RESUMEN
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.
Asunto(s)
Inhibidores de Agregación Plaquetaria , Antagonistas del Receptor Purinérgico P2Y , Humanos , Antagonistas del Receptor Purinérgico P2Y/uso terapéutico , Antagonistas del Receptor Purinérgico P2Y/farmacología , Inhibidores de Agregación Plaquetaria/uso terapéutico , Inhibidores de Agregación Plaquetaria/farmacología , Receptores Purinérgicos P2Y12/metabolismo , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Transducción de Señal/efectos de los fármacos , Activación Plaquetaria/efectos de los fármacos , AnimalesRESUMEN
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.
Asunto(s)
Tauopatías , Humanos , Tauopatías/metabolismo , Tauopatías/patología , Proteínas tau/metabolismo , Microglía/metabolismo , Microglía/patología , Receptores Purinérgicos P2Y12/metabolismo , Animales , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
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.
Asunto(s)
Adenosina Trifosfato , Encéfalo , Receptor 1 de Quimiocinas CX3C , Microglía , Neuronas , Receptores Purinérgicos P2Y12 , Sinapsis , Animales , Microglía/metabolismo , Adenosina Trifosfato/metabolismo , Ratones , Neuronas/metabolismo , Receptor 1 de Quimiocinas CX3C/metabolismo , Receptor 1 de Quimiocinas CX3C/genética , Receptores Purinérgicos P2Y12/metabolismo , Receptores Purinérgicos P2Y12/genética , Encéfalo/metabolismo , Sinapsis/metabolismo , Ratones Endogámicos C57BL , Fenotipo , Masculino , Transducción de SeñalRESUMEN
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.
Asunto(s)
Inflamación , Daño por Reperfusión Miocárdica , Proteína con Dominio Pirina 3 de la Familia NLR , Antagonistas del Receptor Purinérgico P2Y , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Daño por Reperfusión Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/patología , Humanos , Antagonistas del Receptor Purinérgico P2Y/farmacología , Antagonistas del Receptor Purinérgico P2Y/uso terapéutico , Animales , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/antagonistas & inhibidores , Inflamación/tratamiento farmacológico , Inflamación/patología , Inflamación/metabolismo , Receptores Purinérgicos P2Y12/metabolismoRESUMEN
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, 200â¯mg/kg/d) subcutaneously for 12 weeks. TMAO (120â¯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.
Asunto(s)
Regulación hacia Abajo , Galactosa , Metilaminas , Microglía , Receptores Purinérgicos P2Y12 , Animales , Ratas , Envejecimiento/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Galactosa/farmacología , Inflamación/inducido químicamente , Inflamación/metabolismo , Interleucina-1beta/metabolismo , Metilaminas/farmacología , Microglía/efectos de los fármacos , Microglía/metabolismo , Norepinefrina/metabolismo , Núcleo Hipotalámico Paraventricular/efectos de los fármacos , Núcleo Hipotalámico Paraventricular/metabolismo , Ratas Sprague-Dawley , Receptores Purinérgicos P2Y12/metabolismo , Sistema Nervioso Simpático/efectos de los fármacos , Sistema Nervioso Simpático/metabolismoRESUMEN
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 LC3â ¡ 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.
Asunto(s)
Aterosclerosis , Medicamentos Herbarios Chinos , Células Espumosas , Músculo Liso Vascular , Receptores Purinérgicos P2Y12 , Animales , Aterosclerosis/tratamiento farmacológico , Células Espumosas/efectos de los fármacos , Células Espumosas/metabolismo , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Masculino , Ratones , Medicamentos Herbarios Chinos/farmacología , Receptores Purinérgicos P2Y12/metabolismo , Dieta Alta en Grasa , Ratones Endogámicos C57BL , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Ratas , Modelos Animales de Enfermedad , Autofagia/efectos de los fármacos , Ratas Sprague-Dawley , Metabolismo de los Lípidos/efectos de los fármacos , Aorta/efectos de los fármacos , Lipoproteínas LDL/metabolismoRESUMEN
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.
