Safety and Efficacy of Ticagrelor Monotherapy in Patients With Acute Coronary Syndromes Undergoing Percutaneous Coronary Intervention: An Individual Patient Data Meta-Analysis of TWILIGHT and TICO Randomized Trials.

BACKGROUND: Dual antiplatelet therapy with a potent P2Y12 inhibitor coupled with aspirin for 1 year is the recommended treatment for patients with acute coronary syndrome (ACS) undergoing percutaneous coronary intervention (PCI). As an alternative, monotherapy with a P2Y12 inhibitor after a short period of dual antiplatelet therapy has emerged as a bleeding reduction strategy. METHODS: We pooled individual patient data from randomized trials that included patients with ACS undergoing PCI treated with an initial 3-month course of dual antiplatelet therapy followed by ticagrelor monotherapy versus continued ticagrelor plus aspirin. Patients sustaining a major ischemic or bleeding event in the first 3 months after PCI were excluded from analysis. The primary outcome was Bleeding Academic Research Consortium type 3 or 5 bleeding occurring between 3 and 12 months after index PCI. The key secondary end point was the composite of death, myocardial infarction, or stroke. Hazard ratios and 95% CIs were generated using Cox regression with a one-stage approach in the intention-to-treat population. RESULTS: The pooled cohort (n=7529) had a mean age of 62.8 years, 23.2% were female, and 55% presented with biomarker-positive ACS. Between 3 and 12 months, ticagrelor monotherapy significantly reduced Bleeding Academic Research Consortium 3 or 5 bleeding compared with ticagrelor plus aspirin (0.8% versus 2.1%; hazard ratio, 0.37 [95% CI, 0.24-0.56]; P<0.001). Rates of all-cause death, myocardial infarction, or stroke were not significantly different between groups (2.4% versus 2.7%; hazard ratio, 0.91 [95% CI, 0.68-1.21]; P=0.515). Findings were unchanged among patients presenting with biomarker-positive ACS. CONCLUSIONS: Among patients with ACS undergoing PCI who have completed a 3-month course of dual antiplatelet therapy, discontinuation of aspirin followed by ticagrelor monotherapy significantly reduced major bleeding without incremental ischemic risk compared with ticagrelor plus aspirin. REGISTRATION: URL: https://www.crd.york.ac.uk/prospero; Unique identifier: CRD42023449646.

How selective antagonists and genetic modification have helped characterise the expression and functions of vascular P2Y receptors.

Vascular P2Y receptors mediate many effects, but the role of individual subtypes is often unclear. Here we discuss how subtype-selective antagonists and receptor knockout/knockdown have helped identify these roles in numerous species and vessels. P2Y1 receptor-mediated vasoconstriction and endothelium-dependent vasodilation have been characterised using the selective antagonists, MRS2179 and MRS2216, whilst AR-C118925XX, a P2Y2 receptor antagonist, reduced endothelium-dependent relaxation, and signalling evoked by UTP or fluid shear stress. P2Y2 receptor knockdown reduced endothelial signalling and endothelial P2Y2 receptor knockout produced hypertensive mice and abolished vasodilation elicited by an increase in flow. UTP-evoked vasoconstriction was also blocked by AR-C118925XX, but the effects of P2Y2 receptor knockout were complex. No P2Y4 receptor antagonists are available and P2Y4 knockout did not affect the vascular actions of UTP and UDP. The P2Y6 receptor antagonist, MRS2578, identified endothelial P2Y6 receptors mediating vasodilation, but receptor knockout had complex effects. MRS2578 also inhibited, and P2Y6 knockout abolished, contractions evoked by UDP. P2Y6 receptors contribute to the myogenic tone induced by a stepped increase in vascular perfusion pressure and possibly to the development of atherosclerosis. The P2Y11 receptor antagonists, NF157 and NF340, inhibited ATP-evoked signalling in human endothelial cells. Vasoconstriction mediated by P2Y12/P2Y13 and P2Y14 receptors was characterised using the antagonists, cangrelor, ticagrelor, AR-C67085 and MRS2211 or PPTN respectively. This has yet to be backed up by receptor knockout experiments. Thus, subtype-selective antagonists and receptor knockout/knockdown have helped identify which P2Y subtypes are functionally expressed in vascular smooth muscle and endothelial cells and the effects that they mediate.

