or Cys270Ser receptor mutants, but had no effect on Cys17Ser/Cys270Ser P2Y(12) receptor-mediated inhibition of adenylyl cyclase. These results indicate that, unlike the P2Y(1) receptor, which has 2 essential disulfide bridges linking its extracellular domains, the P2Y(12) receptor has 2 free cysteines in its extracellular domains (Cys17 and Cys270), both of which are targets of thiol reagents. We speculate that the active metabolites of clopidogrel and CS-747 form disulfide bridges with both Cys17 and Cys270 in the P2Y(12) receptor, and thereby inactivate the receptor.     

Effects of P2Y 1 and P2Y 12 receptor antagonists on ADP-induced shape change of equine platelets: comparison with human platelets

Platelet activation by adenosine 5' -diphosphate (ADP) is via both P2Y 1 and P2Y 12 receptors and leads to shape change and aggregation. The effects on ADP-induced platelet shape change of two P2Y 1 antagonists, adenosine 3'-phosphate, 5'-phosphosulfate (A3P5PS) and 2-deoxy- N 6 -methyladenosine 3', 5'-diphosphate (MRS-2179) and a P2Y 12 antagonist 2-propylthio- D - g , n -dichloromethylene-adenosine 5'-triphosphate (AR-C67085MX) were determined by turbidimetric aggregometry and scanning electron microscopy (SEM) on equine and human platelets. The platelet aggregation was inhibited during aggregometry by 4-[4-[4(aminoiminomethyl)phenyl]-1-piperazinyl]-1-piperidin acid hydrochloride trihydrate (GR 144053F), an inhibitor of fibrinogen binding. From aggregation profiles, concentration-response curves and SEM we conclude that the shape change of equine platelets was susceptible to inhibition by the P2Y 1 antagonists A3P5PS and MRS-2179, but less so than human platelets. The P2Y 12 antagonist AR-C67085 did not influence significantly the shape change of either equine or human platelets.     

Central role for hydrogen peroxide in P2Y1 ADP receptor-mediated cellular responses in vascular endothelium