Asunto(s)
Enfermedad de Alzheimer , Simulación de Dinámica Molecular , Humanos , Simulación del Acoplamiento Molecular , Receptores Purinérgicos P2Y12/metabolismo , Antagonistas del Receptor Purinérgico P2Y , Enfermedades Neuroinflamatorias , Enfermedad de Alzheimer/metabolismo , Microglía/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas tau/metabolismoRESUMEN
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.
Asunto(s)
Enfermedad de Alzheimer , Microglía , Humanos , Microglía/metabolismo , Receptores Purinérgicos P2Y12/metabolismo , Antagonistas del Receptor Purinérgico P2Y , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Proteínas tau/metabolismoRESUMEN
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.
Asunto(s)
Cuerpo Carotídeo , Humanos , Ratones , Animales , Cuerpo Carotídeo/metabolismo , Oxígeno , Receptores Purinérgicos P2Y12/genética , Receptores Purinérgicos P2Y12/metabolismo , Calcio/metabolismo , Hipoxia/metabolismoRESUMEN
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.
Asunto(s)
Plaquetas , NAD , Humanos , Simulación del Acoplamiento Molecular , NAD/metabolismo , Adenosina Difosfato/farmacología , Adenosina Difosfato/metabolismo , Agregación Plaquetaria , Inflamación/metabolismo , Fibrinógeno/metabolismo , Fibrinógeno/farmacología , Adenosina Difosfato Ribosa/metabolismo , Adenosina Difosfato Ribosa/farmacología , Receptores Purinérgicos P2Y1/metabolismo , Receptores Purinérgicos P2Y12/metabolismoRESUMEN
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.
Asunto(s)
Plaquetas , Sepsis , Humanos , Plaquetas/metabolismo , Inhibidores de Agregación Plaquetaria/farmacología , Inhibidores de Agregación Plaquetaria/uso terapéutico , Antagonistas del Receptor Purinérgico P2Y/farmacología , Antagonistas del Receptor Purinérgico P2Y/uso terapéutico , Inmunidad , Sepsis/tratamiento farmacológico , Sepsis/metabolismo , Receptores Purinérgicos P2Y12/metabolismo , Agregación PlaquetariaRESUMEN
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.
Asunto(s)
Plaquetas , Agregación Plaquetaria , Humanos , Adenosina Difosfato/metabolismo , Plaquetas/fisiología , Transducción de Señal , Inflamación/metabolismo , Receptores Purinérgicos P2Y1/metabolismo , Receptores Purinérgicos P2Y12/metabolismo , Activación PlaquetariaRESUMEN
BACKGROUND AND PURPOSE: 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. EXPERIMENTAL APPROACH: 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. KEY RESULTS: 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. CONCLUSION AND IMPLICATIONS: 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.
Asunto(s)
Síndrome Coronario Agudo , Difosfatos , Humanos , Ticagrelor/farmacología , Ticagrelor/metabolismo , Ticagrelor/uso terapéutico , Difosfatos/metabolismo , Difosfatos/farmacología , Difosfatos/uso terapéutico , Adenosina/farmacología , Agonismo Inverso de Drogas , Antagonistas del Receptor Purinérgico P2Y/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Adenosina Difosfato/farmacología , Adenosina Difosfato/metabolismo , Plaquetas , Síndrome Coronario Agudo/tratamiento farmacológico , Síndrome Coronario Agudo/complicaciones , Receptores Purinérgicos P2Y12/metabolismoRESUMEN
Increasing evidence suggests that immunometabolism has started to unveil the role of metabolism in shaping immune function and autoimmune diseases. In this study, our data show that purinergic receptor P2Y12 (P2RY12) is highly expressed in concanavalin A (ConA)-induced immune hepatitis mouse model and serves as a potential metabolic regulator in promoting metabolic reprogramming from oxidative phosphorylation to glycolysis in T cells. P2RY12 deficiency or inhibition of P2RY12 with P2RY12 inhibitors (clopidogrel and ticagrelor) are proved to reduce the expression of inflammatory mediators, cause CD4+ and CD8+ effector T cells hypofunction and protect the ConA-induced immune hepatitis. A combined proteomics and metabolomics analysis revealed that P2RY12 deficiency causes redox imbalance and leads to reduced aerobic glycolysis by downregulating the expression of hexokinase 2 (HK2), a rate-limiting enzyme of the glycolytic pathway, indicating that HK2 might be a promising candidate for the treatment of diseases associated with T cell activation. Further analysis showed that P2RY12 prevents HK2 degradation by activating the PI3K/Akt pathway and inhibiting lysosomal degradation. Our findings highlight the importance of the function of P2RY12 for HK2 stability and metabolism in the regulation of T cell activation and suggest that P2RY12 might be a pivotal regulator of T cell metabolism in ConA-induced immune hepatitis.
