The P2Y12 Antagonists, 2-Methylthioadenosine 5��-Monophosphate Triethylammonium Salt and Cangrelor (ARC69931MX), Can Inhibit Human Platelet Aggregation through a Gi-independent Increase in cAMP Levels

ADP plays an integral role in the process of hemostasis by signaling through two platelet G-protein-coupled receptors, P2Y₁ and P2Y₁₂. The recent use of antagonists against these two receptors has contributed a substantial body of data characterizing the ADP signaling pathways in human platelets. Specifically, the results have indicated that although P2Y₁ receptors are involved in the initiation of platelet aggregation, P2Y₁₂ receptor activation appears to account for the bulk of the ADP-mediated effects. Based on this consideration, emphasis has been placed on the development of a new class of P2Y₁₂ antagonists (separate from clopidogrel and ticlopidine) as an approach to the treatment of thromboembolic disorders. The present work examined the molecular mechanisms by which two of these widely used adenosine-based P2Y₁₂ antagonists (2-methylthioadenosine 5′-monophosphate triethylammonium salt (2MeSAMP) and ARC69931MX), inhibit human platelet activation. It was found that both of these compounds raise platelet cAMP to levels that substantially inhibit platelet aggregation. Furthermore, the results demonstrated that this elevation of cAMP did not require G_i signaling or functional P2Y₁₂ receptors but was mediated through activation of a separate G protein-coupled pathway, presumably involving G_s. However, additional experiments revealed that neither 2MeSAMP nor ARC69931MX (cangrelor) increased cAMP through activation of A2a, IP, DP, or EP₂ receptors, which are known to couple to G_s. Collectively, these findings indicate that 2MeSAMP and ARC69931MX interact with an unidentified platelet G protein-coupled receptor that stimulates cAMP-mediated inhibition of platelet function. This inhibition is in addition to that derived from antagonism of P2Y₁₂ receptors.Upon damage to the endothelial layer of the blood vessel wall, the underlying subendothelium is exposed to platelets in the blood, initiating a cascade of signaling events resulting in the transformation of “resting” platelets into “activated” platelets (1). One significant characteristic associated with these signaling events is the secretion of ADP from the platelet-dense granules (2). This released ADP acts to further amplify the platelet activation response by interacting with its G-protein-coupled receptors on the platelet surface, namely P2Y₁ (coupled to G_q) and P2Y₁₂ (coupled to G_i) (3–5). The consequence of platelet activation through ADP is a conformational change in the platelet membrane glycoprotein αIIbβ3 (6, 7), which then binds to fibrinogen present in the plasma. The binding of fibrinogen with αIIbβ3 on the surface of adjacent platelets results in fibrinogen-platelet cross-linking and the formation of a hemostatic plug at the site of vascular injury (8).Consequently, ADP is thought to play an integral role in the normal process of hemostasis. Of the two ADP-receptor signaling pathways in platelets, evidence has indicated that ADP-mediated P2Y₁₂ signaling appears to play a more prominent role in platelet activation than ADP-mediated P2Y₁ signaling (9, 10). For the most part, support for this notion derives from the use of the adenosine-based P2Y₁₂ antagonists (i.e. 2MeSAMP⁴ and ARC69931MX), which have a much broader inhibitory profile than P2Y₁ antagonists (e.g. A3P5P (adenosine-3′-phosphate-5′-phosphate) or MRS2179) (9). Thus, 2MeSAMP and ARC69931MX inhibit platelet aggregation in response to multiple agonists, such as thromboxane A₂, collagen, thrombin, etc. (11–13), whereas P2Y₁ antagonists do not. On the other hand, this general requirement for P2Y₁₂ signaling seems to be inconsistent with earlier reports indicating that activation of certain platelet receptors (e.g. thromboxane A₂ receptor) can cause aggregation through ADP-independent mechanisms (14, 15). Based on this apparent inconsistency in the contribution of P2Y₁₂ signaling to the overall platelet activation response, the present study investigated the possibility that the broad spectrum of inhibitory activity of this new generation of P2Y₁₂ antagonists (i.e. MeSAMP and ARC69931MX) may derive from an elevation in platelet cAMP levels.Our data demonstrated that both 2MeSAMP and ARC69931MX do in fact significantly raise human platelet cAMP. Furthermore, this pharmacological effect is independent of P2Y₁₂-G_i signaling and appears to proceed through activation of a separate G_s-coupled platelet receptor. Taken together, the results therefore indicate that these adenosine-based P2Y₁₂ antagonists can produce their inhibition of platelet function through a cAMP-mediated mechanism.     

