The effects of CYP2B6 inactivators on the metabolism of ciprofol.

Ciprofol is a novel short-acting intravenous anaesthetic developed in China that is mainly metabolized by cytochrome P450 2B6 (CYP2B6) and uridine diphosphate glucuronosyltransferase 1A9 (UGT1A9). Currently, insufficient evidence is available to support drug‒drug interactions between ciprofol and CYP2B6 inactivators. Here, we established a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method to assess the concentration of ciprofol and investigated the effects of psoralen and clopidogrel on the metabolism of ciprofol in liver microsomes and rats. In rat and human liver microsomes, the median inhibitory concentration (IC50) values of psoralen were 63.31 µmol·L-1 and 34.05 µmol·L-1, respectively, showing mild inhibitory effects on ciprofol metabolism, whereas the IC50 values of clopidogrel were 6.380 µmol·L-1 and 2.565 µmol·L-1, respectively, with moderate inhibitory effects. SD rats were randomly divided into three groups: psoralen (27 mg·kg-1), clopidogrel (7.5 mg·kg-1), and the same volume of 0.5% carboxy methyl cellulose. After 7 days, all rats were injected with 2.4 mg·kg-1 ciprofol. Compared with the control group, the AUC and MRT values of ciprofol in the psoralen and clopidogrel groups were significantly greater, whereas the CL values were significantly lower. In addition, the durations of loss of righting reflex (LORR) in the psoralen and clopidogrel groups were 16.1% and 23.0% longer than that in the control group, respectively. In conclusion, psoralen and clopidogrel inhibit ciprofol metabolism to different degrees and prolong the duration of LORR in rats.

The polygenic implication of clopidogrel responsiveness: Insights from platelet reactivity analysis and next-generation sequencing.

Clopidogrel is widely used worldwide as an antiplatelet therapy in patients with acute coronary disease. Genetic factors influence interindividual variability in response. Some studies have explored the polygenic contributions in the drug response, generating pharmacogenomic risk scores (PgxPRS). Importantly, these factors are less explored in underrepresented populations, such as Latin-American countries. Identifying patients at risk of high-on-treatment platelet reactivity (HTPR) is highly valuable in translational medicine. In this study we used a custom next-generation sequencing (NGS) panel composed of 91 single nucleotide polymorphisms (SNPs) and 28 genes related to clopidogrel metabolism, to analyze 70 patients with platelet reactivity values, assessed through closure time (CT). Our results demonstrated the association of SNPs with HTPR and non-HTPR, revealing the strongest associations with rs2286823 (OR: 5,0; 95% CI: 1,02-24,48; p: 0,03), rs2032582 (OR: 4,41; 95% CI: 1,20-16,12; p: 0,019), and rs1045642 (OR: 3,38; 95% CI: 0,96-11,9; p: 0,05). Bivariate regression analysis demonstrated the significant association of several SNPs with the CT value, a "surrogate" biomarker of clopidogrel response. Exploratory results from the LASSO regression model showed a high discriminatory capacity between HTPR and non-HTPR patients (AUC: 0,955), and the generated PgxPRS demonstrated a significant negative association between the risk score, CT value, and the condition of HTPR and non-HTPR. To our knowledge, our study addresses for the first time the analysis of the polygenic contribution in platelet reactivity using NGS and establishes PgxPRS derived from the LASSO model. Our results demonstrate the polygenic implication of clopidogrel response and offer insights applicable to the translational medicine of antiplatelet therapy in an understudied population.

Molecular genomic and epigenomic characteristics related to aspirin and clopidogrel resistance.

