Engineered P2Y12-Overexpressing Cell-Membrane-Wrapped Nanoparticles for the Functional Reversal of Ticagrelor and Clopidogrel.

Antiplatelet agents, particularly P2Y12 receptor inhibitors, are critical medicines in the prevention and treatment of thrombotic diseases in the clinic. However, their long-term use introduces a significant risk of bleeding in patients with cardiovascular diseases. Whether the bleeding is caused by the drug itself or due to surgical procedures or trauma, the need to rapidly reverse the effects of antiplatelet agents in the circulation is essential; however, no such agents are currently available. To address this need, here we describe a strategy that uses cell-membrane-wrapped nanoparticles (CM-NPs) for the rapid reversal of P2Y12 inhibitors. CM-NPs are fabricated with membranes derived from 293T cells genetically engineered to overexpress the P2Y12 receptor. Our findings support the potential of CM-NPs as a strategy for managing bleeding complications associated with P2Y12 receptor inhibitors, offering an approach to improve the safety in the use of these drugs in clinical settings.

Microglia in the hypothalamic paraventricular nucleus sense hemodynamic disturbance and promote sympathetic excitation in hypertension.

Hypertension is usually accompanied by elevated sympathetic tonicity, but how sympathetic hyperactivity is triggered is not clear. Recent advances revealed that microglia-centered neuroinflammation contributes to sympathetic excitation in hypertension. In this study, we performed a temporospatial analysis of microglia at both morphological and transcriptomic levels and found that microglia in the hypothalamic paraventricular nucleus (PVN), a sympathetic center, were early responders to hypertensive challenges. Vasculature analyses revealed that the PVN was characterized by high capillary density, thin vessel diameter, and complex vascular topology relative to other brain regions. As such, the PVN was susceptible to the penetration of ATP released from the vasculature in response to hemodynamic disturbance after blood pressure increase. Mechanistically, ATP ligation to microglial P2Y12 receptor was responsible for microglial inflammatory activation and the eventual sympathetic overflow. Together, these findings identified a distinct vasculature pattern rendering vulnerability of PVN pre-sympathetic neurons to hypertension-associated microglia-mediated inflammatory insults.

P2Y12 Receptor Inhibitor for Antiaggregant Therapies: From Molecular Pathway to Clinical Application.

Platelets play a significant role in hemostasis, forming plugs at sites of vascular injury to limit blood loss. However, if platelet activation is not controlled, it can lead to thrombotic events, such as myocardial infarction and stroke. To prevent this, antiplatelet agents are used in clinical settings to limit platelet activation in patients at risk of arterial thrombotic events. However, their use can be associated with a significant risk of bleeding. An enhanced comprehension of platelet signaling mechanisms should facilitate the identification of safer targets for antiplatelet therapy. Over the past decade, our comprehension of the breadth and intricacy of signaling pathways that orchestrate platelet activation has expanded exponentially. Several recent studies have provided further insight into the regulation of platelet signaling events and identified novel targets against which to develop novel antiplatelet agents. Antiplatelet drugs are essential in managing atherothrombotic vascular disease. The current antiplatelet therapy in clinical practice is limited in terms of safety and efficacy. Novel compounds have been developed in response to patient variability and resistance to aspirin and/or clopidogrel. Recent studies based on randomized controlled trials and systematic reviews have definitively demonstrated the role of antiplatelet therapy in reducing the risk of cardiovascular events. Antiplatelet therapy is the recommended course of action for patients with established atherosclerosis. These studies compared monotherapy with a P2Y12 inhibitor versus aspirin for secondary prevention. However, in patients undergoing percutaneous coronary intervention, it is still unclear whether the efficacy of P2Y12 inhibitor monotherapy after a short course of dual antiplatelet therapy depends on the type of P2Y12 inhibitor. This paper focuses on the advanced-stage evaluation of several promising antiplatelet drugs.