Off-target effect of the Epac agonist 8-pCPT-2′-O-Me-cAMP on P2Y12 receptors in blood platelets

The primary target of the cAMP analogue 8-pCPT-2′-O-Me-cAMP is exchange protein directly activated by cAMP (Epac). Here we tested potential off-target effects of the Epac activator on blood platelet activation signalling. We found that the Epac analogue 8-pCPT-2′-O-Me-cAMP inhibits agonist-induced-GPCR-stimulated, but not collagen-stimulated, P-selectin surface expression on Epac1 deficient platelets. In human platelets, 8-pCPT-2′-O-Me-cAMP inhibited P-selectin expression elicited by the PKC activator PMA. This effect was abolished in the presence of the extracellular ADP scavenger system CP/CPK. In silico modelling of 8-pCPT-2′O-Me-cAMP binding into the purinergic platelet receptor P2Y₁₂ revealed that the analogue docks similar to the P2Y₁₂ antagonist 2MeSAMP. The 8-pCPT-2′-O-Me-cAMP analogue per se, did not provoke Rap 1 (Rap 1-GTP) activation or phosphorylation on the vasodilator-stimulated phosphoprotein (VASP) at Ser-157. In addition, the protein kinase A (PKA) antagonists Rp-cAMPS and Rp-8-Br-cAMPS failed to block the inhibitory effect of 8-pCPT-2′-O-Me-cAMP on thrombin- and TRAP-induced Rap 1 activation, thus suggesting that PKA is not involved. We conclude that the 8-pCPT-2′-O-Me-cAMP analogue is able to inhibit agonist-induced-GPCR-stimulated P-selectin independent from Epac1; the off-target effect of the analogue appears to be mediated by antagonistic P2Y₁₂ receptor binding. This has implications when using cAMP analogues on specialised system involving such receptors. We found, however that the Epac agonist 8-Br-2′-O-Me-cAMP did not affect platelet activation at similar concentrations.     

Stabilizing Role of Platelet P2Y12 Receptors in Shear-Dependent Thrombus Formation on Ruptured Plaques

Background

In most models of experimental thrombosis, healthy blood vessels are damaged. This results in the formation of a platelet thrombus that is stabilized by ADP signaling via P2Y₁₂ receptors. However, such models do not predict involvement of P2Y₁₂ in the clinically relevant situation of thrombosis upon rupture of atherosclerotic plaques. We investigated the role of P2Y₁₂ in thrombus formation on (collagen-containing) atherosclerotic plaques in vitro and in vivo, by using a novel mouse model of atherothrombosis.

Methodology

Plaques in the carotid arteries from Apoe ^−/− mice were acutely ruptured by ultrasound treatment, and the thrombotic process was monitored via intravital fluorescence microscopy. Thrombus formation in vitro was assessed in mouse and human blood perfused over collagen or plaque material under variable conditions of shear rate and coagulation. Effects of two reversible P2Y₁₂ blockers, ticagrelor (AZD6140) and cangrelor (AR-C69931MX), were investigated.

Principal Findings

Acute plaque rupture by ultrasound treatment provoked rapid formation of non-occlusive thrombi, which were smaller in size and unstable in the presence of P2Y₁₂ blockers. In vitro, when mouse or human blood was perfused over collagen or atherosclerotic plaque material, blockage or deficiency of P2Y₁₂ reduced the thrombi and increased embolization events. These P2Y₁₂ effects were present at shear rates >500 s⁻¹, and they persisted in the presence of coagulation. P2Y₁₂-dependent thrombus stabilization was accompanied by increased fibrin(ogen) binding.

Conclusions/Significance

Platelet P2Y₁₂ receptors play a crucial role in the stabilization of thrombi formed on atherosclerotic plaques. This P2Y₁₂ function is restricted to high shear flow conditions, and is preserved in the presence of coagulation.     

The Ratio of ADP- to TRAP-Induced Platelet Aggregation Quantifies P2Y12-Dependent Platelet Inhibition Independently of the Platelet Count

Objective

This study aimed to assess the association of clinical factors with P2Y₁₂-dependent platelet inhibition as monitored by the ratio of ADP- to TRAP-induced platelet aggregation and conventional ADP-induced aggregation, respectively.