BACKGROUND: Mediators, genomic and epigenomic characteristics involving in metabolism of arachidonic acid by cyclooxygenase (COX) and lipoxygenase (ALOX) and hepatic activation of clopidogrel have been individually suggested as factors associated with resistance against aspirin and clopidogrel. The present multi-center prospective cohort study evaluated whether the mediators, genomic and epigenomic characteristics participating in arachidonic acid metabolism and clopidogrel activation could be factors that improve the prediction of the aspirin and clopidogrel resistance in addition to cardiovascular risks. METHODS: We enrolled 988 patients with transient ischemic attack and ischemic stroke who were evaluated for a recurrence of ischemic stroke to confirm clinical resistance, and measured aspirin (ARU) and P2Y12 reaction units (PRU) using VerifyNow to assess laboratory resistance 12 weeks after aspirin and clopidogrel administration. We investigated whether mediators, genotypes, and promoter methylation of genes involved in COX and ALOX metabolisms and clopidogrel activation could synergistically improve the prediction of ischemic stroke recurrence and the ARU and PRU levels by integrating to the established cardiovascular risk factors. RESULTS: The logistic model to predict the recurrence used thromboxane A synthase 1 (TXAS1, rs41708) A/A genotype and ALOX12 promoter methylation as independent variables, and, improved sensitivity of recurrence prediction from 3.4% before to 13.8% after adding the mediators, genomic and epigenomic variables to the cardiovascular risks. The linear model we used to predict the ARU level included leukotriene B4, COX2 (rs20417) C/G and thromboxane A2 receptor (rs1131882) A/A genotypes with the addition of COX1 and ALOX15 promoter methylations as variables. The linear PRU prediction model included G/A and prostaglandin I receptor (rs4987262) G/A genotypes, COX2 and TXAS1 promoter methylation, as well as cytochrome P450 2C19*2 (rs4244285) A/A, G/A, and *3 (rs4986893) A/A genotypes as variables. The linear models for predicting ARU (r = 0.291, R2 = 0.033, p < 0.01) and PRU (r = 0.503, R2 = 0.210, p < 0.001) levels had improved prediction performance after adding the genomic and epigenomic variables to the cardiovascular risks. CONCLUSIONS: This study demonstrates that different mediators, genomic and epigenomic characteristics of arachidonic acid metabolism and clopidogrel activation synergistically improved the prediction of the aspirin and clopidogrel resistance together with the cardiovascular risk factors. TRIAL REGISTRATION: URL: https://www. CLINICALTRIALS: gov ; Unique identifier: NCT03823274.

Analysis of the effect of CYP2C19 gene properties on the anti-platelet aggregation of clopidogrel after carotid artery stenting under network pharmacology.

Antiplatelet therapy is an important factor influencing the postterm patency rate of carotid artery stenting (CAS). Clopidogrel is a platelet aggregation inhibitor mediated by the adenosine diphosphate receptor and is affected by CYP2C19 gene polymorphisms in vivo. When the CYP2C19 gene has a nonfunctional mutation, the activity of the encoded enzyme will be weakened or lost, which directly affects the metabolism of clopidogrel and ultimately weakens its antiplatelet aggregation ability. Therefore, based on network pharmacology, analyzing the influence of CYP2C19 gene polymorphisms on the antiplatelet therapeutic effect of clopidogrel after CAS is highly important for the formulation of individualized clinical drug regimens. The effect of the CYP2C19 gene polymorphism on the antiplatelet aggregation of clopidogrel after CAS was analyzed based on network pharmacology. A total of 100 patients with ischemic cerebrovascular disease who were confirmed by the neurology department and required CAS treatment were studied. CYP2C19 genotyping was performed on all patients via a gene chip. All patients were classified into the wild-type (WT) group (*1/*1), heterozygous mutation (HTM) group (CYP2C19*1/*2, CYP2C19*1/*3), and homozygous mutation (HMM) group (CYP2C19*2/*2, CYP2C19*2/*3, and CYP2C19*3/*3). High-performance liquid chromatography (HPLC) with tandem mass spectrometry (MS/MS) was used to detect the blood concentration of clopidogrel and the plasma clopidogrel clearance (CL) rate in different groups of patients before and after clopidogrel treatment. The platelet aggregation rate of patients with different genotypes was measured by turbidimetry. The incidences of clopidogrel resistance (CR) and stent thrombosis in different groups after three months of treatment were analyzed. The results showed that among the different CYP2C19 genotypes, patients from the HTM group accounted for the most patients, while patients from the HTM group accounted for the least patients. Similarly, the clopidogrel CL of patients in the HMM group was lower than that of patients in the WT group and HTM group (P < 0.01). The platelet inhibition rate of patients in the HMM group was evidently inferior to that of patients in the WT group and HTM group (P < 0.01). The incidence of CR and stent thrombosis in the WT group was notably lower than that in the HTM and HMM groups (P < 0.01). These results indicate that the CYP2C19 gene can affect CR occurrence and stent thrombosis after CAS by influencing clopidogrel metabolism and platelet count.

Comparison of thrombogenicity in different types of drug-eluting stents during transition from DAPT to SAPT.