Asunto(s)
Hepatitis Autoinmune , Receptores Purinérgicos P2Y12 , Animales , Ratones , Glucólisis , Hexoquinasa/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Receptores Purinérgicos P2Y12/metabolismo , Linfocitos T/metabolismoRESUMEN
P2Y12 is a G-protein-coupled receptor that is activated upon ADP binding. Considering its well-established role in platelet activation, blocking P2Y12 has been used as a therapeutic strategy for antiplatelet aggregation in cardiovascular disease patients. However, receptor studies have shown that P2Y12 is functionally expressed not only in platelets and the microglia but also in other cells of the immune system, such as in monocytes, dendritic cells, and T lymphocytes. As a result, studies were carried out investigating whether therapies targeting P2Y12 could also ameliorate inflammatory conditions, such as sepsis, rheumatoid arthritis, neuroinflammation, cancer, COVID-19, atherosclerosis, and diabetes-associated inflammation in animal models and human subjects. This review reports what is known about the expression of P2Y12 in the cells of the immune system and the effect of P2Y12 activation and/or inhibition in inflammatory conditions. Lastly, we will discuss the major problems and challenges in studying this receptor and provide insights on how they can be overcome.
Asunto(s)
COVID-19 , Receptores Purinérgicos P2 , Animales , Humanos , Antagonistas del Receptor Purinérgico P2Y/farmacología , Antagonistas del Receptor Purinérgico P2Y/uso terapéutico , COVID-19/metabolismo , Plaquetas/metabolismo , Transducción de Señal , Sistema Inmunológico , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2Y12/genética , Receptores Purinérgicos P2Y12/metabolismo , Agregación Plaquetaria , Inhibidores de Agregación Plaquetaria/farmacología , Adenosina Difosfato/metabolismoRESUMEN
Alzheimer's disease (AD) is a progressive neurodegenerative disease that is associated with protein misfolding, plaque accumulation, neuronal dysfunction, synaptic loss, and cognitive decline. The pathological cascade of AD includes the intracellular Tau hyperphosphorylation and its subsequent aggregation, extracellular Amyloid-ß plaque formation and microglia-mediated neuroinflammation. The extracellular release of aggregated Tau is sensed by surveilling microglia through the involvement of various cell surface receptors. Among all, purinergic P2Y12R signaling is involved in microglial chemotaxis towards the damaged neurons. Microglial migration is highly linked with membrane-associated actin remodeling leading to the phagocytosis of extracellular Tau species. Here, we studied the formation of various actin structures such as podosome, lamellipodia and filopodia, in response to extracellular Tau monomers and aggregates. Microglial podosomes are colocalized with actin nucleator protein WASP, Arp2 and TKS5 adaptor protein during Tau-mediated migration. Moreover, the P2Y12 receptors were associated with F-actin-rich podosome structures, which signify the potential of Tau aggregates in microglial chemotaxis through the involvement of actin remodeling.
