P2Y Receptors Research Articles

P2Y12 receptor residues crucial for thrombosis regulation.

Thrombosis, or the formation of blood clots, can lead to serious medical conditions such as stroke, heart attack, and deep vein thrombosis. The purinoreceptor P2Y12 plays a critical role in the thrombotic pathway and is targeted for therapy to prevent clot formation. However, it is essential to balance the regulation of thrombosis to avoid adverse situations. This study focuses on the P2Y12 receptor and aims to discern the protein residue network and differentiate residues based on their intramolecular interactions. The study utilized a statistical analysis to characterize the significant residues involved in ligand interaction, which helps to identify critical residues that are essential for the function of the receptor. A parametric analysis of interactions of residues in the intraprotein interaction was conducted, which revealed significant residue-based contacts that facilitate protein interactions. By examining the interactions between residues, the mechanisms underlying protein interactions were studied and the importance of specific residues in facilitating these interactions was determined. This research provides important information on P2Y12, and the findings based on the networkbased significance of interacting residues may contribute to the development of new therapies that target the receptor to prevent clot formation while maintaining a balance in thrombosis regulation to avoid adverse outcomes. Ultimately, this study could lead to improved treatments for thrombotic disorders and better patient outcomes.

Mind the Gap: Model-Based Switching from Selatogrel to Maintenance Therapy with Oral P2Y12 Receptor Antagonists.

Background: The P2Y12 receptor antagonist selatogrel is being developed for subcutaneous self-administration with a ready-to-use autoinjector at the onset of acute myocardial infarction (AMI) symptoms. The unique pharmacological profile of selatogrel (fast, potent, and short-acting) can bridge the time gap between the onset of AMI and first medical care. A clinical Phase 1 study showed a time-dependent pharmacodynamic interaction between selatogrel and loading doses of clopidogrel and prasugrel. As treatment switching is a common clinical practice, the assessment of subsequent switching from a clopidogrel loading dose to the first maintenance dose of oral P2Y12 receptor antagonists is highly relevant. Objectives: Model-based predictions of inhibition of platelet aggregation (IPA) for the drugs triggering pharmacodynamic interactions were to be derived to support clinical guidance on the transition from selatogrel to oral P2Y12 receptor antagonists. Methods: Scenarios with selatogrel 16 mg administration or placebo followed by a clopidogrel loading dose and, in turn, prasugrel or ticagrelor maintenance doses at different times of administration were studied. Population pharmacokinetic/pharmacodynamic modeling and simulations of different treatment scenarios were used to derive quantitative estimates for IPA over time. Results: Following selatogrel/placebo and a clopidogrel loading dose, maintenance treatment with ticagrelor or a prasugrel loading dose followed by maintenance treatment quickly achieved sustained IPA levels above 80%. Prior to maintenance treatment, a short time span from 18 to 24 h was identified where IPA levels were predicted to be lower with selatogrel than with placebo if clopidogrel was administered 12 h after selatogrel or placebo. Predicted IPA levels reached with placebo alone and a clopidogrel loading dose at 4 h were consistently lower than with selatogrel administration, followed by a clopidogrel loading dose at 12 h. If a clopidogrel loading dose is administered at 12 h, selatogrel maintains higher IPA levels up to 16 h. IPA levels are subsequently lower than on the placebo until the administration of the first maintenance dose. Conclusions: Model-based predictions informed the transition from selatogrel subcutaneous administration to oral P2Y12 therapy. The application of modeling techniques illustrates the value of employing pharmacokinetic and pharmacodynamic modeling for the simulation of various clinical scenarios of switching therapies.

Astrocytic Extracellular Vesicles Regulated by Microglial Inflammatory Responses Improve Stroke Recovery.

There are no effective treatments for post-stroke glial scar formation, which inhibits axonal outgrowth and functional recovery after stroke. We investigated whether astrocytic extracellular vesicles (AEVs) regulated by microglia modulate glial scars and improve stroke recovery. We found that peri-infarct glial scars comprised reactive astrocytes with proliferating C3d and decreased S100A10 expression in chronic stroke. In cultured astrocytes, microglia-conditioned media and treatment with P2Y1 receptor antagonists increased and reduced the area of S100A10- and C3d-expressing reactive astrocytes, respectively, by suppressing mitogen-activated protein kinase/nuclear factor-κβ (NF-κB)/tumor necrosis factor-α (TNF-α)/interleukin-1β signaling after oxygen-glucose deprivation. Intracerebral administrations of AEVs enriched miR-146a-5p, downregulated NF-κB, and suppressed TNF-α expressions, by transforming reactive astrocytes to those with S100A10 preponderance, causing functional recovery in rats subjected to middle cerebral artery occlusion. Modulating neuroinflammation in post-stroke glial scars could permit axonal outgrowth, thus providing a basis for stroke recovery with neuroprotective AEVs.