Platelet P2Y1 receptor exhibits constitutive G protein signaling and ß-arrestin 2 recruitment.

BACKGROUND: Purinergic P2Y1 and P2Y12 receptors (P2Y1-R and P2Y12-R) are G protein-coupled receptors (GPCR) activated by adenosine diphosphate (ADP) to mediate platelet activation, thereby playing a pivotal role in hemostasis and thrombosis. While P2Y12-R is the major target of antiplatelet drugs, no P2Y1-R antagonist has yet been developed for clinical use. However, accumulating data suggest that P2Y1-R inhibition would ensure efficient platelet inhibition with minimal effects on bleeding. In this context, an accurate characterization of P2Y1-R antagonists constitutes an important preliminary step. RESULTS: Here, we investigated the pharmacology of P2Y1-R signaling through Gq and ß-arrestin pathways in HEK293T cells and in mouse and human platelets using highly sensitive resonance energy transfer-based technologies (BRET/HTRF). We demonstrated that at basal state, in the absence of agonist ligand, P2Y1-R activates Gq protein signaling in HEK293T cells and in mouse and human platelets, indicating that P2Y1-R is constitutively active in physiological conditions. We showed that P2Y1-R also promotes constitutive recruitment of ß-arrestin 2 in HEK293T cells. Moreover, the P2Y1-R antagonists MRS2179, MRS2279 and MRS2500 abolished the receptor dependent-constitutive activation, thus behaving as inverse agonists. CONCLUSIONS: This study sheds new light on P2Y1-R pharmacology, highlighting for the first time the existence of a constitutively active P2Y1-R population in human platelets. Given the recent interest of P2Y12-R constitutive activity in patients with diabetes, this study suggests that modification of constitutive P2Y1-R signaling might be involved in pathological conditions, including bleeding syndrome or high susceptibility to thrombotic risk. Thus, targeting platelet P2Y1-R constitutive activation might be a promising and powerful strategy for future antiplatelet therapy.

Purinergic signaling: a potential therapeutic target for depression and chronic pain.

The comorbid mechanism of depression and chronic pain has been a research hotspot in recent years. Until now, the role of purinergic signals in the comorbid mechanism of depression and chronic pain has not been fully understood. This review mainly summarizes the research results published in PubMed during the past 5 years and concludes that purinergic signaling is a potential therapeutic target for comorbid depression and chronic pain, and the purinergic receptors A1, A2A, P2X3, P2X4, and P2X7and P2Y6, P2Y1, and P2Y12 may be important factors. The main potential pathways are as follows: A1 receptor-related G protein-dependent activation of introverted K+ channels (GIRKs), A2A receptor-related effects on the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) and MAPK/nuclear factor-κB (NF-κB) pathways, P2X3 receptor-related effects on dorsal root ganglia (DRG) excitability, P2X4 receptor-related effects on proinflammatory cytokines and inflammasome activation, P2X7 receptor-related effects on ion channels, the NLRP3 inflammasome and brain-derived neurotrophic factor (BDNF), and P2Y receptor-related effects on the phospholipase C (PLC)/inositol triphosphate (IP3)/Ca2+ signaling pathway. We hope that the conclusions of this review will provide key ideas for future research on the role of purinergic signaling in the comorbid mechanism of depression and chronic pain.

Transactivation of receptor tyrosine kinases by purinergic P2Y and adenosine receptors.

Transactivation of receptor tyrosine kinases (RTK) is a crosstalk mechanism exhibited by G-protein-coupled receptors (GPCR) to activate signaling pathways classically associated with growth factors. The discovery of RTK transactivation was a breakthrough in signal transduction that contributed to developing current concepts in intracellular signaling. RTK transactivation links GPCR signaling to important cellular processes, such as cell proliferation and differentiation, and explains the functional diversity of these receptors. Purinergic (P2Y and adenosine) receptors belong to class A of GPCR; in the present work, we systematically review the experimental evidence showing that purinergic receptors have the ability to transactivate RTK in multiple tissues and physiopathological conditions resulting in the modulation of cellular physiology. Of particular relevance, the crosstalk between purinergic receptors and epidermal growth factor receptor is a redundant pathway that participates in multiple pathophysiological processes. Specific and detailed knowledge of purinergic receptor-regulated pathways advances our understanding of the complexity of GPCR signal transduction and opens the way for pharmacologic intervention in the pathological context.