Asunto(s)
Enfermedad de Alzheimer , Enfermedades Neurodegenerativas , Podosomas , Humanos , Microglía/metabolismo , Actinas/metabolismo , Podosomas/metabolismo , Receptores Purinérgicos P2Y12/genética , Receptores Purinérgicos P2Y12/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Enfermedad de Alzheimer/metabolismoRESUMEN
BACKGROUND: The hemostatic plug formation at sites of vascular injury is strongly dependent on rapid platelet activation and integrin-mediated adhesion and aggregation. However, to prevent thrombotic complications, platelet aggregate formation must be a self-limiting process. The second-wave mediator adenosine diphosphate (ADP) activates platelets via Gq-coupled P2Y1 and Gi-coupled P2Y12 receptors. After ADP exposure, the P2Y1 receptor undergoes rapid phosphorylation-induced desensitization, a negative feedback mechanism believed to be critical for limiting thrombus growth. OBJECTIVE: The objective of this study was to examine the role of rapid P2Y1 receptor desensitization on platelet function and thrombus formation in vivo. METHODS: We analyzed a novel knock-in mouse strain expressing a P2Y1 receptor variant that cannot be phosphorylated beyond residue 340 (P2Y1340-0P), thereby preventing the desensitization of the receptor. RESULTS: P2Y1340-0P mice followed a Mendelian inheritance pattern, and peripheral platelet counts were comparable between P2Y1340-0P/340-0P and control mice. In vitro, P2Y1340-0P/340-0P platelets were hyperreactive to ADP, showed a robust activation response to the P2Y1 receptor-selective agonist, MRS2365, and did not desensitize in response to repeated ADP challenge. We observed increased calcium mobilization, protein kinase C substrate phosphorylation, alpha granule release, activation of the small GTPase Rap1, and integrin inside-out activation/aggregation. This hyperreactivity, however, did not lead to increased platelet adhesion or excessive plug formation under physiological shear conditions. CONCLUSION: Our studies demonstrate that receptor phosphorylation at the C-terminus is critical for P2Y1 receptor desensitization in platelets and that impaired desensitization leads to increased P2Y1 receptor signaling in vitro. Surprisingly, desensitization of the P2Y1 receptor is not required for limiting platelet adhesion/aggregation at sites of vascular injury, likely because ADP is degraded quickly or washed away in the bloodstream.
Asunto(s)
Trombosis , Lesiones del Sistema Vascular , Ratones , Animales , Agregación Plaquetaria , Plaquetas/metabolismo , Hemostasis , Trombosis/genética , Trombosis/prevención & control , Trombosis/metabolismo , Adenosina Difosfato/farmacología , Integrinas/metabolismo , Receptores Purinérgicos P2Y1/genética , Receptores Purinérgicos P2Y1/metabolismo , Receptores Purinérgicos P2Y12/genética , Receptores Purinérgicos P2Y12/metabolismoRESUMEN
Pharmacological inhibition of the platelet ADP-receptor P2Y12 is a cornerstone in the prevention of atherothrombotic events in adult patients with acute coronary syndrome (ACS). Thienopyridines such as clopidogrel and prasugrel exert their antithrombotic effect by means of active metabolites that irreversibly inhibit P2Y12. Due to the short half-life of these metabolites, a subpopulation of ADP-responsive platelets will form in between dosing. With increased platelet turnover rate or poor patient compliance, the fraction of ADP-responsive platelets will increase, potentially increasing the risk for new thrombotic events. In contrast, the reversible P2Y12 inhibition produced by direct-acting ADP blockers such as ticagrelor and cangrelor inhibit the entire platelet population. In this study, we evaluated the impact of these pharmacological differences on thrombus formation in an ex vivo flow chamber model. A customized image analysis pipeline was used for automatized, large-scale identification and tracking of single platelets incorporated into the thrombus, enabling quantitative analysis of the relative contribution of inhibited and uninhibited platelets to thrombus growth and consolidation. Comparative experiments were conducted using the irreversible and reversible P2Y12 inhibitors prasugrel active metabolite (PAM) and ticagrelor, respectively. Our results show that PAM inhibited thrombus platelet recruitment more gradually than ticagrelor, with a slower onset of inhibition. Further, we show that the presence of a small fraction (<10%) of uninhibited platelets did not abrogate the antithrombotic effect of PAM to any significant extent. Finally, we demonstrate a gradual enrichment of inhibited platelets in the thrombus shell due to selective recruitment of inhibited platelets to the thrombus periphery.