Effects of Early Exposure to Isoflurane on Susceptibility to Chronic Pain Are Mediated by Increased Neural Activity Due to Actions of the Mammalian Target of the Rapamycin Pathway.

Patients who have undergone surgery in early life may be at elevated risk for suffering neuropathic pain in later life. The risk factors for this susceptibility are not fully understood. Here, we used a mouse chronic pain model to test the hypothesis that early exposure to the general anesthetic (GA) Isoflurane causes cellular and molecular alterations in dorsal spinal cord (DSC) and dorsal root ganglion (DRG) that produces a predisposition to neuropathic pain via an upregulation of the mammalian target of the rapamycin (mTOR) signaling pathway. Mice were exposed to isoflurane at postnatal day 7 (P7) and underwent spared nerve injury at P28 which causes chronic pain. Selected groups were treated with rapamycin, an mTOR inhibitor, for eight weeks. Behavioral tests showed that early isoflurane exposure enhanced susceptibility to chronic pain, and rapamycin treatment improved outcomes. Immunohistochemistry, Western blotting, and q-PCR indicated that isoflurane upregulated mTOR expression and neural activity in DSC and DRG. Accompanying upregulation of mTOR and rapamycin-reversible changes in chronic pain-associated markers, including N-cadherin, cAMP response element-binding protein (CREB), purinergic P2Y12 receptor, glial fibrillary acidic protein (GFAP) in DSC; and connexin 43, phospho-extracellular signal-regulated kinase (p-ERK), GFAP, Iba1 in DRG, were observed. We concluded that early GA exposure, at least with isoflurane, alters the development of pain circuits such that mice are subsequently more vulnerable to chronic neuropathic pain states.

Plasma protein signatures for high on-treatment platelet reactivity to aspirin and clopidogrel in peripheral artery disease

BackgroundA significant proportion of patients with peripheral artery disease (PAD) displays a poor response to aspirin and/or the platelet P2Y12 receptor antagonist clopidogrel. This phenomenon is reflected by high on-treatment platelet reactivity (HTPR) in platelet function assays in vitro and is associated with an increased risk of adverse cardiovascular events. ObjectiveThis study aimed to elucidate specific plasma protein signatures associated with HTPR to aspirin and clopidogrel in PAD patients. Methods and resultsBased on targeted plasma proteomics, 184 proteins from two cardiovascular Olink panels were measured in 105 PAD patients. VerifyNow ASPI- and P2Y12-test values were transformed to a continuous variable representing HTPR as a spectrum instead of cut-off level-defined HTPR. Using the Boruta random forest algorithm, the importance of 3 plasma proteins for HTPR in the aspirin, six in clopidogrel and 10 in the pooled group (clopidogrel or aspirin) was confirmed. Network analysis demonstrated clusters with CD84, SLAMF7, IL1RN and THBD for clopidogrel and with F2R, SELPLG, HAVCR1, THBD, PECAM1, TNFRSF10B, MERTK and ADM for the pooled group. F2R, TNFRSF10B and ADM were higher expressed in Fontaine III patients compared to Fontaine II, suggesting their relation with PAD severity. ConclusionsA plasma protein signature, including eight targets involved in proatherogenic dysfunction of blood cell-vasculature interaction, coagulation and cell death, is associated with HTPR (aspirin and/or clopidogrel) in PAD. This may serve as important systems-based determinants of poor platelet responsiveness to aspirin and/or clopidogrel in PAD and other cardiovascular diseases and may contribute to identify novel treatment strategies.

Ticagrelor inverse agonist activity at the P2Y12 receptor is non-reversible versus its endogenous agonist adenosine 5´-diphosphate.