Background

Controversial findings to identify and overcome high platelet reactivity (HPR) after coronary stent-implantation and to improve clinical outcome by tailored anti-platelet therapy exist. Monitoring anti-platelet therapy ex vivo underlies several confounding parameters causing that ex vivo platelet aggregation might not reflect in vivo platelet inhibition.

Methods

In a single centre observational study, multiple electrode aggregometry was performed in whole blood of patients after recent coronary stent-implantation. Relative ADP-induced aggregation (r-ADP-agg) was defined as the ratio of ADP- to TRAP- induced aggregation reflecting the individual degree of P2Y₁₂-mediated platelet reactivity.

Results

Platelet aggregation was assessed in 359 patients. Means (± SD) of TRAP-, ADP-induced aggregation and r-ADP-agg were 794 ± 239 AU*min, 297 ± 153 AU*min and 37 ± 14%, respectively. While ADP- and TRAP-induced platelet aggregation correlated significantly with platelet count (ADP: r = 0.302; p<0.001; TRAP: r = 0.509 p<0.001), r-ADP-agg values did not (r = -0.003; p = 0.960). These findings were unaltered in multivariate analyses adjusting for a range of factors potentially influencing platelet aggregation. The presence of an acute coronary syndrome and body weight were found to correlate with both ADP-induced platelet aggregation and r-ADP-agg.

Conclusion

The ratio of ADP- to TRAP-induced platelet aggregation quantifies P2Y₁₂-dependent platelet inhibition independently of the platelet count in contrast to conventional ADP-induced aggregation. Furthermore, r-ADP-agg was associated with the presence of an acute coronary syndrome and body weight as well as ADP-induced aggregation. Thus, the r-ADP-agg is a more valid reflecting platelet aggregation and potentially prognosis after coronary stent-implantation in P2Y₁₂-mediated HPR than conventional ADP-induced platelet aggregation.     

The Gi-coupled P2Y12 receptor regulates diacylglycerol-mediated signaling in human platelets

Stimulation of G(q)-coupled receptors activates phospholipase C and is supposed to promote both intracellular Ca(2+) mobilization and protein kinase C (PKC) activation. We found that ADP-induced phosphorylation of pleckstrin, the main platelet substrate for PKC, was completely inhibited not only by an antagonist of the G(q)-coupled P2Y1 receptor but also upon blockade of the G(i)-coupled P2Y12 receptor. The role of G(i) on PKC regulation required stimulation of phosphatidylinositol 3-kinase rather than inhibition of adenylyl cyclase. P2Y12 antagonists also inhibited pleckstrin phosphorylation, Rap1b activation, and platelet aggregation induced upon G(q) stimulation by the thromboxane A(2) analogue U46619. Importantly, activation of phospholipase C and intracellular Ca(2+) mobilization occurred normally. Phorbol 12-myristate 13-acetate overcame the inhibitory effect of P2Y12 receptor blockade on PKC activation but not on Rap1b activation and platelet aggregation. By contrast, inhibition of diacylglycerol kinase restored both PKC and Rap1b activity and caused platelet aggregation. Stimulation of P2Y12 receptor or direct inhibition of diacylglycerol kinase potentiated the effect of membrane-permeable sn-1,2-dioctanoylglycerol on platelet aggregation and pleckstrin phosphorylation, in association with inhibition of its phosphorylation to phosphatidic acid. These results reveal a novel and unexpected role of the G(i)-coupled P2Y12 receptor in the regulation of diacylglycerol-mediated events in activated platelets.     

P2 receptors and platelet function

Following vessel wall injury, platelets adhere to the exposed subendothelium, become activated and release mediators such as TXA₂ and nucleotides stored at very high concentration in the so-called dense granules. Released nucleotides and other soluble agents 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 ADP receptors, namely the P2Y₁ and P2Y₁₂ receptor subtypes, while the P2X₁ receptor ligand-gated cation channel is activated by ATP. The P2Y₁ receptor initiates platelet aggregation but is not sufficient for a full platelet aggregation in response to ADP, while the P2Y₁₂ receptor is responsible for completion of the aggregation to ADP. The latter receptor, the molecular target of the antithrombotic drugs clopidogrel, prasugrel and ticagrelor, is responsible for most of the potentiating effects of ADP when platelets are stimulated by agents such as thrombin, collagen or immune complexes. The P2X₁ 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 the sequential events involved in platelet function and haemostasis. As such, they represent potential targets for antithrombotic drugs.     