The developmental journey of therapies targeting purine receptors: from basic science to clinical trials.

Since the discovery of ATP as an extracellular signalling molecule in 1972, purinergic signalling, mediated by extracellular purines and pyrimidines has been identified in virtually all mammalian tissues and is implicated in regulating fundamental cellular processes. In recent years, there has been an increasing focus on the pathophysiology and potential therapeutic interventions based on purinergic signalling. A vast range of compounds targeting purine receptors are in clinical development, and many more are in preclinical studies, which highlights the fast growth in this research field. As a tribute to Professor Geoffrey Burnstock's legacy in purinergic signalling, we present here a brief review of compounds targeting purine receptors that are in different stages of clinical trials. The review highlights the 50-year journey from basic research on purinergic receptors to clinical applications of therapies targeting purine receptors.

Synthesis and pharmacological characterization of multiply substituted 2H-chromene derivatives as P2Y6 receptor antagonists.

P2Y6 receptor (P2Y6R) antagonists represent potential drugs for treating cancer, pain, neurodegeneration, asthma, diabetes, colitis and other disorders. However, there are few chemical classes of known competitive antagonists. We recently explored the structure activity relationship (SAR) of 2H-chromene derivatives as P2Y6R antagonists of moderate affinity. New analogues in this series modified at five positions were synthesized and shown to antagonize Ca2+ transients induced by the native agonist UDP in human (h) P2Y6R-expressing (but not turkey P2Y1R-, hP2Y2R- or hP2Y4R-expressing) astrocytoma cells. Alternatives to the reported 2-(trifluoromethyl)- and 3-nitro- substitutions of this scaffold were not identified. However, 6­fluoro 11 and 6­chloro 12 analogues displayed enhanced potency compared to other halogens, although still in the 1 - 2 µM range. Similar halogen substitution at 5, 7 or 8 positions reduced affinity. 5- or 8­Triethylsilylethynyl extension maintained hP2Y6R affinity, with IC50 0.46 µM for 26 (MRS4853). The 6,8­difluoro analogue 27 (IC50 2.99 µM) lacked off-target activities among 45 sites examined, unlike earlier analogues that bound to biogenic amine receptors. 11 displayed only one weak off-target activity (σ2). Mouse P2Y6R IC50s of 5, 25, 26 and 27 were 4.94, 17.6, 6.15 and 17.8 µM, respectively, but most other analogues had reduced affinity (>20 µM) compared to the hP2Y6R. These analogues are suitable for evaluation in in vivo inflammation and cancer models, which will be performed in the future studies.

Influence of Genetic and Epigenetic Factors of P2Y12 Receptor on the Safety and Efficacy of Antiplatelet Drugs.

PURPOSE: P2Y12 receptor inhibitors are drugs that decrease the risk of stent thrombosis and lower the long-term risk of non-stent-related myocardial infarction and stroke. They inhibit the binding of adenosine diphosphate (ADP) to the P2Y12 receptor and effectively reduce platelet reactivity. However, considerable variability in the pharmacodynamics response contributes to a failure of antiplatelet therapy; this phenomenon is especially notorious for older drugs, such as clopidogrel. Some genetic polymorphisms associated with these drugs' metabolic pathway, especially in the CYP2C19 gene, can significantly decrease antiplatelet efficacy. There are few reports on the variability stemming from the target of this drug class that is the P2Y12 receptor itself. RESULTS AND CONCLUSION: This review summarizes the results of research that focus on the influence of P2Y12 genetic polymorphisms on the pharmacodynamics and the efficacy of P2Y12 inhibitors. We found that the conclusions of the studies are unequivocal, and despite several strong candidates, such as G52T (rs6809699) or T744C (rs2046934), they may not be independent predictors of the inadequate response to the drug. Most probably, P2Y12 genetic polymorphisms contribute to the effect exerted by other gene variants (such as CYP2C19*2/*3/*17), drug interactions, or patient habits, such as smoking. Also, epigenetic modifications, such as methylation or miRNA levels, may play a role in the efficacy of antiplatelet treatment.