Ticagrelor is labelled as a reversible, direct-acting platelet P2Y12 receptor (P2Y12 R) antagonist that is indicated clinically for the prevention of thrombotic events in patients with acute coronary syndrome (ACS). As with many antiplatelet drugs, ticagrelor therapy increases bleeding risk in patients, which may require platelet transfusion in emergency situations. The aim of this study was to further examine the reversibility of ticagrelor at the P2Y12 R. Studies were performed in human platelets, with P2Y12 R-stimulated GTPase activity and platelet aggregation assessed. Cell-based bioluminescence resonance energy transfer (BRET) assays were undertaken to assess G protein-subunit activation downstream of P2Y12 R activation. Initial studies revealed that a range of P2Y12 R ligands, including ticagrelor, displayed inverse agonist activity at P2Y12 R. Only ticagrelor was resistant to washout and, in human platelet and cell-based assays, washing failed to reverse ticagrelor-dependent inhibition of ADP-stimulated P2Y12 R function. The P2Y12 R agonist 2MeSADP, which was also resistant to washout, was able to effectively compete with ticagrelor. In silico docking revealed that ticagrelor and 2MeSADP penetrated more deeply into the orthosteric binding pocket of the P2Y12 R than other P2Y12 R ligands. Ticagrelor binding to P2Y12 R is prolonged and more akin to that of an irreversible antagonist, especially versus the endogenous P2Y12 R agonist ADP. This study highlights the potential clinical need for novel ticagrelor reversal strategies in patients with spontaneous major bleeding, and for bleeding associated with urgent invasive procedures.

Understanding clopidogrel resistance: Mechanisms, detections, implications and surmount the clopidogrel resistant in order to potential solutions

Clopidogrel, a drug belonging to the thienopyridine class of antiplatelet agents, has been utilized either as a standalone medication or in combination with aspirin for averting vascular complications in patients afflicted with atherothrombotic conditions. The selective and irreversible blockade of the P2Y12 receptor by clopidogrel makes it a potent inhibitor of platelet activation and aggregation. In patients with acute coronary syndromes or undergoing percutaneous interventions, combining clopidogrel with aspirin is a crucial approach to antiplatelet therapy. The aim of this review article is to investigate the factors that contribute to clopidogrel resistance, mechanisms involved in clopidogrel resistance, evaluate it through laboratory methods and clinical implications of clopidogrel resistance. Additionally, the article explores the clinical importance of Clopidogrel Resistance and its correlation with harmful cardiovascular events. The paper concludes by proposing potential solutions to this resistance and suggesting the usage of alternative antiplatelet from the new generation.

Platelet P2Y12 signalling pathway in the dysregulated immune response during sepsis.

Sepsis is a complicated pathological condition in response to severe infection. It is characterized by a strong systemic inflammatory response, where multiple components of the immune system are involved. Currently, there is no treatment for sepsis. Blood platelets are known for their role in haemostasis, but they also participate in inflammation through cell-cell interaction and the secretion of inflammatory mediators. Interestingly, an increase in platelet activation, secretion, and aggregation with other immune cells (such as monocytes, T-lymphocytes and neutrophils) has been detected in septic patients. Therefore, antiplatelet therapy in terms of P2Y12 antagonists has been evaluated as a possible treatment for sepis. It was found that blocking P2Y12 receptors decreased platelet marker expression and limited attachment to immune cells in some studies, but not in others. This review addresses the role of platelets in sepsis and discusses whether antagonizing P2Y12 signalling pathways can alter the disease outcome. Challenges in studying P2Y12 antagonists in sepsis also are discussed. LINKED ARTICLES: This article is part of a themed issue on Platelet purinergic receptor and non-thrombotic disease. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v181.4/issuetoc.

Sex-specific effect of P2Y2 purinergic receptor on glucose metabolism during acute inflammation.