G-protein coupled receptors regulates Tauopathy in neurodegeneration.

In Alzheimer's disease, the microtubule-associated protein, Tau misfolds to form aggregates and filaments in the intra- and extracellular region of neuronal cells. Microglial cells are the resident brain macrophage cells involved in constant surveillance and activated by the extracellular deposits. Purinergic receptors are involved in the chemotactic migration of microglial cells towards the site of inflammation. From our recent study, we have observed that the microglial P2Y12 receptor is involved in phagocytosis of full-length Tau species such as monomers, oligomers and aggregates by actin-driven chemotaxis. This study shows the interaction of repeat-domain of Tau (TauRD) with the microglial P2Y12 receptor and the corresponding residues for interaction have been analyzed by various in-silico approaches. In the cellular studies, TauRD was found to interact with microglial P2Y12R and induces its cellular expression confirmed by co-immunoprecipitation and western blot analysis. Furthermore, the P2Y12R-mediated TauRD internalization has demonstrated activation of microglia with an increase in the Iba1 level, and TauRD becomes accumulated at the peri-nuclear region for the degradation.

Microglia contribute to neuronal synchrony despite endogenous ATP-related phenotypic transformation in acute mouse brain slices.

Acute brain slices represent a workhorse model for studying the central nervous system (CNS) from nanoscale events to complex circuits. While slice preparation inherently involves tissue damage, it is unclear how microglia, the main immune cells and damage sensors of the CNS react to this injury and shape neuronal activity ex vivo. To this end, we investigated microglial phenotypes and contribution to network organization and functioning in acute brain slices. We reveal time-dependent microglial phenotype changes influenced by complex extracellular ATP dynamics through P2Y12R and CX3CR1 signalling, which is sustained for hours in ex vivo mouse brain slices. Downregulation of P2Y12R and changes of microglia-neuron interactions occur in line with alterations in the number of excitatory and inhibitory synapses over time. Importantly, functional microglia modulate synapse sprouting, while microglial dysfunction results in markedly impaired ripple activity both ex vivo and in vivo. Collectively, our data suggest that microglia are modulators of complex neuronal networks with important roles to maintain neuronal network integrity and activity. We suggest that slice preparation can be used to model time-dependent changes of microglia-neuron interactions to reveal how microglia shape neuronal circuits in physiological and pathological conditions.

Initial Despair and Current Hope of Identifying a Clinically Useful Treatment of Myocardial Reperfusion Injury: Insights Derived from Studies of Platelet P2Y12 Antagonists and Interference with Inflammation and NLRP3 Assembly.

Myocardial necrosis following the successful reperfusion of a coronary artery occluded by thrombus in a patient presenting with ST-elevation myocardial infarction (STEMI) continues to be a serious problem, despite the multiple attempts to attenuate the necrosis with agents that have shown promise in pre-clinical investigations. Possible reasons include confounding clinical risk factors, the delayed application of protective agents, poorly designed pre-clinical investigations, the possible effects of routinely administered agents that might unknowingly already have protected the myocardium or that might have blocked protection, and the biological differences of the myocardium in humans and experimental animals. A better understanding of the pathobiology of myocardial infarction is needed to stem this reperfusion injury. P2Y12 receptor antagonists minimize platelet aggregation and are currently part of the standard treatment to prevent thrombus formation and propagation in STEMI protocols. Serendipitously, these P2Y12 antagonists also dramatically attenuate reperfusion injury in experimental animals and are presumed to provide a similar protection in STEMI patients. However, additional protective agents are needed to further diminish reperfusion injury. It is possible to achieve additive protection if the added intervention protects by a mechanism different from that of P2Y12 antagonists. Inflammation is now recognized to be a critical factor in the complex intracellular response to ischemia and reperfusion that leads to tissue necrosis. Interference with cardiomyocyte inflammasome assembly and activation has shown great promise in attenuating reperfusion injury in pre-clinical animal models. And the blockade of the executioner protease caspase-1, indeed, supplements the protection already seen after the administration of P2Y12 antagonists. Importantly, protective interventions must be applied in the first minutes of reperfusion, if protection is to be achieved. The promise of such a combination of protective strategies provides hope that the successful attenuation of reperfusion injury is attainable.