ATP induces contraction of cultured brain capillary pericytes via activation of P2Y-type purinergic receptors.

Brain capillary pericytes have been suggested to play a role in the regulation of cerebral blood flow under physiological and pathophysiological conditions. ATP has been shown to cause constriction of capillaries under ischemic conditions and suggested to be involved in the "no-reflow" phenomenon. To investigate the effects of extracellular ATP on pericyte cell contraction, we studied purinergic receptor activation of cultured bovine brain capillary pericytes. We measured intracellular Ca2+ concentration ([Ca2+]i) responses to purinergic agonists with the fluorescent indicators fura-2 and Cal-520 and estimated contraction of pericytes as relative change in cell area, using real-time confocal imaging. Addition of ATP caused an increase in cytosolic calcium and contraction of the brain capillary pericytes, both reversible and inhibited by the purinergic receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). Furthermore, we demonstrated that ATP-induced contraction could be eliminated by intracellular calcium chelation with BAPTA, indicating that the contraction was mediated via purinergic P2-type receptor-mediated [Ca2+]i signaling. ATP stimulation induced inositol triphosphate signaling, consistent with the notion of P2Y receptor activation. Receptor profiling studies demonstrated the presence of P2Y1 and P2Y2 receptors, using ATP, UTP, ADP, and the subtype specific agonists MRS2365 (P2Y1) and 2-thio-UTP (P2Y2). Addition of specific P2X agonists only caused an [Ca2+]i increase at high concentrations, attributed to activation of inositol triphosphate signaling. Our results suggest that contraction of brain capillary pericytes in vitro by activation of P2Y-type purinergic receptors is caused by intracellular calcium release. This adds more mechanistic understanding of the role of pericytes in vessel constriction and points toward P2Y receptors as potential therapeutic targets.NEW & NOTEWORTHY The study concerns brain capillary pericytes, which have been suggested to play a role in the regulation of cerebral blood flow. We show that extracellular ATP causes contraction of primary brain pericytes by stimulation of purinergic receptors and subsequent release of intracellular Ca2+ concentration ([Ca2+]i). The contraction is mainly mediated through activation of P2Y-receptor subtypes, including P2Y1 and P2Y2. These findings add more mechanistic understanding of the role of pericytes in regulation of capillary blood flow. ATP was earlier suggested to be involved in capillary constriction in brain pathologies, and our study gives a detailed account of a part of this important mechanism.

Recommended tool compounds and drugs for blocking P2X and P2Y receptors.

This review article presents a collection of tool compounds that selectively block and are recommended for studying P2Y and P2X receptor subtypes, investigating their roles in physiology and validating them as future drug targets. Moreover, drug candidates and approved drugs for P2 receptors will be discussed.

Burnstock and the legacy of the inhibitory junction potential and P2Y1 receptors.

The synaptic event called the inhibitory junction potential (IJP) was arguably one of the more important discoveries made by Burnstock and arguably one of his finer legacies. The discovery of the IJP fundamentally changed how electromechanical coupling was visualised in gastrointestinal smooth muscle. Its discovery also set in motion the search for novel inhibitory neurotransmitters in the enteric nervous system, eventually leading to proposal that ATP or a related nucleotide was a major inhibitory transmitter. The subsequent development of purinergic signalling gave impetus to expanding the classification of surface receptors for extracellular ATP, not only in the GI tract but beyond, and then led to successive phases of medicinal chemistry as the P2 receptor field developed. Ultimately, the discovery of the IJP led to the successful cloning of the first P2Y receptor (chick P2Y1) and expansion of mammalian ATP receptors into two classes: metabotropic P2Y receptors (encompassing P2Y1, P2Y2, P2Y4, P2Y6, P2Y11-14 receptors) and ionotropic P2X receptors (encompassing homomeric P2X1-P2X7 receptors). Here, the causal relationship between the IJP and P2Y1 is explored, setting out the milestones reached and achievements made by Burnstock and his colleagues.