The sex of an animal impacts glucose sensitivity, but little information is available regarding the mechanisms causing that difference, especially during acute inflammation. We examined sex-specific differences in the role of the P2Y2 receptor (P2Y2R) in glucose flux with and without LPS challenge. Male and female wild-type and P2Y2R knockout mice (P2Y2R-/-) were injected with LPS or saline and glucose tolerance tests (GTT) were performed. P2Y2R, insulin receptor, and GLUT4 transporter gene expression was also evaluated. Female mice had reduced fasting plasma glucose and females had reduced glucose excursion times compared to male mice during GTT. P2Y2R-/- males had significantly decreased glucose flux throughout the GTT as compared to all female mice. Acute inflammation reduced fasting plasma glucose and the GTT area under the curve in both sexes. While both wild-type and P2Y2R-/- male animals displayed reduced fasting glucose in LPS treatment, female mice did not have significant difference in glucose tolerance, suggesting that the effects of P2Y2R are specific to male mice, even under inflammatory conditions. Overall, we conclude that the role for the purinergic receptor, P2Y2R, in regulating glucose metabolism is minimal in females but plays a large role in male mice, particularly in the acute inflammatory state.

Proton pump inhibitors and gastroprotection in patients treated with antithrombotic drugs: A cardiologic point of view.

Aspirin, other antiplatelet agents, and anticoagulant drugs are used across a wide spectrum of cardiovascular and cerebrovascular diseases. A concomitant proton pump inhibitor (PPI) treatment is often prescribed in these patients, as gastrointestinal complications are relatively frequent. On the other hand, a potential increased risk of cardiovascular events has been suggested in patients treated with PPIs; in particular, it has been discussed whether these drugs may reduce the cardiovascular protection of clopidogrel, due to pharmacodynamic and pharmacokinetic interactions through hepatic metabolism. Previously, the concomitant use of clopidogrel and omeprazole or esomeprazole has been discouraged. In contrast, it remains less known whether PPI use may affect the clinical efficacy of ticagrelor and prasugrel, new P2Y12 receptor antagonists. Current guidelines recommend PPI use in combination with antiplatelet treatment in patients with risk factors for gastrointestinal bleeding, including advanced age, concurrent use of anticoagulants, steroids, or non-steroidal anti-inflammatory drugs, and Helicobacter pylori (H. pylori) infection. In patients taking oral anticoagulant with risk factors for gastrointestinal bleeding, PPIs could be recommended, even if their usefulness deserves further data. H. pylori infection should always be investigated and treated in patients with a history of peptic ulcer disease (with or without complication) treated with antithrombotic drugs. The present review summarizes the current knowledge regarding the widespread combined use of platelet inhibitors, anticoagulants, and PPIs, discussing consequent clinical implications.

Intracellular cAMP Signaling Pathway via Gs Protein-Coupled Receptor Activation in Rat Primary Cultured Trigeminal Ganglion Cells.

G protein-coupled receptors in trigeminal ganglion (TG) neurons are often associated with sensory mechanisms, including nociception. We have previously reported the expression of P2Y12 receptors, which are Gi protein-coupled receptors, in TG cells. Activating P2Y12 receptors decreased the intracellular free Ca2+ concentration ([Ca2+]i). This indicated that intracellular adenosine 3',5'-cyclic monophosphate (cAMP) levels can mediate Ca2+ signaling in TG cells. Here, we report more extensive-expression patterns of Gs protein-coupled receptors in primary cultured TG neurons isolated from 7-day-old newborn Wistar rats and further examine the roles of these receptors in cAMP signaling using the BacMam sensor in these neurons. To identify TG neurons, we also measured [Ca2+]i using fura-2 in TG cells and measured intracellular cAMP levels. TG neurons were positive for Gαs protein-coupled receptors, beta-2 adrenergic (β2), calcitonin gene-related peptide (CGRP), adenosine A2A (A2A), dopamine 1 (D1), prostaglandin I2 (IP), and 5-hydroxytriptamine 4 (5-HT4) receptor. Application of forskolin (FSK), an activator of adenylyl cyclase, transiently increased intracellular cAMP levels in TG neurons. The application of a phosphodiesterase inhibitor augmented the FSK-elicited intracellular cAMP level increase. These increases were significantly suppressed by the application of SQ22536, an adenylyl cyclase inhibitor, in TG neurons. Application of agonists for β2, CGRP, A2A, D1-like, IP, and 5-HT4 receptors increased intracellular cAMP levels. These increases were SQ22536-sensitive. These results suggested that TG neurons express β2, CGRP, A2A, D1, IP, and 5-HT4 receptors, and the activations of these Gαs protein-coupled receptors increase intracellular cAMP levels by activating adenylyl cyclase.

Silencing of purinergic receptor P2Y2 inhibited enteric neural crest cell proliferation, invasion and migration via suppressing ERK signaling pathway in Hirschsprung disease.