P2Y12 Inhibition in Patients Requiring Oral Anticoagulation After Percutaneous Coronary Intervention: The SWAP-AC-2 Study.

BACKGROUND: Among patients treated with a novel oral anticoagulant (NOAC) undergoing percutaneous coronary intervention (PCI), combination therapy with clopidogrel (ie, known as dual antithrombotic therapy [DAT]) is the treatment of choice. However, there are concerns for individuals with impaired response to clopidogrel. OBJECTIVES: The authors sought to assess the pharmacodynamic (PD) effects of clopidogrel vs low-dose ticagrelor in patients with impaired clopidogrel response assessed by the ABCD-GENE score. METHODS: This was a prospective, randomized PD study of NOAC-treated patients undergoing PCI. Patients with an ABCD-GENE score ≥10 (n = 39), defined as having impaired clopidogrel response, were randomized to low-dose ticagrelor (n = 20; 60 mg twice a day) or clopidogrel (n = 19; 75 mg once a day). Patients with an ABCD-GENE score <10 (n = 42) were treated with clopidogrel (75 mg once a day; control cohort). PD assessments at baseline and 30 days post-randomization (trough and peak) were performed to assess P2Y12 signaling (VerifyNow P2Y12 reaction units [PRU], light transmittance aggregometry, and vasodilator-stimulated phosphoprotein); makers of thrombosis not specific to P2Y12 signaling were also assessed. The primary endpoint was PRU (trough levels) at 30 days. RESULTS: At 30 days, PRU levels were reduced with ticagrelor-based DAT compared with clopidogrel-based DAT at trough (23.0 [Q1-Q3: 3.0-46.0] vs 154.5 [Q1-Q3: 77.5-183.0]; P < 0.001) and peak (6.0 [Q1-Q3: 4.0-14.0] vs 129.0 [Q1-Q3: 66.0-171.0]; P < 0.001). Trough PRU levels in the control arm (104.0 [Q1-Q3: 35.0-167.0]) were higher than ticagrelor-based DAT (P = 0.005) and numerically lower than clopidogrel-based DAT (P = 0.234). Results were consistent by light transmittance aggregometry and vasodilator-stimulated phosphoprotein. Markers measuring other pathways leading to thrombus formation were largely unaffected. CONCLUSIONS: In NOAC-treated patients undergoing PCI with an ABCD-GENE score ≥10, ticagrelor-based DAT using a 60-mg, twice-a-day regimen reduced platelet P2Y12 reactivity compared with clopidogrel-based DAT. (Tailoring P2Y12 Inhibiting Therapy in Patients Requiring Oral Anticoagulation After PCI [SWAP-AC-2]; NCT04483583).

Trimethylamine-N-oxide aggravated the sympathetic excitation in D-galactose induced aging rats by down-regulating P2Y12 receptor in microglia.

