Platelet miR-107 Participates in Clopidogrel Resistance after PCI Treatment by Regulating P2Y12.

INTRODUCTION: High platelet reactivity (HPR) caused by clopidogrel tolerance is an adverse reaction of acute coronary syndrome (ACS) patients who receive clopidogrel antiplatelet therapy after percutaneous coronary intervention (PCI) surgery. Platelet microRNA (miRNA) is related to platelet reactivity. This study explored the mechanism of platelet miRNA in regulating platelet reactivity. METHODS: We recruited 50 ACS/PCI patients and divided them into the HPR group (P2Y12 reaction units [PRU] ≥300) and the LPR group (PRU < 170) according to the PRU through the VerifyNow P2Y12 assay. P2Y12-related miRNAs were screened by TargetScan, miRWalk, and Gene Expression Omnibus. The expressions of P2Y12 and miRNAs in the HPR group and the LPR group were determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Pearson correlation analysis was used to determine the correlation between P2Y12 and miRNAs. The interactions between P2Y12 and miR-107 were predicted by TargetScan and verified by dual-luciferase reporter assay. The regulation of miR-107 mimic or inhibitor on P2Y12 expression was detected by qRT-PCR and Western blot. RESULTS: There were 22 patients in the LPR group and 28 patients in the HPR group. PY212 was highly expressed in the HPR group compared with the LPR group. We screened the P2Y12-related miRNAs (miR-145-5p, miR-4701-3p, miR-107, and miR-15b-5p), but only miR-107 and miR-15b-5p expressions were downregulated in the HPR group and were negatively correlated with PY212 expression. P2Y12 was the target gene of miR-107. PY212 expression was inhibited by miR-107 overexpression but suppressed by miR-107 silencing. CONCLUSION: Platelet miR-107 participated in clopidogrel resistance in ACS/PCI patients by regulating P2Y12 expression.

Microglial P2Y12 Receptor Regulates Seizure-Induced Neurogenesis and Immature Neuronal Projections.

Seizures are common in humans with various etiologies ranging from congenital aberrations to acute injuries that alter the normal balance of brain excitation and inhibition. A notable consequence of seizures is the induction of aberrant neurogenesis and increased immature neuronal projections. However, regulatory mechanisms governing these features during epilepsy development are not fully understood. Recent studies show that microglia, the brain's resident immune cell, contribute to normal neurogenesis and regulate seizure phenotypes. However, the role of microglia in aberrant neurogenic seizure contexts has not been adequately investigated. To address this question, we coupled the intracerebroventricular kainic acid model with current pharmacogenetic approaches to eliminate microglia in male mice. We show that microglia promote seizure-induced neurogenesis and subsequent seizure-induced immature neuronal projections above and below the pyramidal neurons between the DG and the CA3 regions. Furthermore, we identify microglial P2Y12 receptors (P2Y12R) as a participant in this neurogenic process. Together, our results implicate microglial P2Y12R signaling in epileptogenesis and provide further evidence for targeting microglia in general and microglial P2Y12R in specific to ameliorate proepileptogenic processes.SIGNIFICANCE STATEMENT Epileptogenesis is a process by which the brain develops epilepsy. Several processes have been identified that confer the brain with such epileptic characteristics, including aberrant neurogenesis and increased immature neuronal projections. Understanding the mechanisms that promote such changes is critical in developing therapies to adequately restrain epileptogenesis. We investigated the role of purinergic P2Y12 receptors selectively expressed by microglia, the resident brain immune cells. We report, for the first time, that microglia in general and microglial P2Y12 receptors in specific promote both aberrant neurogenesis and increased immature neuronal projections. These results indicate that microglia enhance epileptogenesis by promoting these processes and suggest that targeting this immune axis could be a novel therapeutic strategy in the clinic.

NOD2-mediated P2Y12 upregulation increases platelet activation and thrombosis in sepsis.