The current study aimed to explore the effect and underlying mechanism of the purinergic receptor P2Y2 in regulating the loss of intestinal neurons and the intestinal neural crest in Hirschsprung's disease (HSCR). Western blotting was used to assess the expression levels of P2Y2 in colon tissues. An in vivo HSCR mouse model was established following treatment with benzalkonium chloride (BAC). We overexpressed or silenced P2Y2 in SH-SY5Y cells, and cell proliferation, migration, and invasion were subsequently investigated by CCK-8, wound healing, and transwell assays, respectively. Additionally, we implemented a xenograft model to assess the impact of P2Y2 on tumor growth as well as the expression of extracellular signal-regulated kinase (ERK). The results showed that the expression of P2Y2 protein in the colon tissues of patients with HSCR was lower than that in the normal colon tissues. P2Y2 expression is downregulated in the colon tissues of mice with HSCR. Additionally, P2Y2 silencing inhibited SH-SY5Y cell proliferation, invasion, and migration. Furthermore, adenosine 5'-triphosphate (ATP, a strong agonist of P2Y2)-induced P2Y2 overexpression enhanced the proliferation, invasion, and migration of SH-SY5Y cells. Immunofluorescence staining and western blot analysis revealed that P2Y2 silencing downregulated phosphorylated (p)-ERK in SH-SY5Y cells. In addition, treatment with PD98059, a p-ERK inhibitor, reversed the effects of ATP on SH-SY5Y cell proliferation, invasion, and migration. Finally, we demonstrated that P2Y2 silencing suppressed tumor growth and decreased p-ERK expression. Overall, the results of the present study suggest that P2Y2 plays an important role in HSCR pathogenesis. P2Y2 silencing inhibited the proliferation, invasion, and migration of nerve cells by suppressing the ERK signaling pathway. P2Y2 silencing could be considered an innovative and possible target for treating HSCR.

Cell Surface Platelet Tissue Factor Expression: Regulation by P2Y12 and Link to Residual Platelet Reactivity.

ADP-induced platelet activation leads to cell surface expression of several proteins, including TF (tissue factor). The role of ADP receptors in platelet TF modulation is still unknown. We aimed to assess the (1) involvement of P2Y1 and P2Y12 receptors in ADP-induced TF exposure; (2) modulation of TFpos-platelets in anti-P2Y12-treated patients with coronary artery disease. Based on the obtained results, we revisited the intracellular localization of TF in platelets. The effects of P2Y1 or P2Y12 antagonists on ADP-induced TF expression and activity were analyzed in vitro by flow cytometry and thrombin generation assay in blood from healthy subjects, P2Y12-/-, and patients with gray platelet syndrome. Ex vivo, P2Y12 inhibition of TF expression by clopidogrel/prasugrel/ticagrelor, assessed by VASP (vasodilator-stimulated phosphoprotein) platelet reactivity index, was investigated in coronary artery disease (n=238). Inhibition of open canalicular system externalization and electron microscopy (TEM) were used for TF localization. In blood from healthy subjects, stimulated in vitro by ADP, the percentage of TFpos-platelets (17.3±5.5%) was significantly reduced in a concentration-dependent manner by P2Y12 inhibition only (-81.7±9.5% with 100 nM AR-C69931MX). In coronary artery disease, inhibition of P2Y12 is paralleled by reduction of ADP-induced platelet TF expression (VASP platelet reactivity index: 17.9±11%, 20.9±11.3%, 40.3±13%; TFpos-platelets: 10.5±4.8%, 9.8±5.9%, 13.6±6.3%, in prasugrel/ticagrelor/clopidogrel-treated patients, respectively). Despite this, 15% of clopidogrel good responders had a level of TFpos-platelets similar to the poor-responder group. Indeed, a stronger P2Y12 inhibition (130-fold) is required to inhibit TF than VASP. Thus, a VASP platelet reactivity index <20% (as in prasugrel/ticagrelor-treated patients) identifies patients with TFpos-platelets <20% (92% sensitivity). Finally, colchicine impaired in vitro ADP-induced TF expression but not α-granule release, suggesting that TF is open canalicular system stored as confirmed by TEM and platelet analysis of patients with gray platelet syndrome. Data show that TF expression is regulated by P2Y12 and not P2Y1; P2Y12 antagonists downregulate the percentage of TFpos-platelets. In clopidogrel good-responder patients, assessment of TFpos-platelets highlights those with residual platelet reactivity. TF is stored in open canalicular system, and its membrane exposure upon activation is prevented by colchicine.