This study aimed to determine whether trimethylamine N-oxide (TMAO) was involved in sympathetic activation in aging and the underlying mechanisms. Our hypothesis is TMAO reduces P2Y12 receptor (P2Y12R) and induces microglia-mediated inflammation in the paraventricular nucleus (PVN), then leading to sympathetic activation in aging. This study involved 18 young adults and 16 old adults. Aging rats were established by injecting D-galactose (D-gal, 200 mg/kg/d) subcutaneously for 12 weeks. TMAO (120 mg/kg/d) or 1% 3, 3-dimethyl-l-butanol (DMB) was administrated via drinking water for 12 weeks to investigate their effects on neuroinflammation and sympathetic activation in aging rats. Plasma TMAO, NE and IL-1ß levels were higher in old adults than in young adults. In addition, standard deviation of all normal to normal intervals (SDNN) and standard deviation of the average of normal to normal intervals (SDANN) were lower in old adults and negatively correlated with TMAO, indicating sympathetic activation in old adults, which is associated with an increase in TMAO levels. Treatment of rats with D-gal showed increased senescence-associated protein levels and microglia-mediated inflammation, as well as decreased P2Y12R protein levels in PVN. Plasma TMAO, NE and IL-1ß levels were increased, accompanied by enhanced renal sympathetic nerve activity (RSNA). While TMAO treatment exacerbated the above phenomenon, DMB mitigated it. These findings suggest that TMAO contributes to sympathetic hyperactivity in aging by downregulating P2Y12R in microglia and increasing inflammation in the PVN. These results may provide promising new target for the prevention and treatment of aging and aging-related diseases.

Interaction of Tau with G-Protein-Coupled Purinergic P2Y12 Receptor by Molecular Docking and Molecular Dynamic Simulation.

Alzheimer's disease, a progressive neurological disorder, is characterized by the accumulation of neurofibrillary tangles and senile plaques by Tau and amyloid-ß, respectively, in the brain microenvironment. The misfolded protein aggregates interact with several components of neuronal and glial cells such as membrane lipids, receptors, transporters, enzymes, cytoskeletal proteins, etc. Under pathological conditions, Tau interacts with several G-protein-coupled receptors (GPCRs), which undergoes either receptor signaling or desensitization followed by internalization of the protein complex. The purinergic GPCR, P2Y12 which is expressed in microglial cells, plays a key role in its activation and migration. Microglial cells sense and migrate to the site of injury aided by P2Y12 receptor that interacts with ADP released from damaged cells. P2Y12 receptor also interacts with misfolded Tau accumulated at the extracellular space and promotes receptor-mediated internalization. Immunocolocalization and co-immunoprecipitation studies demonstrated the interaction of Tau species with the P2Y12 receptor. Later, in-silico analyses were carried out with the repeat domain of Tau (TauRD), which has been identified as the interacting partner of P2Y12 receptor by in-vitro studies. Molecular docking and molecular dynamics simulation studies show the stability and the type of interaction in TauRD-receptor complex. Tau interaction with P2Y12 receptor plays a significant role in maintaining the active state of microglia which could lead to neuroinflammation and neuronal damage in AD brain. Hence, blocking P2Y12-Tau interaction and P2Y12-mediated Tau internalization in microglial cells could be possible therapeutic strategies in downregulating the severity of neuroinflammation in AD.

Purinergic Receptor P2Y12-Mediated Tau Internalization in Microglia.

Microglia are the resident brain macrophage cells that are involved in constant surveillance of brain microenvironment. In Alzheimer's disease, microglia get over activated upon the accumulation of Tau and amyloid-ß species in the extracellular space, ultimately leading to neurodegeneration. Microglia phagocytose the extracellular Tau species by several mechanisms among which P2Y12 receptor-mediated internalization of extracellular Tau is recently studied. Extracellular Tau activates microglia and directly interacts with the P2Y12 receptor. Tau-receptor complex is then internalized followed by perinuclear accumulation and lysosomal degradation. Upon microglial activation by extracellular Tau, P2Y12 receptor is also involved in membrane-associated actin remodeling which has its key role in active migration and phagocytosis.

Gualou-Xiebai herb pair and its active ingredients act against atherosclerosis by suppressing VSMC-derived foam cell formation via regulating P2RY12-mediated lipophagy.