BACKGROUND: Platelets from septic patients exhibit increased reactivity. However, the underlying mechanism of sepsis-induced platelet hyperactivity is still not completely understood. OBJECTIVE: P2Y12 is a central receptor for platelet activation. In this study, we investigated the role of platelet P2Y12 in platelet hyperactivity during sepsis. METHODS: We measured platelet P2Y12 expression and aggregation in response to ADP in septic patients and cecal ligation and puncture (CLP)-treated mice. We also detected the downstream signaling of P2Y12 in resting platelets from patients and mice with sepsis. The role of nucleotide-binding oligomerization domain 2 (NOD2)/RIP2/NF-κB/P65 pathway in sepsis-induced platelet P2Y12 high expression was also investigated. Finally, we compared the antiplatelet and antithrombotic effects of clopidogrel, prasugrel, and ticagrelor in experimental sepsis in mice and rats. RESULTS: Compared to healthy subjects, platelets from septic patients exhibit P2Y12 hyperactivity and higher P2Y12 expression. pAkt is enhanced and pVASP is impaired in resting platelets from the patients, indicating the constitutive activation of platelet P2Y12 receptor. Mouse sepsis model recapitulates the findings in septic patients. NOD2 deficiency attenuates sepsis-induced platelet P2Y12 high expression, hyperactivity, and thrombosis. Prasugrel and ticagrelor are potent P2Y12 inverse agonists, and exhibit superior antiplatelet and antithrombotic efficacy over clopidogrel in mice and rats with sepsis. CONCLUSIONS: NOD2 activation upregulates platelet P2Y12 expression, which is constitutively activated and contributes to platelet hyperactivity in septic status. Compared to clopidogrel, prasugrel and ticagrelor are potent P2Y12 inverse agonists with superior antiplatelet and antithrombotic efficacy in experimental sepsis.

Microglia in the RVLM of SHR have reduced P2Y12R and CX3CR1 expression, shorter processes, and lower cell density.

The RVLM of spontaneously hypertensive rats (SHR) contains over-active C1 neurons, which model the pathology of essential hypertension. Hypertension involves chronic low-grade neuroinflammation. Inflammation in the brain is produced and maintained primarily by microglia. We assessed microglial gene expression (P2Y12R and CX3CR1) and morphology in the RVLM of SHR compared to normotensive Wistar-Kyoto rats (WKY). The gene expression of the metabotropic purinergic receptor P2Y12 and the fractalkine receptor CX3CR1 was downregulated in the RVLM of SHR compared to WKY (by 37.3% and 30.9% respectively). P2Y12R and CX3CR1 are required for normal microglial function, and reduced P2Y12R expression is associated with changes in microglial activity. Histological analysis showed a 22.9% reduction in microglial cell density, along with 18.7% shorter microglial processes, a phenotypic indicator of activation, in the RVLM of SHR compared to WKY. These results indicate a subtle loss of function, or a mild state of inflammation, in the RVLM microglia of SHR.

Effects of endurance training on hsa-miR-223, P2RY12 receptor expression and platelet function in type 2 diabetic patients.

BACKGROUND: Type 2 diabetes mellitus (T2DM) is associated with thrombotic events due to platelets' defects. OBJECTIVE: The aim of this study was to investigate the effects of endurance training on expressions of platelet hsa-miR-223 and P2RY12 receptor, as well as platelet function in T2DM patients. METHODS: Twenty female T2DM patients (age, 62.25±3.81 yr; weight, 73.53±9.04 kg; height, 154.7±4.3 cm) were randomly divided into two groups (Control (CONT) and endurance training (ET)). Subjects in ET group performed eight weeks of running on treadmill at 60-75% of VO2peak (moderate intensity), 3 sessions per week, while, the subjects in control group were not involved in any kind of exercise training. Two blood samples were taken before and 48 h after the training and were analyzed for platelet aggregation, and hsa-miR-223 and P2RY12 expressions. RESULTS: Although platelet aggregation decreased significantly in ET group (P <  0.05), these changes were not significantly different between two groups. Expression of platelet hsa-miR-223 increased and P2RY12 mRNA reduced following ET non-significantly. However, decreases in fasting blood glucose, glycated hemoglobin and body weight, and increases in VO2peak following ET were significantly different when compared to control group (P <  0.05). CONCLUSIONS: Short-term endurance training dose not induce up-regulation of hsa-miR-223 and down-regulation of P2RY12, while it has a positive impact on platelet function, glycemic indices, physical fitness and body composition in female T2DM patients.