ADP activates a family of cell surface receptors that modulate signaling pathways in a broad range of cells. ADP receptor antagonists are widely used to treat cardiovascular disease states. These studies identify a critical role for the stable reactive oxygen species hydrogen peroxide (H₂O₂) in mediating cellular responses activated by the G protein-coupled P2Y1 receptor for ADP. We found that ADP-dependent phosphorylation of key endothelial signaling proteins—including endothelial nitric oxide synthase, AMP-activated protein kinase, and the actin-binding MARCKS protein—was blocked by preincubation with PEG-catalase, which degrades H₂O₂. ADP treatment promoted the H₂O₂-dependent phosphorylation of c-Abl, a nonreceptor tyrosine kinase that modulates the actin cytoskeleton. Cellular imaging experiments using fluorescence resonance energy transfer-based biosensors revealed that ADP-stimulated activation of the cytoskeleton-associated small GTPase Rac1 was independent of H₂O₂. However, Rac1-dependent activation of AMP-activated protein kinase, the signaling phospholipid phosphatidylinositol-(4, 5)-bisphosphate, and the c-Abl–interacting protein CrkII are mediated by H₂O₂. We transfected endothelial cells with differentially targeted HyPer2 H₂O₂ biosensors and found that ADP promoted a marked increase in H₂O₂ levels in the cytosol and caveolae, and a smaller increase in mitochondria. We performed a screen for P2Y1 receptor-mediated receptor tyrosine kinase transactivation and discovered that ADP transactivates Fms-like tyrosine kinase 3 (Flt3), a receptor tyrosine kinase expressed in these cells. Our observation that P2Y1 receptor-mediated responses involve Flt3 transactivation may identify a unique mechanism whereby cancer chemotherapy with receptor tyrosine kinase inhibitors promotes vascular dysfunction. Taken together, these findings establish a critical role for endogenous H₂O₂ in control of ADP-mediated signaling responses in the vascular wall.Beyond their established roles in intracellular energy flux and nucleic acid metabolism, purine nucleotides also serve as intercellular messenger molecules that regulate signal transduction pathways in a broad range of cells and tissues (1–3). The purine nucleotide ADP binds to G protein-coupled P2Y purinergic cell surface receptors, which are expressed in diverse mammalian cells, including blood platelets and vascular endothelial cells (reviewed in refs. 2 and 4). ADP is a critical determinant of platelet aggregation, blood vessel tone, and vascular wall integrity. Platelet granules contain high concentrations of ADP, which is released during platelet aggregation. The released ADP binds to P2Y12 and P2Y1 cell surface receptors for ADP on platelets and further potentiates platelet aggregation. P2Y receptor antagonists play a central role in cardiovascular therapeutics (2, 4): The P2Y12 blocker clopidogrel is one of the most commonly prescribed drugs in the United States, and other P2Y1 and P2Y12 blockers are being actively developed and tested for treatment of cardiovascular and cerebrovascular disease states. ADP also binds to P2Y1 receptors in vascular endothelial cells and rapidly activates endothelial nitric oxide synthase (eNOS) (5). Endothelium-generated nitric oxide (NO) inhibits platelet aggregation (3, 6) and provides an important feedback loop between endothelial cells and platelets that serves to attenuate the direct proaggregatory effects of ADP on platelets. ADP may also be released from the vascular endothelium and act in an autocrine or paracrine fashion to exert longer-term effects on vascular cell migration and barrier function (1, 3, 7–9). Clearly, a deeper understanding of P2Y receptor pharmacodynamics could inform current efforts in the development of novel purinergic antagonist drugs.Purinergic receptors for ADP can be classified by their structure and mode of action into two distinct receptor families, P2X and P2Y. P2X receptors are ligand-gated ion channels, whereas members of the P2Y receptor family are G protein-coupled receptors. ADP signaling pathways in platelets have been extensively characterized, yet the roles of ADP in the modulation of endothelial responses are less well understood. The current studies have focused on exploring the signaling pathways activated by P2Y1 receptors in vascular endothelial cells. We have shown (6) that ADP acts via P2Y1 receptors to activate the endothelial isoform of nitric oxide synthase (eNOS) in cultured endothelial cells and also modulates the activation of key signaling protein kinases including the AMP-activated protein kinase (AMPK). We also found that ADP promotes the P2Y1 ADP receptor-dependent endothelial cell migration through activation of the small GTPase Rac1 (6, 10). Discovering the involvement of Rac1 provided an important clue to the mechanisms whereby ADP exerts its influence on endothelial cell responses.Rac1 is an actin-binding cytoskeletal regulatory protein and is a member of the Rho GTPase protein family. The activation of eNOS by P2Y1 receptors for ADP depends on Rac1 (1, 6). Rac1 has been identified as a critical determinant of endothelial cell migration and barrier function, at least in part by modulating the levels of intracellular NO and hydrogen peroxide (H₂O₂) (11–15). H₂O₂ is a stable reactive oxygen species (ROS) that has been identified in recent years as a physiologically important intracellular messenger molecule (11–14), belying the classical concept of ROS functioning solely as deleterious molecules responsible for pathological states such as aging and neurodegeneration (14, 16). We reported (17, 18) that endogenous H₂O₂ regulates endothelial cell migration via dynamic signaling pathways involving the MARCKS protein, a ubiquitous phosphoprotein that translocates from the cell membrane to the actin cytoskeleton. The MARCKS protein also reversibly sequesters the signaling phospholipid phosphatidylinositol-(4, 5)-bisphosphate (PIP₂). PIP₂ is an important activator of proteins that initiate actin nucleation, including the phosphoprotein c-Abl, a nonreceptor tyrosine kinase that has been implicated in the dynamic cytoskeletal rearrangements that modulate endothelial barrier function. Endogenous H₂O₂ induces changes in cellular phospholipid metabolism via the phosphorylation and translocation of MARCKS in endothelial cells, yet the connections between receptor activation and intracellular modulation of H₂O₂ levels are incompletely understood.The roles of H₂O₂ as a physiological intracellular messenger molecule were initially discovered through studies of growth factor-dependent activation of their cognate receptor tyrosine kinases (19, 20), which then signal to redox-regulated phosphoprotein phosphatases via H₂O₂ (20). In contrast to the widespread involvement of H₂O₂ in receptor tyrosine kinase signaling, only a handful of G protein-coupled receptors have been shown to directly modulate H₂O₂ levels (11, 16, 21, 22). Indeed, the roles of H₂O₂ in modulation of physiological responses have not yet been clearly defined for G protein-coupled receptors. In these studies, we present observations that establish that the G protein-coupled P2Y1 receptor for ADP modulates key H₂O₂-dependent signaling responses in the vascular endothelium via transactivation of the receptor tyrosine kinase Flt3.     