Tribute to Prof. Geoffrey Burnstock: transition of purinergicsignaling to drug discovery.

Geoffrey Burnstock made a chance observation early in his research career that did not fit the conventional scientific dogma-non-noradrenergic, non-cholinergic (NANC) nerves. Instead of rejecting these as an artifact, he followed their logical course to characterize the actions of extracellular ATP on nerves and muscles, eventually founding a large branch of pharmacology around purinergic signaling. The solid proof that validated his concept and dismissed many detractors was the cloning of seven ionotropic P2X receptors and eight metabotropic P2Y receptors, which are expressed in some combination in every tissue and organ. Given the broad importance of this signaling system in biology, medicinal chemists, inspired by Burnstock, began creating synthetic agonists and antagonists for these purinergic receptors. Various ligands have advanced to clinical trials, for disorders of the immune, nervous, cardiovascular, and other systems, and a few are already approved. Thus, medically important approaches have been derived from Burnstock's original pharmacological concepts and his constant guiding of the course of the field. The therapeutic potential of modulators of purinergic signaling is vast.

Structure activity relationship of 3-nitro-2-(trifluoromethyl)-2H-chromene derivatives as P2Y6 receptor antagonists.

Various 6-alkynyl analogues of a known 3-nitro-2-(trifluoromethyl)-2H-chromene antagonist 3 of the Gq-coupled P2Y6 receptor (P2Y6R) were synthesized using a Sonogashira reaction to replace a 6-iodo group. The analogues were tested in a functional assay consisting of inhibition of calcium mobilization in P2Y6R-expressing astrocytoma cells elicited by native P2Y6R agonist UDP. 6-Ethynyl and 6-cyano groups were installed, and the alkynes were extended through both alkyl and aryl spacers. The most potent antagonists, with IC50 of ~1 µM, were found to be trialkylsilyl-ethynyl 7 and 8 (3-5 fold greater affinity than reference 3), t-butyl prop-2-yn-1-ylcarbamate 14 and p-carboxyphenyl-ethynyl 16 derivatives, and 3 and 8 displayed surmountable antagonism of UDP-induced production of inositol phosphates. Other chain-extended terminal carboxylate derivatives were less potent than the corresponding methyl ester derivatives. Thus, the 6 position in this chromene series is suitable for derivatization with flexibility of substitution, even with sterically extended chains, without losing P2Y6R affinity. However, a 3-carboxylic acid or 3-ester substitution did not serve as a nitro bioisostere, as the affinity was eliminated. These compounds provide additional ligand tools for the underexplored P2Y6R, which is a target for inflammatory, neurodegenerative and metabolic diseases.

Synthesis, characterization and biological evaluation of indomethacin derived thioureas as purinergic (P2Y1, P2Y2, P2Y4, and P2Y6) receptor antagonists.

G-protein-coupled receptors for extracellular nucleotides are known as P2Y receptors and are made up of eight members that are encoded by distinct genes and can be classified into two classes based on their affinity for specific G-proteins. P2Y receptor modulators have been studied extensively, but only a few small-molecule P2Y receptor antagonists have been discovered so far and approved by drug agencies. Derivatives of indole carboxamide have been identified as P2Y12 and P2X7 antagonist, as a result, we developed and tested a series of indole derivatives4a-lhaving thiourea moiety as P2Y receptor antagonist by using a fluorescence-based assay to measure the inhibition of intracellular calcium release in 1321N1 astrocytoma cells that had been stably transfected with the P2Y1, P2Y2, P2Y4 and P2Y6 receptors. Most of the compounds exhibited moderate to excellent inhibition activity against P2Y1 receptor subtype. The series most potent compound, 4h exhibited an IC50 value of 0.36 ± 0.01 µM selectivity against other subtypes of P2Y receptor. To investigate the ligand-receptor interactions, the molecular docking studies were carried out. Compound 4h is the most potent P2Y1 receptor antagonist due to interaction with an important amino acid residue Pro105, in addition to Ile108, Phe119, and Leu102.

Purinergic Signalling in Allogeneic Haematopoietic Stem Cell Transplantation and Graft-versus-Host Disease.