Neural targets of the enteric dopaminergic system in regulating motility of rat proximal colon.

In isolated segments of the rat proximal colon, the dopamine reuptake inhibitor GBR 12909 (GBR) causes a dilatation, while the D1-like receptor antagonist SCH 23390 (SCH) induces a tonic constriction, suggesting that neurally released dopamine tonically stimulates enteric inhibitory efferent neurons. Here, the targets of the enteric dopaminergic neurons were investigated. Cannulated segments of rat proximal colon were bathed in physiological salt solution and luminally perfused with 0.9% saline, while all drugs were applied to the bath. Spatio-temporal maps of colonic motility were constructed from video recordings of peristaltic contractions, and the maximum diameter was measured as an index of colonic contractility. GBR (1μM)-induced dilatations of colonic segments were prevented by SCH (5μM), L-nitro arginine (L-NA; 100μM), a nitric oxide synthase inhibitor, or tetrodotoxin (0.6μM). In contrast, constrictions induced by a higher concentration of SCH (20μM) were unaffected by either L-NA or tetrodotoxin. The vasoactive intestinal peptide (VIP) receptor antagonist VIP10-28 (3μM) or P2Y1 receptor antagonist MRS 2500 (1μM) had no effect on either the GBR-induced dilatation or the SCH-induced constriction. In colonic segments that had been pretreated with 6-hydroxydopamine (100μM, 3h) to deplete enteric dopamine, GBR failed to increase the colonic diameter, while SCH was still capable of constricting colonic segments. Enteric dopaminergic neurons appear to project to nitrergic neurons to dilate the proximal colon by activating neuronal D1-like receptors. In addition, constitutively activated D1-like receptors expressed in cells yet to be determined may provide a tonic inhibition on colonic constrictions.

Racial/Ethnic Disparities in Outcomes After Percutaneous Coronary Intervention

Asian American/Pacific Islanders (AAPIs) and Hispanics are growing minority United States populations, but are poorly represented in the cardiovascular literature. This study examines guideline adherence and outcomes in AAPIs and Hispanics compared with non-Hispanic Whites (NHWs) in a quaternary care center after inpatient percutaneous coronary intervention (PCI). The primary end points were inpatient post-PCI bleed, heart failure, cardiogenic shock, and all-cause mortality, whereas the secondary end point was the prescription rate of post-PCI guideline-directed medical therapy including aspirin, statins, P2Y12 receptor blockers, and cardiopulmonary rehabilitation. Intergroup differences were assessed through analysis of variance or two-way chi-square tests, and the association of race with binary outcomes was examined through logistic regression with NHW as the reference group. Compared with NHW, AAPIs, and Hispanics had higher odds of diabetes mellitus, and AAPIs had higher odds of hypertension and being on dialysis. Hispanics had higher odds of post-PCI mortality versus NHW, both in acute coronary syndrome (odds ratio [OR] 2.04, p=0.03) and elective PCI (OR2.51, p=0.04). AAPI also trended toward higher mortality than NHW in both categories. AAPIs were found to have higher odds of statin prescription (OR1.91, p=0.04). Hispanics had lower odds of ticagrelor prescription versus NHW (OR0.65, p=0.04), and AAPIs trended toward such. No differences were found for cardiopulmonary rehabilitation prescriptions in groups. This study suggests that despite quality improvement efforts, disparities remain in postprocedural outcomes in minority groups in comparison with NHW.

Tumoral P2Y2 receptor modulates tumor growth and host anti-tumor immune responses in a syngeneic murine model of oral cancer.