BACKGROUND: During the transition from dual antiplatelet therapy (DAPT) to single antiplatelet therapy (SAPT), previous studies have raised concerns about a rebound effect. We compared platelet and inflammatory cell adhesion on different types of stents in the setting of clopidogrel presence and withdrawal. METHODS: In Experiment 1, three pigs were administered with DAPT, that is, clopidogrel and acetylsalicylic acid (ASA), for 7 days. Each animal underwent an extracorporeal carotid arteriovenous shunt model implanted with fluoropolymer-coated everolimus-eluting stent (FP-EES), biodegradable-polymer sirolimus-eluting stent (BP-SES), and biodegradable-polymer everolimus-eluting stents (BP-EES). In Experiment 2, two pigs were administered DAPT, clopidogrel was then withdrawn at day 7, and SAPT with ASA was continued for next 21 days. Then flow-loop experiments with the drawn blood from each time point were performed for FP-EES, BioLinx-polymer zotarolimus-eluting stents (BL-ZES), and BP-EES. The rebound effect was defined as the statistical increase of inflammation and platelet adhesion assessed with immunohistochemistry on the stent-strut level basis from baseline to day-14 or 28. RESULTS: Both experiments showed platelet adhesion value was highest in BP-EES, while the least in FP-EES during DAPT therapy. There was no increase in platelet or inflammatory cell adhesion above baseline values (i.e., no therapy) due to the cessation of clopidogrel on the stent-strut level. Monocyte adhesion was the least for FP-EES with the same trend observed for neutrophil adhesion. CONCLUSIONS: No evidence of rebound effect was seen after the transition from DAPT to SAPT. FP-EES demonstrated the most favorable antithrombotic and anti-inflammatory profile regardless of the different experimental designs.

Superoxide Dismutase and Clopidogrel: A Potential Role in Peripheral Arterial Disease Treatment.

Oxidative stress plays a crucial role in the pathogenesis of peripheral artery disease (PAD). This study aimed to investigate the effect of clopidogrel on oxidative stress in PAD patients. Seventy subjects were divided into three groups: PAD patients before treatment (B-PAD), PAD patients after treatment with clopidogrel (A-PAD), and healthy controls. Serum levels of superoxide dismutase (SOD), copper (Cu), zinc (Zn), manganese (Mn), and oxidized protein were measured. SOD activities were also determined. The results showed that SOD activities, and SOD specific activities were significantly decreased in PAD patients compared to healthy individuals. After treatment with clopidogrel, SOD activities, and SOD specific activities were continuously decrease in PAD patients. The SOD and oxidized protein concentrations were significantly increased in PAD patients compared to healthy individuals. After treatment with clopidogrel, the oxidized protein concentration was significantly decreased, while SOD concentration was significantly increased in PAD patients. These findings suggest that the treatment by clopidogrel stimulated the production of the enzyme but the ratio of active enzyme remained low. The decrease in oxidized protein can be explained by the treatment having antioxidant efficacy that may have compensated for the deficiency in enzyme activity and led to a decrease in oxidized protein. Additionally, the results of this study provide promising evidence that oxidative stress biomarkers including SOD concentration, T-SOD activity, Mn-SOD activity, and oxidized protein levels have potential utility in the diagnosis and management of PAD.

Assessment of platelet functional activity in healthy individuals and patients receiving antiplatelet therapy. Possible inconsistencies between aggregation and flow cytometry tests.

Platelet functional activity was assessed in healthy volunteers (HV, n=92), patients with stable angina pectoris (SA, n=42) and acute coronary syndrome (ACS, n=73), treated with acetylsalicylic acid (ASA) + clopidogrel and ASA + ticagrelor, respectively. In all HV and patients we have compared parameters of platelet aggregation (maximum light transmission and velocity, Tmax and Vmax) and parameters, characterizing exposure of platelet activation markers, evaluated by flow cytometry. HV platelets were activated by 10 µM, 1 µM TRAP, and 20 µM, 5 µM, 2.5 µM ADP; patient platelets were activated by 10 µM TRAP and by 20 µM and 5 µM ADP. Strong and significant correlations between the aggregation and flow cytometry parameters (the r correlation coefficient from 0.4 up to >0.6) most frequently were registered in HV platelet during activation by 1 µM TRAP and in SA patients during platelet activation by 20 µM and 5 µM ADP. However, in many other cases these correlations were rather weak (r < 0.3) and sometimes statistically insignificant. In HV the differences in PAC-1 binding parameters between platelets activated by 10 µM TRAP (the strongest agonist) and all ADP concentrations were negligible (≤ 10%), while CD62P binding (at all ADP concentrations) and LTA parameters for (5 µM and 2.5 µM ADP) were significantly lower (by 40-60%). Antiplatelet therapy in patients decreased all parameters as compared to HV, but to varying extents. For 10 µM TRAP the MFI index for PAC-1 binding (40-50% decrease) and for both ADP concentrations the Tmax values (60-85% decrease) appeared to be the most sensitive in comparison with the other parameters that decreased to a lesser extent. The data obtained indicate a possibility of inconsistency between different LTA and flow cytometry parameters in assessing platelet activity and efficacy of antiplatelet drugs.