Allogeneic haematopoietic stem cell transplantation (allo-HSCT) is a curative therapy for blood cancers and other haematological disorders. However, allo-HSCT leads to graft-versus-host disease (GVHD), a severe and often lethal immunological response, in the majority of transplant recipients. Current therapies for GVHD are limited and often reduce the effectiveness of allo-HSCT. Therefore, pro- and anti-inflammatory factors contributing to disease need to be explored in order to identify new treatment targets. Purinergic signalling plays important roles in haematopoiesis, inflammation and immunity, and recent evidence suggests that it can also affect haematopoietic stem cell transplantation and GVHD development. This review provides a detailed assessment of the emerging roles of purinergic receptors, most notably P2X7, P2Y2 and A2A receptors, and ectoenzymes, CD39 and CD73, in GVHD.

The P2Y6 receptor signals through Gαq /Ca2+ /PKCα and Gα13 /ROCK pathways to drive the formation of membrane protrusions and dictate cell migration.

Cell migration is a ubiquitous process necessary to maintain and restore tissue functions. However, in cancer, cell migration leads to metastasis development and thus worsens the prognosis. Although the mechanism of cell migration is well understood, the identification of new targets modulating cell migration and deciphering their signaling events could lead to new therapies to restore tissue functions in diseases, such as inflammatory bowel disease, or to block metastatic development in different forms of cancer. Previous research has identified the G-protein-coupled P2Y6 receptor as an innovative target that could dictate cell migration under normal and pathological conditions. Surprisingly, there is little information on the cellular events triggered by activated P2Y6 during cell migration. Here, we demonstrated that P2Y6 activation stimulated A549 human lung cancer cells and Caco-2 colorectal cancer cell migration. Activated P2Y6 increased the number of filopodia and focal adhesions; two migratory structures required for cell migration. The generation of these structures involved Gαq /calcium/protein kinases C (PKC) and Gα13 /RHO-associated protein kinase-dependent pathways that dictate the formation of the migratory structures. These pathways led to the stabilization of the actin cytoskeleton through a PKC-dependent phosphorylation of cofilin. These results support the idea that the P2Y6 receptor represents a target of interest to modulate cell migration and revealed an intricate dialogue between two Gα-protein signaling pathways.

P2 Purinergic Signaling in the Distal Lung in Health and Disease.

The distal lung provides an intricate structure for gas exchange in mammalian lungs. Efficient gas exchange depends on the functional integrity of lung alveoli. The cells in the alveolar tissue serve various functions to maintain alveolar structure, integrity and homeostasis. Alveolar epithelial cells secrete pulmonary surfactant, regulate the alveolar surface liquid (ASL) volume and, together with resident and infiltrating immune cells, provide a powerful host-defense system against a multitude of particles, microbes and toxicants. It is well established that all of these cells express purinergic P2 receptors and that purinergic signaling plays important roles in maintaining alveolar homeostasis. Therefore, it is not surprising that purinergic signaling also contributes to development and progression of severe pathological conditions like pulmonary inflammation, acute lung injury/acute respiratory distress syndrome (ALI/ARDS) and pulmonary fibrosis. Within this review we focus on the role of P2 purinergic signaling in the distal lung in health and disease. We recapitulate the expression of P2 receptors within the cells in the alveoli, the possible sources of ATP (adenosine triphosphate) within alveoli and the contribution of purinergic signaling to regulation of surfactant secretion, ASL volume and composition, as well as immune homeostasis. Finally, we summarize current knowledge of the role for P2 signaling in infectious pneumonia, ALI/ARDS and idiopathic pulmonary fibrosis (IPF).

Nucleotide receptor P2RY4 is required for head formation via induction and maintenance of head organizer in Xenopus laevis.