The role of agonist-induced activation and inhibition for the regulation of purinergic receptor expression in human platelets.

INTRODUCTION: Adenosine diphosphate (ADP) as physiological activator of human platelets mediates its effects via three purinergic receptors: P2Y1, P2Y12 and P2X1. The inhibition of P2Y12 is used pharmacologically to suppress aggregation underlining the physiological significance of this receptor. Since the regulation of purinergic receptor expression has not thoroughly been investigated yet, this study analyzed the content of purinergic receptors on the platelet surface membrane upon activation and inhibition. MATERIALS AND METHODS: The surface expression of purinergic receptors was measured by flow cytometry using two different polyclonal antibodies as basal values and after incubation with thrombin receptor activating peptide (TRAP-6) or with inhibitors DEA/NO, MAHMA/NO or Prostaglandin E1 (PGE1). Western blot analysis was used to confirm inhibitory effects. RESULTS: Both investigated antibodies revealed a significant increase of purinergic receptor expression upon TRAP-6 stimulation. The NO donors, DEA/NO and MAHMA/NO, did not influence basal or TRAP-6 stimulated values. PGE1 did not affect basal receptor expression, but diminished TRAP-6 stimulated purinergic receptor expression in a dose-dependent manner. CONCLUSIONS: In summary, TRAP-6 induced platelet activation leads to an elevation of purinergic receptor expression. In contrast to other surface ligands, this effect is not suppressed by cGMP-mediated inhibition, but almost completely abrogated by enhanced cAMP-mediated signaling as induced by PGE1.

Oral Antiplatelet Therapy for Secondary Prevention of Acute Coronary Syndrome.

Patients surviving an acute coronary syndrome (ACS) remain at increased risk of ischemic events long term. This paper reviews current evidence and guidelines for oral antiplatelet therapy for secondary prevention following ACS, with respect to decreased risk of ischemic events versus bleeding risk according to individual patient characteristics and risk factors. Specifically, data are reviewed from clinical studies of clopidogrel, prasugrel, ticagrelor and vorapaxar, as well as the results of systematic reviews and meta-analyses looking at the benefits and risks of oral antiplatelet therapy, and the relative merits of shorter versus longer duration of dual antiplatelet therapy, in different patient groups.

Biological effects of leukotriene E4 on eosinophils.

Studies demonstrate the existence of novel receptors for cysteinyl leukotrienes (CysLTs) that are responsive to leukotriene (LT) E4 and might be pathogenic in asthma. Given the eosinophilic infiltration in this disorder, we investigated eosinophil expression of P2Y12 and gpr99 and their capacity to respond to LTE4. Receptor transcript expression was investigated via quantitative PCR and surface protein expression via flow cytometry. We investigated LTE4 influences on eosinophils including Ca(+2) flux, cAMP induction, modulation of adhesion molecule expression, apoptosis and degranulation. Eosinophils displayed both transcript and surface protein expression of P2Y12 and gpr99. We could not find evidence of LTE4 activation of eosinophils, however, LTE4 induced cAMP expression, and preincubation of eosinophils with LTE4 inhibited degranulation. Even though eosinophils are an important source of CysLTs in AERD, eosinophils are not themselves the pro-inflammatory biological target and, in contrast, LTE4 via cAMP primarily elicits anti-inflammatory responses.

P2Y12 receptor inhibition augments cytotoxic effects of cisplatin in breast cancer.

Expression of P2Y12 receptors has been documented in some cancer cell lines like C6 glioma, renal carcinoma and colon carcinoma. However, its direct role in altering response to chemotherapeutics has not been studied. In this study, we characterize the expression of P2Y12 receptor in breast cancer cell lines and evaluate its role in enhancing the cytotoxic effects of cisplatin. We observed a significant upregulation in P2Y12 expression in 4T1 breast cancer cell line with cisplatin treatment. Co-administration of P2Y12 inhibitor with cisplatin resulted in significantly higher cytotoxic response in 4T1 cancer cell line. This was mediated by HIF1α-dependent upregulation of cellular apoptotic pathways. These findings identify P2Y12 receptor as a potential target to enhance antitumor efficacy of chemotherapeutic agents like cisplatin.