Comparison of a New P2Y12 Receptor Specific Platelet Aggregation Test with Other Laboratory Methods in Stroke Patients on Clopidogrel Monotherapy

Background

Clinical studies suggest that 10-50% of patients are resistant to clopidogrel therapy. ADP induced platelet aggregation, a widely used test to monitor clopidogrel therapy, is affected by aspirin and is not specific for the P2Y12 receptor inhibited by clopidogrel.

Objectives

To develop a P2Y12-specific platelet aggregation test and to compare it with other methods used for monitoring clopidogrel therapy.

Patients/Methods

Study population included 111 patients with the history of ischemic stroke being on clopidogrel monotherapy and 140 controls. The effect of clopidogrel was tested by a newly developed ADP(PGE1) aggregation test in which prostaglandin E1 treated platelets are used. Results of conventional ADP induced platelet aggregation, VerifyNow P2Y12 assay and ADP(PGE1) aggregation were compared to those obtained by flow cytometric analysis of vasodilator stimulated phosphoprotein (VASP) phosphorylation. Reference intervals for all assays were determined according to the guidelines of Clinical Laboratory Standards Institute.

Results

The P2Y12-specificity of ADP(PGE1) test was proven by comparing it with ADP aggregation in the presence of P2Y1 antagonist, adenosine 3’, 5’-diphosphate. The method was not influenced by aspirin treatment. Approximately 50% of patients were clopidogrel resistant by conventional ADP aggregation and VerifyNow tests. The ADP(PGE1) method and the VASP phosphorylation assay identified 25.9% and 11.7% of patients as non-responders, respectively. ADP(PGE1) aggregation showed good correlation with VASP phosphorylation and had high diagnostic efficiency.

Conclusion

The new ADP(PGE1) method is a reliable test for monitoring P2Y12 receptor inhibition by platelet aggregation. As a subset of patients are non-responders, monitoring clopidogrel therapy by adequate methods is essential.     

Differential Endosomal Sorting of a Novel P2Y12 Purinoreceptor Mutant

P2Y₁₂ receptor internalization and recycling play an essential role in ADP‐induced platelet activation. Recently, we identified a patient with a mild bleeding disorder carrying a heterozygous mutation of P2Y₁₂ (P341A) whose P2Y₁₂ receptor recycling was significantly compromised. Using human cell line models, we identified key proteins regulating wild‐type (WT) P2Y₁₂ recycling and investigated P2Y₁₂‐P341A receptor traffic. Treatment with ADP resulted in delayed Rab5‐dependent internalization of P341A when compared with WT P2Y₁₂. While WT P2Y₁₂ rapidly recycled back to the membrane via Rab4 and Rab11 recycling pathways, limited P341A recycling was observed, which relied upon Rab11 activity. Although minimal receptor degradation was evident, P341A was localized in Rab7‐positive endosomes with considerable agonist‐dependent accumulation in the trans‐Golgi network (TGN). Rab7 activity is known to facilitate recruitment of retromer complex proteins to endosomes to transport cargo to the TGN. Here, we identified that P341A colocalized with Vps26; depletion of which blocked limited recycling and promoted receptor degradation. This study has identified key points of divergence in the endocytic traffic of P341A versus WT‐P2Y₁₂. Given that these pathways are retained in human platelets, this research helps define the molecular mechanisms regulating P2Y₁₂ receptor traffic and explain the compromised receptor function in the platelets of the P2Y₁₂‐P341A‐expressing patient.     