Vertebrates have unique head structures that are mainly composed of the central nervous system, the neural crest, and placode cells. These head structures are brought about initially by the neural induction between the organizer and the prospective neuroectoderm at early gastrula stage. Purinergic receptors are activated by nucleotides released from cells and influence intracellular signaling pathways, such as phospholipase C and adenylate cyclase signaling pathways. As P2Y receptor is vertebrate-specific and involved in head formation, we expect that its emergence may be related to the acquisition of vertebrate head during evolution. Here, we focused on the role of p2ry4 in early development in Xenopus laevis and found that p2ry4 was required for the establishment of the head organizer during neural induction and contributed to head formation. We showed that p2ry4 was expressed in the head organizer region and the prospective neuroectoderm at early gastrula stage, and was enriched in the head components. Disruption of p2ry4 function resulted in the small head phenotype and the reduced expression of marker genes specific for neuroectoderm and neural border at an early neurula stage. Furthermore, we examined the effect of p2ry4 disruption on the establishment of the head organizer and found that a reduction in the expression of head organizer genes, such as dkk1 and cerberus, and p2ry4 could also induce the ectopic expression of these marker genes. These results suggested that p2ry4 plays a key role in head organizer formation. Our study demonstrated a novel role of p2ry4 in early head development.

Functional characterization of purinergic receptor P2Y14 in the Japanese flounder (Paralichthys olivaceus) head kidney macrophages.

Extracellular nucleotides and nucleotide sugars are important danger-associated signaling molecules that play critical roles in regulation of immune responses in mammals through activation of purinergic receptors located on the cell surface. However, the immunological role of extracellular UDP-glucose-activated P2Y14 receptor (P2Y14R) in fish still remains unknown. In this study, we identified and characterized a P2Y14R paralog in the Japanese flounder (Paralichthys olivaceus). The mRNA transcripts of P2Y14R are detected in all examined Japanese flounder tissues. Compared with the UDP-activated P2Y6 receptor, however, P2Y14R gene is highly expressed in Japanese flounder head kidney macrophages (HKMs). In addition, P2Y14R is significantly upregulated following inflammatory stimulation with LPS and poly (I:C) in the HKMs, suggesting a role of P2Y14R in response to inflammation in fish. Furthermore, activation of P2Y14 receptor with its potent and selective agonist MRS 2905 resulted in a decreased expression of LPS-induced pro-inflammatory cytokine IL-1beta gene in the HKMs. In contrast, inhibition of P2Y14 receptor activity or down-regulation of the endogenous expression of P2Y14R by small interfering RNA significantly upregulates the LPS-induced pro-inflammatory cytokine IL-1beta gene expression in the HKMs, demonstrating that P2Y14R is involved in inflammation regulation in fish. Moreover, stimulation of the Japanese flounder HKMs with UDP-glucose evoked a rapid increase of extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation in a dose- and time-dependent manner, indicating the involvement of P2Y14R in activation of ERK1/2 signaling in fish immune cells. Taken together, we demonstrated that the inducible P2Y14R plays an important role in regulation of fish innate immunity.

Extended dual antiplatelet therapy for Asian patients with acute coronary syndrome: expert recommendations.

BACKGROUND: Recently, new evidence from large scale trials and updated guidelines have emerged on the risks and benefits of extended dual antiplatelet therapy (DAPT) for patients with acute coronary syndrome (ACS). AIMS: To discuss, clarify and advise on the application of the evidences and guidelines on individual patient selection for extended DAPT, with regard to balancing risk factors, particularly in Asian populations. METHODS: A total of 14 local cardiologists from Hong Kong with extensive experience in cardiology and cardiac interventions convened in a series of 3 advisory board meetings from October 2016 to September 2017, which included reviews of new evidence in the literature and discussions of the latest clinical trends, using an anonymous, electronic voting system for arriving at consensuses. RESULTS: Recommendations were produced for the following nine risk factors: old age (>65), chronic kidney disease (CKD), diabetes mellitus (DM), recurrent myocardial infarction (MI), multi-vessel disease (MVD), multiple stents, bioresorbable vascular scaffold (BVS) stent, left main stenting and peripheral artery disease (PAD). Strong ischaemic risk factors include DM, recurrent MI, MVD and/or >3 stents; less-strong ischaemic factors include CKD, left main stenting, BVS stent and PAD. Old age can be an unclear risk factor due to variations in physical fitness even among patients of identical age. DISCUSSION: The strengths and limitations of the results were acknowledged. CONCLUSION: ACS patients with ischaemic risk factors could be considered for extended DAPT beyond 12 months, while balancing the risk of bleeding.