BACKGROUND: Atherosclerosis (AS) is a chronic disease characterized by lipid accumulation in the aortic wall and the formation of foam cells overloaded with large lipids inclusions. Currently, Western medicine is primarily used to improve lipid metabolism disorders and reduce inflammatory reactions to delay AS progression, but these medicines come with serious side effects and drug resistance. Gualou-Xiebai (GLXB) is a renowned herb pair that has been proven effective against AS. However, the potential molecular mechanism through which GLXB exerts the anti-atherosclerotic effects of increasing lipophagy in vascular smooth muscle cells (VSMCs) remains unknown. PURPOSE: This study aims to explore the role of lipophagy and the therapeutic mechanism of GLXB in AS. METHODS: UPLC-Q-TOF-MS for the determination of the main components of GLXB-containing serum. An AS mouse model was established by feeding a high-fat diet (HFD) to ApoE-/- mice for 12 weeks. Ultrasonography monitoring was used to confirm the successful establishment of the AS model. Plaque areas and lipid deposition were evaluated using HE staining and aorta imagingafter GLXB treatment. Immunofluorescence staining and Western blotting were utilized to observe the P2RY12 and lipophagy levels in AS mice. VSMCs were stimulated with oxidized low-density lipoprotein (ox-LDL) to induce foam cell formation. The degree of lipophagy and the related molecular mechanisms were assessed after treating the VSMCs with GLXB-containing serum or si-P2RY12 transfection. The active components of GLXB-containing serum that act on P2RY12 were screened and verified by molecular docking and dual-luciferase reporter assays. RESULTS: Seventeen components of GLXB were identified in rat serum by UPLC-Q-TOF-MS. GLXB significantly reduced lipid deposition in HFD-fed ApoE-/- mice and ox-LDL-induced VSMCs. GLXB strikingly increased lipophagy levels by downregulating P2RY12, p62, and plin2, upregulating LC3Ⅱ protein expression, and increasing the number of autophagosomes. Notably, the lipophagy inhibitor CQ and the P2RY12 receptor agonist ADPß abolished the GLXB-induced increase in lipophagy. Last, we confirmed that albiflorin, apigenin, luteolin, kaempferol, 7,8-dihydroxyflavone, and hesperetin from GLXB significantly inhibited P2RY12. CONCLUSION: GLXB activates lipophagy and inhibits lipid accumulation-associated VSMC-derived foam cell formation through suppressing P2RY12 activation, resulting in anti-atherosclerotic effects. The GLXB components albiflorin, apigenin, luteolin, kaempferol, 7,8-dihydroxyflavone, and hesperetin are the potential active effectors against P2RY12.

Immunohistochemical localization of P2Y12 purinoceptors in the rat carotid body.

The present study investigated the localization of the adenosine 5'-diphosphate (ADP)-selective P2Y12 purinoceptors in the rat carotid body using multilabeling immunofluorescence. Punctate immunoreactive products for P2Y12 were distributed in chemoreceptive type I cells immunoreactive to vesicular nucleotide transporter (VNUT) or dopamine beta-hydroxylase, but not in S100B-immunoreactive glial-like type II cells. P2Y12 immunoreactivity was localized in cell clusters containing VNUT-immunoreactive type I cells surrounded by the perinuclear cytoplasm and cytoplasmic processes of type II cells immunoreactive for ectonucleoside triphosphate diphosphohydrolase 2 (NTPDase2) and NTPDase3, which hydrolyze extracellular nucleotide tri- and/or di-phosphates. In ATP bioluminescence assays using carotid bodies, the degradation of extracellular ATP was attenuated in the presence of the selective NTPDases inhibitor ARL67156, suggesting ATP-degrading activity by NTPDases in the tissue. These results suggest that ATP released from type I cells is degraded into ADP and adenosine 5'-monophosphate by NTPDases expressed in type II cells, and that ADP modulates type I cells via P2Y12 purinoceptors.

Antithrombotic Stewardship: Evaluation of Platelet Reactivity-Guided Cangrelor Dosing Using the VerifyNow Assay.