Pharmacokinetic/Pharmacodynamic Assessment of the Structural Refinement of Clopidogrel Focusing on the Balance between Bioactivation and Deactivation.

The delicate balance between ischemic and bleeding risks is a critical factor in antiplatelet therapy administration. Clopidogrel and prasugrel, belonging to the thienopyridine class of antiplatelet drugs, are known for their variability in individual responsiveness and high incidence of bleeding events, respectively. The present study is centered on the development and assessment of a range of deuterated thienopyridine derivatives, leveraging insights from structure-pharmacokinetic relationships of clopidogrel and prasugrel. Our approaches were grounded in the molecular framework of clopidogrel and incorporated the C2-pharmacophore design from prasugrel. The selection of ester or carbamate substituents at the C2-position facilitated the generation of the 2-oxointermediate through hydrolysis, akin to prasugrel, thereby bypassing the issue of CYP2C19 dependency. The bulky C2-pharmacophore in our approach distinguishes itself from prasugrel's acetyloxy substituent by exhibiting a moderated hydrolysis rate, resulting in a more gradual formation of the active metabolite. Excessive and rapid release of the active metabolite, believed to be linked with an elevated risk of bleeding, is thus mitigated. Our proposed structural modification retains the hydrolysis-sensitive methyl ester of clopidogrel but substitutes it with a deuterated methyl group, shown to effectively reduce metabolic deactivation. Three promising compounds demonstrated a pharmacokinetic profile similar to that of clopidogrel at four times the dose, while also augmenting its antiplatelet activity. SIGNIFICANCE STATEMENT: Inspired by the structure-pharmacokinetic relationship of clopidogrel and prasugrel, a range of clopidogrel derivatives were designed, synthesized, and assessed. Among them, three promising compounds have been identified, striking a delicate balance between efficacy and safety for antiplatelet therapy. Additionally, the ozagrel prodrug conjugate was discovered to exert a synergistic therapeutic effect alongside clopidogrel.

Ticagrelor downregulates the expression of proatherogenic and proinflammatory miR125-b compared to clopidogrel: A randomized, controlled trial.

BACKGROUND: Platelet P2Y12 antagonist ticagrelor reduces cardiovascular mortality after acute myocardial infarction (AMI) compared to clopidogrel, but the underlying mechanism is unknown. Because activated platelets release proatherogenic and proinflammatory microRNAs, including miR-125a, miR-125b and miR-223, we hypothesized that the expression of these miRNAs is lower on ticagrelor, compared to clopidogrel. OBJECTIVES: We compared miR-125a, miR-125b and miR-223 expression in plasma of patients after AMI treated with ticagrelor or clopidogrel. METHODS: After percutaneous coronary intervention on acetylsalicylic acid and clopidogrel, 60 patients with first AMI were randomized to switch to ticagrelor or to continue with clopidogrel. Plasma expression of miR-223, miR-125a-5p, miR-125b was measured using quantitative polymerase chain reaction at baseline and after 72 h and 6 months of treatment with ticagrelor or clopidogrel in patients and one in 30 healthy volunteers. Multiple electrode aggregometry using ADP test was used to determine platelet reactivity in response to P2Y12 inhibitors. RESULTS: Expression of miR-125b was higher in patients with AMI 72 h and 6 months, compared to healthy volunteers (p = 0.001), whereas expression of miR-125a-5p and miR-223 were comparable. In patients randomized to ticagrelor, expression of miR-125b decreased at 72 h (p = 0.007) and increased back to baseline at 6 months (p = 0.005). Expression of miR-125a-5p and miR-223 was not affected by the switch from clopidogrel to ticagrelor. CONCLUSIONS: Ticagrelor treatment leads to lower plasma expression of miR-125b after AMI, compared to clopidogrel. Higher expression of miR-125b might explain recurrent thrombotic events and worse clinical outcomes in patients treated with clopidogrel, compared to ticagrelor.