Characterisation of P2Y12 Receptor Responsiveness to Cysteinyl Leukotrienes

Leukotriene E₄ (LTE₄), the most stable of the cysteinyl leukotrienes (cysLTs), binds poorly to classical type 1 and 2 cysLT receptors although in asthmatic individuals it may potently induce bronchial constriction, airway hyperresponsiveness and inflammatory cell influx to the lung. A recent study has suggested that the purinergic receptor P2Y₁₂ is required for LTE₄ mediated pulmonary inflammation in a mouse model of asthma and signals in response to cysLTs. The aim of the study was to characterise the responsiveness of human P2Y₁₂ to cysteinyl leukotrienes. Models of human CysLT₁, CysLT₂ and P2Y₁₂ overexpressed in HEK293, CHO cells and human platelets were used and responsiveness to different agonists was measured using intracellular calcium, cAMP and β-arrestin recruitment assays. CysLTs induced concentration dependent calcium mobilisation in cells overexpressing CysLT₁ and CysLT₂ but failed to induce any calcium response in cells expressing P2Y₁₂ or P2Y₁₂+ Gα₁₆. In contrast, selective P2Y₁₂ agonists ADP and 2-MeS-ADP induced specific calcium flux in cells expressing P2Y₁₂+ Gα₁₆. Similarly, specific response to 2-MeS-ADP, but not to cysLTs was also observed in cells expressing P2Y₁₂ when intracellular cAMP and β-arrestin signalling were analysed. Platelets were used as a model of human primary cells expressing P2Y₁₂ to analyse potential signalling and cell activation through P2Y₁₂ receptor or receptor heterodimers but no specific LTE₄ responses were observed. These results show that LTE₄ as well as other cysLTs do not activate intracellular signalling acting through P2Y₁₂ and suggest that another LTE₄ specific receptor has yet to be identified.     

Functional interaction between purinergic receptors: effect of ligands for A2A and P2Y12 receptors on P2Y1 receptor function

A_2A adenosine receptor (A_2AR), P2Y₁ receptor (P2Y₁R) and P2Y₁₂ receptor (P2Y₁₂R) are predominantly expressed on human platelets. The individual role of each of these receptors in platelet aggregation has been actively reported. Previously, hetero-oligomerization between these three receptors has been shown to occur. Here, we show that Ca²⁺ signaling evoked by the P2Y₁R agonist, 2-methylthioladenosine 5’ diphosphate (2MeSADP) was significantly inhibited by the A_2AR antagonist (ZM241385 and SCH442416) and the P2Y₁₂R antagonist (ARC69931MX) using HEK293T cells expressing the three receptors. It was confirmed that inhibition of P2Y₁R signaling by A_2AR and P2Y₁₂R antagonists was indeed mediated through A_2AR and P2Y₁₂R using 1321N1 human astrocytoma cells which do not express P2Y receptors. We expect that intermolecular signal transduction and specific conformational changes occur among components of hetero-oligomers formed by these three receptors.     

Thienopyrimidine-based P2Y12 platelet aggregation inhibitors

Herein we describe the design and synthesis of a novel series of potent thienopyrimidine P2Y₁₂ inhibitors and the negative impact protein binding has on the inhibition of platelet aggregation.     

6-Amino-2-mercapto-3H-pyrimidin-4-one derivatives as new candidates for the antagonism at the P2Y12 receptors

P2Y₁₂ plays an important role in platelet aggregation, which makes it an interesting target for antithrombotic agents. Compounds that antagonize P2Y₁₂ include the active metabolites of thienopyridines and molecules that are structurally related to ATP, which is an antagonist of P2Y₁₂. During the last few years, our group has been working on the development of P2Y₁₂ receptors antagonists that are based on an extremely simple chemical structure, the 6-amino-2-mercapto-3H-pyrimidin-4-one, variously substituted at the sulfur and oxygen functions. This nucleus represents the simplified combination of two known P2Y₁₂ antagonists: the active metabolite of the thienopyridines and ATP derivatives. The effects of the synthesized compounds were tested on ADP-induced human platelet aggregation, using light transmission aggregometry. None of the tested compounds induced platelet aggregation, while some of them, at concentration of 10^?4 M, partially inhibited platelet aggregation induced by ADP 10^?6 M. The most potent compound, 6b, antagonized the inhibitory effect of 2-methylthio-ADP on the forskolin-induced accumulation of cyclic-AMP in CHO FlpIN cells expressing recombinant human P2Y₁₂-receptors. In addition, none of the tested compounds, including 6b, interfered with ligand binding to P1 receptors. Our results suggest that some of the synthesized compounds are specific antagonists of P2 receptors, and in particular of P2Y₁₂ and suggest that further development of this structurally new series of compounds as P2Y₁₂ receptors antagonists is recommended.     