ABSTRACT: Cangrelor may be used as a bridge when temporary interruption of dual antiplatelet therapy is necessary. However, the optimal dose and monitoring of cangrelor in patients remains unknown, especially in the setting of mechanical circulatory support (MCS). We conducted an observational, single-center, retrospective cohort study of patients who had percutaneous coronary intervention within 3 months and received cangrelor while admitted to any intensive care unit. The primary outcome was the incidence of any major adverse cardiovascular event. Secondary outcomes included VerifyNow platelet reactivity units (PRUs) measured while on cangrelor and any bleeding events while on cangrelor. A total of 92 patients were included. The most common reason for cangrelor use was in the periprocedural setting, with or without MCS (42%-45%), followed by NPO status (26%-28%) and MCS alone (22%-24%). The primary outcome of major adverse cardiovascular event occurred in 1 patient (1.1%). Of 92 patients, 77% had a P2Y12 level collected within 24 hours, and 89% of the cohort was able to achieve the goal P2Y12 PRU of <194. The median P2Y12 value within 24 hours of cangrelor initation was 115 PRU (40-168 PRU). We observed a bleed event rate of 23% (21/92). We found a standardized protocol of cangrelor dosing in critically ill patients who received a drug-eluting stent in the past 3 months to be successful in achieving a goal P2Y12 PRU. Although the optimal PRU remains unknown, cardiovascular clinicians may monitor these levels to help guide decisions regarding cangrelor management. Future randomized controlled trials should evaluate the optimal PRU threshold to balance risks of ischemia and bleeding.

Blood oxygen regulation via P2Y12R expressed in the carotid body.

BACKGROUND: Peripheral blood oxygen monitoring via chemoreceptors in the carotid body (CB) is an integral function of the autonomic cardiorespiratory regulation. The presence of the purinergic P2Y12 receptor (P2Y12R) has been implicated in CB; however, the exact role of the receptor in O2 sensing and signal transduction is unknown. METHODS: The presence of P2Y12R was established by immunoblotting, RT qPCR and immunohistochemistry. Primary glomus cells were used to assess P2Y12R function during hypoxia and hypercapnia, where monoamines were measured by HPLC; calcium signal was recorded utilizing OGB-1 and N-STORM Super-Resolution System. Ingravescent hypoxia model was tested in anaesthetized mice of mixed gender and cardiorespiratory parameters were recorded in control and receptor-deficient or drug-treated experimental animals. RESULTS: Initially, the expression of P2Y12R in adult murine CB was confirmed. Hypoxia induced a P2Y12R-dependent release of monoamine transmitters from isolated CB cells. Receptor activation with the endogenous ligand ADP promoted release of neurotransmitters under normoxic conditions, while blockade disrupted the amplitude and duration of the intracellular calcium concentration. In anaesthetised mice, blockade of P2Y12R expressed in the CB abrogated the initiation of compensatory cardiorespiratory changes in hypoxic environment, while centrally inhibited receptors (i.e. microglial receptors) or receptor-deficiency induced by platelet depletion had limited influence on the physiological adjustment to hypoxia. CONCLUSIONS: Peripheral P2Y12R inhibition interfere with the complex mechanisms of acute oxygen sensing by influencing the calcium signalling and the release of neurotransmitter molecules to evoke compensatory response to hypoxia. Prospectively, the irreversible blockade of glomic receptors by anti-platelet drugs targeting P2Y12Rs, propose a potential, formerly unrecognized side-effect to anti-platelet medications in patients with pulmonary morbidities.

The role of platelet P2Y12 receptors in inflammation.