Head and neck squamous cell carcinomas (HNSCCs) are a heterogenous group of tumors and among the top 10 most common cancers and they arise from the epithelial tissues of the mucosal surfaces of the oral cavity, oropharynx, and larynx. Aberrant purinergic signaling has been associated with various cancer types. Here, we studied the role ofthe P2Y2 purinergic receptor (P2Y2R) in the context of oral cancer. We utilized bioinformatics analysis of deposited datasets to examine purinome gene expression in HNSCC tumors and cells lines and functionally characterized nucleotide-induced P2 receptor signaling in human FaDu and Cal27 and murine MOC2 oral cancer cell lines. Utilizing tumorigenesis assays with wild-type or P2ry2 knockout MOC2 cells we evaluated the role of P2Y2Rs in tumor growth and the host anti-tumor immune responses. Our data demonstrate that human and murine oral cancer cell lines express numerous P2 receptors, with the P2Y2R being highly expressed. Using syngeneic tumor graftsin wild-type mice, we observed that MOC2 tumors expressing P2Y2R were larger than P2Y2R-/- tumors. Wild-type MOC2 tumors contained a lower population of tumor-infiltrating CD11b+F4/80+ macrophages and CD3+cells, which were revealed to be CD3+CD4+IFNγ+ T cells, compared to P2Y2R-/- tumors. These results were mirrored when utilizing P2Y2R-/- mice, indicating that the changes in MOC2 tumor growth and to the host anti-tumor immune response were independent of host derived P2Y2Rs. Results suggest that targeted suppression of the P2Y2R in HNSCC cells in vivo, rather than systemic P2Y2R antagonism, may be a more effective treatment strategy for HNSCCs.

Comparison of the effects of the GPIIb-IIIa antagonist Zalunfiban and the P2Y12 antagonist Selatogrel on Platelet Aggregation

Understanding the pharmacodynamic effects of platelet inhibitors is standard for developing more effective antithrombotic therapies. An example is the antithrombotic treatment of acute coronary syndrome (ACS), in particular ST-elevated myocardial infarction (STEMI) patients who are in need for rapid acting strong antithrombotic therapy despite the use of aspirin and oral P2Y12-inhibitors. In this study, we evaluated two injectable platelet inhibitors under clinical development (the P2Y12 antagonist selatogrel and the GPIIb-IIIa antagonist zalunfiban) that may be amenable to pre-hospital treatment of STEMI patients. Platelet reactivity was assessed at inhibitor concentrations that represent clinically relevant levels of platelet inhibition (IC20-50%, 1/2Cmax, and Cmax). Light transmission aggregometry (LTA), was used to evaluate the initial rate of aggregation (primary slope, PS) and maximal aggregation (MA). Both adenosine diphosphate (ADP) and thrombin receptor agonist peptide (TRAP) were used as agonists. Zalunfiban demonstrated similar inhibition of platelet aggregation when blood was collected in PPACK or TSC, whereas selatogrel demonstrated greater inhibition in PPACK. In this study, using PPACK anticoagulant, selatogrel and zalunfiban affected PS in response to ADP equivalently at all drug concentrations tested. In contrast, zalunfiban had significantly greater potency at its Cmax concentration compared to selatogrel using TRAP as agonist. Upon evaluation of MA responses at lower doses, selatogrel had greater inhibition of MA in response to ADP than zalunfiban; however, at concentrations that represent Cmax, the drugs were equivalent. Zalunfiban also had greater inhibition of MA in response to TRAP at the Cmax dose. These data suggest that zalunfiban may provide greater protection in reducing thrombus formation than selatogrel, especially since thrombin is an early, key primary agonist in the pathophysiology of thrombotic events.

Exploring bias in platelet P2Y1 signalling: Host defence versus haemostasis.

Platelets are necessary for maintaining haemostasis. Separately, platelets are important for the propagation of inflammation during the host immune response against infection. The activation of platelets also causes inappropriate inflammation in various disease pathologies, often in the absence of changes to haemostasis. The separate functions of platelets during inflammation compared with haemostasis are therefore varied and this will be reflected in distinct pathways of activation. The activation of platelets by the nucleotide adenosine diphosphate (ADP) acting on P2Y1 and P2Y12 receptors is important for the development of platelet thrombi during haemostasis. However, P2Y1 stimulation of platelets is also important during the inflammatory response and paradoxically in scenarios where no changes to haemostasis and platelet aggregation occur. In these events, Rho-GTPase signalling, rather than the canonical phospholipase Cβ (PLCβ) signalling pathway, is necessary. We describe our current understanding of these differences, reflecting on recent advances in knowledge of P2Y1 structure, and the possibility of biased agonism occurring from activation via other endogenous nucleotides compared with ADP. Knowledge arising from these different pathways of P2Y1 stimulation of platelets during inflammation compared with haemostasis may help therapeutic control of platelet function during inflammation or infection, while preserving essential haemostasis. LINKED ARTICLES: This article is part of a themed issue on Platelet purinergic receptor and non-thrombotic disease. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v181.4/issuetoc.