Antiplatelet Activity,P2Y1 and P2Y12 Inhibition,and Metabolism in Plasma of Stereoisomers of Diadenosine 5′,5′″-P1,P4-dithio-P2,P3-chloromethylenetetraphosphate

Background

Diadenosine tetraphosphate (Ap₄A), a constituent of platelet dense granules, and its P¹,P⁴-dithio and/or P²,P³-chloromethylene analogs, inhibit adenosine diphosphate (ADP)-induced platelet aggregation. We recently reported that these compounds antagonize both platelet ADP receptors, P2Y₁ and P2Y₁₂. The most active of those analogs, diadenosine 5′,5″″-P¹,P⁴-dithio-P²,P³-chloromethylenetetraphosphate, (compound 1), exists as a mixture of 4 stereoisomers.

Objective

To separate the stereoisomers of compound 1 and determine their effects on platelet aggregation, platelet P2Y₁ and P2Y₁₂ receptor antagonism, and their metabolism in human plasma.

Methods

We separated the 4 diastereomers of compound 1 by preparative reversed-phase chromatography, and studied their effect on ADP-induced platelet aggregation, P2Y₁-mediated changes in cytosolic Ca²⁺, P2Y₁₂-mediated changes in VASP phosphorylation, and metabolism in human plasma.

Results

The inhibition of ADP-induced human platelet aggregation and human platelet P2Y₁₂ receptor, and stability in human plasma strongly depended on the stereo-configuration of the chiral P¹- and P⁴-phosphorothioate groups, the S_PS_P diastereomer being the most potent inhibitor and completely resistant to degradation in plasma, and the R_PR_P diastereomer being the least potent inhibitor and with the lowest plasma stability. The inhibitory activity of S_PR_P diastereomers depended on the configuration of the pseudo-asymmetric carbon of the P²,P³-chloromethylene group, one of the configurations being significantly more active than the other. Their plasma stability did not differ significantly, being intermediate to that of the S_PS_P and the R_PR_P diastereomers.

Conclusions

The presently-described stereoisomers have utility for structural, mechanistic, and drug development studies of dual antagonists of platelet P2Y₁ and P2Y₁₂ receptors.     

Another “String to the Bow” of PJ34, a Potent Poly(ADP-Ribose)Polymerase Inhibitor: An Antiplatelet Effect through P2Y12 Antagonism?

Background

Neuro- and vasoprotective effects of poly(ADP-ribose)polymerase (PARP) inhibition have been largely documented in models of cerebral ischemia, particularly with the potent PARP inhibitor PJ34. Furthermore, after ischemic stroke, physicians are faced with incomplete tissue reperfusion and reocclusion, in which platelet activation/aggregation plays a key role. Data suggest that certain PARP inhibitors could act as antiplatelet agents. In that context, the present in vitro study investigated on human blood the potential antiplatelet effect of PJ34 and two structurally different PARP inhibitors, DPQ and INO-1001.

Methods and results

ADP concentrations were chosen to induce a biphasic aggregation curve resulting from the successive activation of both its receptors P2Y₁ and P2Y₁₂. In these experimental conditions, PJ34 inhibited the second phase of aggregation; this effect was reduced by incremental ADP concentrations. In addition, in line with a P2Y₁₂ pathway inhibitory effect, PJ34 inhibited the dephosphorylation of the vasodilator stimulated phosphoprotein (VASP) in a concentration-dependent manner. Besides, PJ34 had no effect on platelet aggregation induced by collagen or PAR1 activating peptide, used at concentrations inducing a strong activation independent on secreted ADP. By contrast, DPQ and INO-1001 were devoid of any effect whatever the platelet agonist used.

Conclusions

We showed that, in addition to its already demonstrated beneficial effects in in vivo models of cerebral ischemia, the potent PARP inhibitor PJ34 exerts in vitro an antiplatelet effect. Moreover, this is the first study to report that PJ34 could act via a competitive P2Y₁₂ antagonism. Thus, this antiplatelet effect could improve post-stroke reperfusion and/or prevent reocclusion, which reinforces the interest of this drug for stroke treatment.     

Decreased platelet aggregation, increased bleeding time and resistance to thromboembolism in P2Y1-deficient mice.