The plaque lipid lysophosphatidic acid stimulates platelet activation and platelet-monocyte aggregate formation in whole blood: involvement of P2Y1 and P2Y12 receptors

Despite the fact that lysophosphatidic acid (LPA) has been identified as a main platelet-activating lipid of mildly oxidized low-density lipoprotein (LDL) and human atherosclerotic lesions, it remains unknown whether it is capable of activating platelets in blood. We found that LPA at concentrations slightly above plasma levels induces platelet shape change, aggregation, and platelet-monocyte aggregate formation in blood. 1-alkyl-LPA (16:0 fatty acid) was almost 20-fold more potent than 1-acyl-LPA (16:0). LPA directly induced platelet shape change in blood and platelet-rich plasma obtained from all blood donors. However, LPA-stimulated platelet aggregation in blood was donor dependent. It could be completely blocked by apyrase and antagonists of the platelet adenosine diphosphate (ADP) receptors P2Y1 and P2Y12. These substances also inhibited LPA-induced aggregation of platelet-rich plasma and aggregation and serotonin secretion of washed platelets. These results indicate a central role for ADP-mediated P2Y1 and P2Y12 receptor activation in supporting LPA-induced platelet aggregation. Platelet aggregation and platelet-monocyte aggregate formation stimulated by LPA was insensitive to inhibition by aspirin. We conclude that LPA at concentrations approaching those found in vivo can induce platelet shape change, aggregation, and platelet-monocyte aggregate formation in whole blood and suggest that antagonists of platelet P2Y1 and P2Y12 receptors might be useful preventing LPA-elicited thrombus formation in patients with cardiovascular diseases.     

Effects of P2Y(1) and P2Y(12) receptor antagonists on ADP-induced shape change of equine platelets: comparison with human platelets

Platelet activation by adenosine 5' -diphosphate (ADP) is via both P2Y(1 )and P2Y(12) receptors and leads to shape change and aggregation. The effects on ADP-induced platelet shape change of two P2Y(1) antagonists, adenosine 3'-phosphate, 5'-phosphosulfate (A3P5PS) and 2-deoxy-N(6)-methyladenosine 3', 5'-diphosphate (MRS-2179) and a P2Y(12) antagonist 2-propylthio-D-beta,gamma-dichloromethylene-adenosine 5'-triphosphate (AR-C67085MX) were determined by turbidimetric aggregometry and scanning electron microscopy (SEM) on equine and human platelets. The platelet aggregation was inhibited during aggregometry by 4-[4-[4(aminoiminomethyl)phenyl]-1-piperazinyl]-1-piperidin acid hydrochloride trihydrate (GR 144053F), an inhibitor of fibrinogen binding. From aggregation profiles, concentration-response curves and SEM we conclude that the shape change of equine platelets was susceptible to inhibition by the P2Y(1) antagonists A3P5PS and MRS-2179, but less so than human platelets. The P2Y(12) antagonist AR-C67085 did not influence significantly the shape change of either equine or human platelets.     