Ticlopidine induces embryonic development toxicity and hepatotoxicity in zebrafish by upregulating the oxidative stress signaling pathway

Ticlopidine exerts its anti-platelet effects mainly by antagonizing platelet p2y12 receptors. Previously, a few studies have shown that ticlopidine can induce liver injury, but the exact mechanism of hepatotoxicity remains unclear. Oxidative stress, metabolic disorders, hepatocyte apoptosis, lipid peroxidation, and inflammatory responses can all lead to hepatic liver damage, which can cause hepatotoxicity. In this study, in order to deeply explore the potential molecular mechanisms of ticlopidine -induced hepatotoxicity, we used zebrafish as a model organism to comprehensively evaluate the hepatotoxicity of ticlopidine and its associated mechanism. Three days post-fertilization, zebrafish larvae were exposed to varying concentrations (1.5, 1.75 and 2 μg/mL) of ticlopidine for 72 h, in contrast, adult zebrafish were exposed exposure to 4 μg/mL of ticlopidine for 28 days. Ticlopidine-exposed zebrafish larvae showed changes in liver morphology, shortened body length, and delayed development of the swim bladder development. Liver tissues of ticlopidine-exposed zebrafish larvae and adults stained with Hematoxylin & Eosin revealed vacuolization and increased cellular interstitial spaces in liver tissues. Furthermore, using Oil Red O and periodic acid-Schiff staining methods and evaluating different metabolic enzymes of ticlopidine-exposed zebrafish larvae and adults suggested abnormal liver metabolism and liver injury in both ticlopidine-exposed zebrafish larvae and adults. Ticlopidine also significantly elevated inflammation and oxidative stress and reduced hepatocyte proliferation. During the rescue intervention using N-acetylcysteine, we observed significant improvement in ticlopidine-induced morphological changes in the liver, shortened body length, delayed swim bladder development, and proliferation of liver tissues showed significant improvement. In conclusion, ticlopidine might inhibit normal development and liver proliferation in zebrafish by upregulation of oxidative stress levels, thus leading to embryonic developmental toxicity and hepatotoxicity. In this study, we used zebrafish as a model organism to elucidate the developmental toxicity and hepatotoxicity induced by ticlopidine upregulation of oxidative stress signaling pathway in zebrafish, providing a theoretical basis for clinical application.

ShlA toxin of Serratia induces P2Y2- and α5β1-dependent autophagy and bacterial clearance from host cells

Serratia marcescens is an opportunistic human pathogen involved in antibiotic-resistant hospital acquired infections. Upon contact with the host epithelial cell and prior to internalization, Serratia induces an early autophagic response that is entirely dependent on the ShlA toxin. Once Serratia invades the eukaryotic cell and multiples inside an intracellular vacuole, ShlA expression also promotes an exocytic event that allows bacterial egress from the host cell without compromising its integrity. Several toxins, including ShlA, were shown to induce ATP efflux from eukaryotic cells. Here, we demonstrate that ShlA triggered a nonlytic release of ATP from Chinese hamster ovary (CHO) cells. Enzymatic removal of accumulated extracellular ATP (eATP) or pharmacological blockage of the eATP-P2Y2 purinergic receptor inhibited the ShlA-promoted autophagic response in CHO cells. Despite the intrinsic ecto-ATPase activity of CHO cells, the effective concentration and kinetic profile of eATP was consistent with the established affinity of the P2Y2 receptor and the known kinetics of autophagy induction. Moreover, eATP removal or P2Y2 receptor inhibition also suppressed the ShlA-induced exocytic expulsion of the bacteria from the host cell. Blocking α5β1 integrin highly inhibited ShlA-dependent autophagy, a result consistent with α5β1 transactivation by the P2Y2 receptor. In sum, eATP operates as the key signaling molecule that allows the eukaryotic cell to detect the challenge imposed by the contact with the ShlA toxin. Stimulation of P2Y2-dependent pathways evokes the activation of a defensive response to counteract cell damage and promotes the nonlytic clearance of the pathogen from the infected cell.