Platelet activation is characterized by shape change, induction of fibrinogen receptor expression and release of granular contents, leading to aggregation and plug formation. While this response is essential for hemostasis, it is also important in the pathogenesis of a broad spectrum of diseases, including myocardial infarction, stroke and unstable angina. Adenosine 5'-diphosphate (ADP) induces platelet aggregation, but the mechanism for this has not been established, and the relative contribution of ADP in hemostasis and the development of arterial thrombosis is poorly understood. We show here that the purinoceptor P2Y1 is required for platelet shape change in response to ADP and is also a principal receptor mediating ADP-induced platelet aggregation. Activation of P2Y1 resulted in increased intracellular calcium but no alteration in cyclic adenosine monophosphate (cAMP) levels. P2Y1-deficient platelets partially aggregated at higher ADP concentrations, and the lack of P2Y1 did not alter the ability of ADP to inhibit cAMP, indicating that platelets express at least one additional ADP receptor. In vivo, the lack of P2Y1 expression increased bleeding time and protected from collagen- and ADP-induced thromboembolism. These findings support the hypothesis that the ATP receptor P2Y1 is a principal receptor mediating both physiologic and pathological ADP-induced processes in platelets.     

The platelet P2Y12 receptor under normal and pathological conditions. Assessment with the radiolabeled selective antagonist [3H]PSB-0413

Various radioligands have been used to characterize and quantify the platelet P2Y₁₂ receptor, which share several weaknesses: (a) they are metabolically unstable and substrates for ectoenzymes, (b) they are agonists, and (c) they do not discriminate between P2Y₁ and P2Y₁₂. We used the [³H]PSB-0413 selective P2Y₁₂ receptor antagonist radioligand to reevaluate the number of P2Y₁₂ receptors in intact platelets and in membrane preparations. Studies in humans showed that: (1) [³H]PSB-0413 bound to 425 ± 50 sites/platelet (K_D = 3.3 ± 0.6 nM), (2) 0.5 ± 0.2 pmol [³H]PSB-0413 bound to 1 mg protein of platelet membranes (K_D = 6.5 ± 3.6 nM), and (3) competition studies confirmed the known features of P2Y₁₂, with the expected rank order of potency: AR-C69931MX > 2MeSADP ≫ ADPβS > ADP, while the P2Y₁ ligand MRS2179 and the P2X₁ ligand α,β-Met-ATP did not displace [³H]PSB-0413 binding. Patients with severe P2Y₁₂ deficiency displayed virtually no binding of [³H]PSB-0413 to intact platelets, while a patient with a dysfunctional P2Y₁₂ receptor had normal binding. Studies in mice showed that: (1) [³H]PSB-0413 bound to 634 ± 87 sites/platelet (K_D = 14 ± 4.5 nM) and (2) 0.7 pmol ± 0.3 [³H]PSB-0413 bound to 1 mg protein of platelet membranes (K_D = 9.1 ± 5.3 nM). Clopidogrel and other thiol reagents like pCMBS or DTT abolished the binding both to intact platelets and membrane preparations. Therefore, [³H]PSB-0413 is an accurate and selective tool for radioligand binding studies aimed at quantifying P2Y₁₂ receptors, to identify patients with P2Y₁₂ deficiencies or quantify the effect of P2Y₁₂ targeting drugs.     

Plant extracts inhibit ADP-induced platelet activation in humans: their potential therapeutic role as ADP antagonists

Adenosine diphosphate (ADP) plays a pivotal role in platelet activation. Platelet hyperactivity is associated with vascular disease and also has a key role in haemostasis and thrombosis. ADP activates platelets through three purinoceptor subtypes, the G_q-coupled P2Y₁ receptor, G_i-coupled P2Y₁₂ receptor and P2X₁ ligand-gated cation channel. Platelet ADP purinergic receptors are therefore suitable targets for antiplatelet drugs. Thienopyridines such as clopidogrel and ticlopidine, as well as other ADP receptor antagonists like prasugrel, ticagrelor, cangrelor and elinogrel have demonstrated clinical benefits via the inhibition of the selective purinergic ADP receptor, P2Y₁₂. However, they still have limitations in their mode of action and efficacy, like increased risk of bleeding. Thus, the ongoing pursuit to develop newer and more effective antiplatelet agents continues. There is a growing interest in the purinergic antiplatelet properties exhibited by plant extracts. This article considers the following: pomolic acid isolated from Licania pittieri, brazilin isolated from the heartwood of Caesalpinia sappan L, phylligenin isolated from the twigs of Muraltia vulpina, bark oil of Gonystylus velutinus, seed and bark extracts from Aesculus hippocastanum L. and red wine phenolics and catechins isolated from green tea. Moreover, the method used to investigate platelet purinergic receptors should be considered, since using a more sensitive, high-resolution platelet sizer can sometimes detect platelet variations when the light transmission method was not able to do so. The exact mechanisms by which these plant extracts work need further investigation. They all however inhibit ADP-induced activation in human platelets. This could explain, at least in part, the protective effect of plant extracts as antiplatelet agents.     