The P2Y1 receptor is essential for ADP-induced shape change and aggregation in mouse platelets

Adenosine diphosphate (ADP) is an important platelet agonist, causing the shape change and aggregation required for physiological hemostasis. We have recently demonstrated that the P2Y1 receptor plays an important role in ADP-induced shape change and aggregation in human platelets. The role of the P2Y1 receptor in these physiological responses can be conclusively delineated with gene-knockout approaches in transgenic mice. However, before proceeding to the P2Y1 gene-knockout mice generation, it is important to demonstrate that the P2Y1 receptor plays an essential role in ADP-induced shape change and aggregation in mouse platelets. We examined platelets pooled from twenty 129J mice, a strain used in the generation of knockout mice. Immunofluorescence experiments using P2Y1 specific antiserum detected the presence of the P2Y1 receptor on mouse platelets. ARL 66096, a potent P2T(AC) receptor antagonist, caused a dose-dependent inhibition of both ADP-induced aggregation and ADP-induced inhibition of adenylyl cyclase, without affecting shape change or calcium mobilization. On the other hand, adenosine-2'-phosphate-5'-phosphate (A2P5P), a P2Y1 receptor-selective antagonist, caused a dose-dependent inhibition of ADP-induced aggregation and shape change, as well as inhibiting the mobilization of calcium from intracellular stores. A2P5P had no effect on the inhibition of adenylyl cyclase by ADP. These findings clearly demonstrate the existence of two distinct ADP receptors, the P2Y1 and P2T(AC), in mouse platelets with similar function as in human platelets.     

Specific impairment of human platelet P2Y(AC) ADP receptor-mediated signaling by the antiplatelet drug clopidogrel.

Clopidogrel is an effective new antiplatelet agent useful for the treatment of ischemic cerebrovascular, cardiac, and peripheral arterial disease. However, the mechanism of clopidogrel action is not well understood, although it is known to inhibit ADP-evoked platelet aggregation. In the current study, the effect of clopidogrel on recently identified human platelet ADP receptors and their signaling pathways was investigated by using platelets from clopidogrel-treated subjects, 6 healthy volunteers (2 females and 4 males) who received 75 mg of clopidogrel daily for 7 days. Blood was taken and various platelet receptor signaling pathways were analyzed before treatment, after 7 days of medication, and 4 weeks after treatment had ceased. Platelet tests included the analysis of aggregation, rapid calcium influx, calcium mobilization from intracellular stores, adenylyl cyclase, and phosphorylation of vasodilator-stimulated phosphoprotein (VASP). The data indicate that clopidogrel does not affect those platelet ADP receptors coupled to cation influx (P2X1 ADP receptors) or calcium mobilization (P2Y1 ADP receptors). In contrast, clopidogrel treatment specifically impairs the ADP receptor coupled to G(i)/adenylyl cyclase (P2Y(AC) ADP receptors). Clopidogrel abolishes the inhibitory P2Y(AC) receptor-mediated ADP effects on prostaglandin E(1)-stimulated, cAMP-dependent phosphorylation of VASP without affecting epinephrine, thrombin, and thromboxane signaling. VASP phosphorylation is known to be closely correlated with the inhibition of platelet and fibrinogen receptor (glycoprotein IIb/IIIa) activation. Therefore, inhibition of the platelet P2Y(AC) ADP receptor and its intracellular signaling, including decreased VASP phosphorylation, is suggested as a molecular mechanism of clopidogrel action.     