Inflammation is a complex pathophysiological process underlying many clinical conditions. Platelets contribute to the thrombo-inflammatory response. Platelet P2Y12 receptors amplify platelet activation, potentiating platelet aggregation, degranulation and shape change. The contents of platelet alpha granules, in particular, act directly on leucocytes, including mediating platelet-leucocyte aggregation and activation via platelet P-selectin. Much evidence for the role of platelet P2Y12 receptors in inflammation comes from studies using antagonists of these receptors, such as the thienopyridines clopidogrel and prasugrel, and the cyclopentyltriazolopyrimidine ticagrelor, in animal and human experimental models. These suggest that antagonism of P2Y12 receptors decreases markers of inflammation with some evidence that this reduces incidence of adverse clinical sequelae during inflammatory conditions. Interpretation is complicated by pleiotropic effects such as those of the thienopyridines on circulating leucocyte numbers and of ticagrelor on adenosine reuptake. The available evidence suggests that P2Y12 receptors are prominent mediators of inflammation and P2Y12 receptor antagonism as a potentially powerful strategy in a broad range of inflammatory conditions. 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.

Pharmacological characterization of P2Y receptor subtypes - an update.

P2Y receptors are G-protein-coupled receptors (GPCRs) for extracellular nucleotides. There are eight mammalian P2Y receptor subtypes (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14). The widely expressed P2Y receptors play important roles in physiology and pathophysiology. This review summarizes the use of pharmacological tools to characterize the P2Y receptor subtypes involved in these responses. MRS2500 is a potent and selective antagonist acting at the P2Y1 receptor. AR-C118925 is useful for the selective antagonism of the P2Y2 receptor. PSB16133 blocks the P2Y4 receptor, MRS2578 is an antagonist at the P2Y6 receptor and NF157 as well as NF340 block the P2Y11 receptor. ADP-induced platelet aggregation is mediated by P2Y1 and P2Y12 receptors. A number of compounds or their active metabolites reduce ADP-induced platelet aggregation by blocking the P2Y12 receptor. These include the active metabolites of the thienopyridine compounds clopidogrel and prasugrel, the nucleoside analogue ticagrelor and the nucleotide analogue cangrelor. PSB0739 is also a potent antagonist at the P2Y12 receptor useful for both in vitro and in vivo studies. MRS2211 and MRS2603 inhibit P2Y13 mediated responses. PPTN is a very potent antagonist at the P2Y14 receptor.

Stimulation of platelet P2Y1 receptors by different endogenous nucleotides leads to functional selectivity via biased signalling.

BACKGROUND AND PURPOSE: Platelet function during inflammation is dependent on activation by endogenous nucleotides. Non-canonical signalling via the P2Y1 receptor is important for these non-thrombotic functions of platelets. However, apart from ADP, the role of other endogenous nucleotides acting as agonists at P2Y1 receptors is unknown. This study compared the effects of ADP, Ap3A, NAD+ , ADP-ribose, and Up4A on platelet functions contributing to inflammation or haemostasis. EXPERIMENTAL APPROACH: Platelets obtained from healthy human volunteers were incubated with ADP, Ap3A, NAD+ , ADP-ribose, or Up4A, with aggregation and fibrinogen binding measured (examples of function during haemostasis) or before exposure to fMLP to measure platelet chemotaxis (an inflammatory function). In silico molecular docking of these nucleotides to the binding pocket of P2Y1 receptors was then assessed. KEY RESULTS: Platelet aggregation and binding to fibrinogen induced by ADP was not mimicked by NAD+ , ADP-ribose, and Up4A. However, these endogenous nucleotides induced P2Y1 -dependent platelet chemotaxis, an effect that required RhoA and Rac-1 activity, but not canonical PLC activity. Analysis of molecular docking of the P2Y1 receptor revealed distinct differences of amino acid interactions and depth of fit within the binding pocket for Ap3A, NAD+ , ADP-ribose, or Up4A compared with ADP. CONCLUSION AND IMPLICATIONS: Platelet function (aggregation vs motility) can be differentially modulated by biased-agonist activation of P2Y1 receptors. This may be due to the character of the ligand-binding pocket interaction. This has implications for future therapeutic strategies aimed to suppress platelet activation during inflammation without affecting haemostasis as is the requirement of current ant-platelet drugs. 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.

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 Cß (PLCß) 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.

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.