A novel small molecule 1,2,3,4,6-penta-O-galloyl-α-D-glucopyranose mimics the antiplatelet actions of insulin

Background

We have shown that 1,2,3,4,6-penta-O-galloyl-α-D-glucopyranose (α-PGG), an orally effective hypoglycemic small molecule, binds to insulin receptors and activates insulin-mediated glucose transport. Insulin has been shown to bind to its receptors on platelets and inhibit platelet activation. In this study we tested our hypothesis that if insulin possesses anti-platelet properties then insulin mimetic small molecules should mimic antiplatelet actions of insulin.

Principal Findings

Incubation of human platelets with insulin or α-PGG induced phosphorylation of insulin receptors and IRS-1 and blocked ADP or collagen induced aggregation. Pre-treatment of platelets with α-PGG inhibited thrombin-induced release of P-selectin, secretion of ATP and aggregation. Addition of ADP or thrombin to platelets significantly decreased the basal cyclic AMP levels. Pre-incubation of platelets with α-PGG blocked ADP or thrombin induced decrease in platelet cyclic AMP levels but did not alter the basal or PGE₁ induced increase in cAMP levels. Addition of α-PGG to platelets blocked agonist induced rise in platelet cytosolic calcium and phosphorylation of Akt. Administration of α-PGG (20 mg kg⁻¹) to wild type mice blocked ex vivo platelet aggregation induced by ADP or collagen.

Conclusions

These data suggest that α-PGG inhibits platelet activation, at least in part, by inducing phosphorylation of insulin receptors leading to inhibition of agonist induced: (a) decrease in cyclic AMP; (b) rise in cytosolic calcium; and (c) phosphorylation of Akt. These findings taken together with our earlier reports that α-PGG mimics insulin signaling suggest that inhibition of platelet activation by α-PGG mimics antiplatelet actions of insulin.     

The Src Family Kinases and Protein Kinase C Synergize to Mediate Gq-dependent Platelet Activation

The Src family kinases (SFKs) play essential roles in collagen- and von Willebrand factor (VWF)-mediated platelet activation. However, the roles of SFKs in G protein-coupled receptor-mediated platelet activation and the molecular mechanisms whereby SFKs are activated by G protein-coupled receptor stimulation are not fully understood. Here we show that the thrombin receptor protease-activated receptor 4 agonist peptide AYPGKF elicited SFK phosphorylation in P2Y₁₂ deficient platelets but stimulated minimal SFK phosphorylation in platelets lacking G_q. We have previously shown that thrombin-induced SFK phosphorylation was inhibited by the calcium chelator 5,5′-dimethyl-bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (dimethyl-BAPTA). The calcium ionophore A23187 induced SFK phosphorylation in both wild-type and G_q deficient platelets. Together, these results indicate that SFK phosphorylation in response to thrombin receptor stimulation is downstream from G_q/Ca²⁺ signaling. Moreover, A23187-induced thromboxane A₂ synthesis, platelet aggregation, and secretion were inhibited by preincubation of platelets with a selective SFK inhibitor, PP2. AYPGKF-induced thromboxane A₂ production in wild-type and P2Y₁₂ deficient platelets was abolished by PP2, and AYPGKF-mediated P-selectin expression, integrin α_IIbβ₃ activation, and aggregation of P2Y₁₂ deficient platelets were partially inhibited by the PKC inhibitor Ro-31-8220, PP2, dimethyl-BAPTA, or {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, but were abolished by Ro-31-8220 plus PP2, dimethyl-BAPTA, or {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. These data indicate that Ca²⁺/SFKs/PI3K and PKC represent two alternative signaling pathways mediating G_q-dependent platelet activation.