Asunto(s)
Síndrome Coronario Agudo , Inhibidores de Agregación Plaquetaria , Antagonistas del Receptor Purinérgico P2Y , Trombosis , Humanos , Síndrome Coronario Agudo/tratamiento farmacológico , Plaquetas/metabolismo , Fibrinolíticos/uso terapéutico , Inhibidores de Agregación Plaquetaria/uso terapéutico , Clorhidrato de Prasugrel/farmacología , Clorhidrato de Prasugrel/uso terapéutico , Antagonistas del Receptor Purinérgico P2Y/uso terapéutico , Receptores Purinérgicos P2Y12/metabolismo , Trombosis/tratamiento farmacológico , Trombosis/metabolismo , Ticagrelor/farmacología , Ticagrelor/uso terapéuticoRESUMEN
GPI anchorless prion diseases (GPIALPs) show numerous coarse prion protein (PrP) deposits in the CNS but neuropil spongiform changes are mild and the incidence of dementia is low. Here, we examined differences in resident microglial phenotypes between GPIALP (D178fs25) and the other prion diseases Gerstmann-Sträussler-Scheinker (GSS) disease and sporadic Creutzfeldt-Jakob disease (sCJD) with respect to homeostasis and activation. Immunohistochemistry was performed on 2 GPIALP (D178fs25), 4 GSS (P102L), and 4 sCJD cases. Homeostatic microglia expressing TMEM119 and P2RY12 were preserved in GPIALP compared to GSS and sCJD. Microglia/macrophage activation in GSS and sCJD was associated with the extent of spongiform change. Immunoelectron microscopy revealed TMEM119 and P2RY12 in PrP plaque cores. Activated microglia/macrophages expressing HLA-DR and CD68 were predominant in GSS and sCJD whereas in GPIALP, homeostatic microglia were retained and activated microglia/macrophages were rarely observed. These data suggest that PrP deposition in GPIALP is less toxic and that microglia may be immune-tolerant to PrP deposition. This may be associated with milder tissue damage and a low incidence of dementia. Whereas microglia/macrophage activation is considered to be a reaction to tissue injury, this study shows that the degree of microglia/macrophage activity might influence the extent of tissue damage.
Asunto(s)
Síndrome de Creutzfeldt-Jakob , Enfermedad de Gerstmann-Straussler-Scheinker , Proteínas de la Membrana , Microglía , Receptores Purinérgicos P2Y12 , Humanos , Síndrome de Creutzfeldt-Jakob/metabolismo , Enfermedad de Gerstmann-Straussler-Scheinker/genética , Microglía/metabolismo , Proteínas Priónicas/genética , Proteínas Priónicas/metabolismo , Receptores Purinérgicos P2Y12/genética , Receptores Purinérgicos P2Y12/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismoRESUMEN
Platelets are anucleate cells that mediate hemostasis. This occurs via a primary signal that is reinforced by secreted products such as ADP that bind purinergic receptors (P2Y1 and P2Y12) on the platelet surface. We recently identified a human subject, whom we termed platelet defect subject 25 (PDS25) with a platelet functional disorder associated with the P2Y12 receptor. PDS25 has normal blood cell counts and no history of bleeding diathesis. However, platelets from PDS25 have virtually no response to 2-MeSADP (a stable analogue of ADP). Genetic analysis of P2Y12 from PDS25 revealed a heterozygous mutation of D121N within the DRY motif. Rap1b activity was reduced in platelets from PDS25, while VASP phosphorylation was enhanced, suggesting that signaling from the P2Y12 receptor was interrupted by the heterozygous mutation. To explore this further, we produced knock-in mice that mimic our subject. Bleeding failed to cease in homozygous KI mice during tail bleeding assays, while tail bleeding times did not differ between WT and heterozygous KI mice. Furthermore, occlusions failed to form in most homozygous KI mice following carotid artery injury via FeCl3. These data indicate that the aspartic acid residue found in the DRY motif of P2Y12 is essential for P2Y12 function.