The P2Y1 receptor is essential for ADP-induced shape change and aggregation in mouse platelets

Adenosine diphosphate (ADP) is an important platelet agonist, causing the shape change and aggregation required for physiological hemostasis. We have recently demonstrated that the P2Y1 receptor plays an important role in ADP-induced shape change and aggregation in human platelets. The role of the P2Y1 receptor in these physiological responses can be conclusively delineated with gene-knockout approaches in transgenic mice. However, before proceeding to the P2Y1 gene-knockout mice generation, it is important to demonstrate that the P2Y1 receptor plays an essential role in ADP-induced shape change and aggregation in mouse platelets. We examined platelets pooled from twenty 129J mice, a strain used in the generation of knockout mice. Immunofluorescence experiments using P2Y1 specific antiserum detected the presence of the P2Y1 receptor on mouse platelets. ARL 66096, a potent P2T AC receptor antagonist, caused a dose-dependent inhibition of both ADP-induced aggregation and ADP-induced inhibition of adenylyl cyclase, without affecting shape change or calcium mobilization. On the other hand, adenosine-2'-phosphate-5'-phosphate (A2P5P), a P2Y1 receptorselective antagonist, caused a dose-dependent inhibition of ADP-induced aggregation and shape change, as well as inhibiting the mobilization of calcium from intracellular stores. A2P5P had no effect on the inhibition of adenylyl cyclase by ADP. These findings clearly demonstrate the existence of two distinct ADP receptors, the P2Y1 and P2T AC , in mouse platelets with similar function as in human platelets.     

The platelet P2 receptors in thrombosis

Adenosine diphosphate (ADP) and adenosine triphosphate (ATP) play a crucial role in hemostasis and thrombosis, and their receptors are potential targets for antithrombotic drugs. The ATP-gated channel P2X (1) and the two G protein-coupled P2Y (1) and P2Y (12) ADP receptors selectively contribute to platelet aggregation. Because of its central role in the formation and stabilization of a thrombus, the P2Y (12) receptor is a well-established target of antithrombotic drugs such as clopidogrel, which has proven efficacy in many clinical trials and experimental models of thrombosis. Competitive P2Y (12) antagonists have also been shown to be effective in experimental thrombosis as well as in several clinical trials. Studies in P2Y (1) and P2X (1) knockout mice and experimental thrombosis models using selective P2Y (1) and P2X (1) antagonists have shown that, depending on the conditions, these receptors could also be potential targets for new antithrombotic drugs. Because both P2X (1) and P2Y (1) receptor inhibition result in milder prolongation of the bleeding time as compared with P2Y (12) inhibition, the idea is put forward that combinations of P2 receptor antagonists could improve efficacy with diminished hemorrhagic risk. However, further studies with stronger and more selective P2 receptor antagonists are required to validate such a point of view.     

Role of ADP receptor P2Y(12) in platelet adhesion and thrombus formation in flowing blood

ADP plays a central role in regulating platelet function. It induces platelet aggregation via the activation of 2 major ADP receptors, P2Y(1) and P2Y(12). We have investigated the role of P2Y(12) in platelet adhesion and thrombus formation under physiological flow by using blood from a patient with a defect in the gene encoding P2Y(12). Anticoagulated blood from the patient and from healthy volunteers was perfused over collagen-coated coverslips. The patient's thrombi were smaller and consisted of spread platelets overlying platelets that were not spread, whereas control thrombi were large and densely packed. Identical platelet surface coverage, aggregate size, and morphology were found when a P2Y(12) antagonist, N(6)-(2-methylthioethyl)-2-(3,3,3-trifluoropropylthio)-beta,gamma-dichloromethylene ATP (also known as AR-C69931 MX), was added to control blood. The addition of a P2Y(1) antagonist (adenosine-3',5'-diphospate) to control blood resulted in small, but normally structured, thrombi. Thus, the ADP-P2Y(12) interaction is essential for normal thrombus buildup on collagen. The patient's blood also showed reduced platelet adhesion on fibrinogen, which was not due to changes in morphology. Comparable results were found by using control blood with AR-C69931 MX and also with adenosine-3',5'-diphospate. This suggested that P2Y(12) and P2Y(1) were both involved in platelet adhesion on immobilized fibrinogen, thereby revealing it as ADP dependent. This was confirmed by complete inhibition on the addition of creatine phosphate/creatine phosphokinase.