Ticagrelor, but Not Clopidogrel, Attenuates Hepatic Steatosis in a Model of Metabolic Dysfunction-Associated Steatotic Liver Disease.

BACKGROUND: Previous studies have suggested that platelets are associated with inflammation and steatosis and may play an important role in liver health. Therefore, we evaluated whether antiplatelet agents can improve metabolic disorder-related fatty liver disease (MASLD). METHODS: The mice used in the study were fed a high-fat-diet (HFD) and were stratified through liver biopsy at 18 weeks. A total of 22 mice with NAFLD activity scores (NAS) ≥ 4 were randomly divided into three groups (HFD-only, clopidogrel (CLO; 35 mg/kg/day), ticagrelor (TIC; 40 mg/kg/day) group). And then, they were fed a feed mixed with the respective drug for 15 weeks. Blood and tissue samples were collected and used in the study. RESULTS: The TIC group showed a significantly lower degree of NAS and steatosis than the HFD group (p = 0.0047), but no effect on the CLO group was observed. Hepatic lipogenesis markers' (SREBP1c, FAS, SCD1, and DGAT2) expression and endoplasmic reticulum (ER) stress markers (CHOP, Xbp1, and GRP78) only reduced significantly in the TIC treatment group. Inflammation genes (MCP1 and TNF-α) also decreased significantly in the TIC group, but not in the CLO group. Nile red staining intensity and hepatic lipogenesis markers were reduced significantly in HepG2 cells following TIC treatment. CONCLUSION: Ticagrelor attenuated NAS and hepatic steatosis in a MASLD mice model by attenuating lipogenesis and inflammation, but not in the CLO group.

Assessment of Aspirin and Clopidogrel Resistance in Patients Undergoing Cardiovascular Surgery: A Single-Center Cross-Sectional Study

Objective: We aimed to investigate antiplatelet drug resistance utilizing light transmission-lumiaggregometry (LT-LA) and the Platelet Function Analyzer-100 (PFA-100) in patients undergoing cardiovascular surgery. Materials and Methods: The study included 60 patients diagnosed with stable coronary artery disease and peripheral vascular diseases that required surgery. Participants were divided into three groups: patients receiving aspirin (ASA) (n=21), patients receiving clopidogrel (CLO) (n=19), and patients receiving dual therapy (ASA+CLO) (n=20). Aggregation and secretion tests by LT-LA and closure time by the PFA-100 were used to measure antiplatelet drug resistance. Results: Based on the adenosine diphosphate (ADP)-induced aggregation test, 43% of patients were resistant to ASA, 22% to CLO, and 15% to dual therapy. Diabetes, hypertension, and hyperlipidemia were the most commonly identified comorbid disorders. In patients with comorbid risk factors, the median value of platelet aggregation response to ADP was significantly higher in the ASA group than in the CLO and dual therapy groups (p=0.0001). In patients receiving ASA monotherapy, the maximum amplitude of aggregation response to platelet agonists was ≥70% in 43% of patients for ADP and 28% for collagen by LT-LA. Elevated ADP (≥0.29 nmol) and collagen (≥0.41 nmol)-induced adenosine triphosphate release were found by LT-LA in 66% of patients utilizing an ADP agonist and 80% of patients using a collagen agonist undergoing ASA therapy. Closure times obtained with the PFA-100 were normal in 28% of patients using collagen-ADP cartridges and 62% of patients using collagen-epinephrine (CEPI) cartridges who received ASA. Recurrent thrombosis and bleeding were observed in 12 (20%) patients with cardiovascular disease. Three of these individuals (25%) showed ASA resistance with normal responses to ADP-induced aggregation (≥70%) and secretion (≥0.29 nmol), as well as normal CEPI closure times. Conclusion: Our findings suggest that antiplatelet drug monitoring by LT-LA and PFA-100 may be useful for high-risk and complicated cardiovascular patients.

Low on-clopidogrel ADP- and TRAP-6-induced platelet aggregation in patients with atrial fibrillation undergoing percutaneous coronary intervention: an observational pilot study.

High on-clopidogrel platelet reactivity (HPR) associates with ischemic risk in patients after percutaneous intervention (PCI). This study aimed to evaluate the association of HPR as assessed by multiple electrode aggregometry (MEA) with ischemic, thromboembolic, and bleeding risk in patients with atrial fibrillation (AF) undergoing PCI. Patients with AF and an indication for oral anticoagulation (OAC) were included in this prospective cohort study on day 1-3 after PCI. Platelet aggregation [U] was analyzed by MEA. HPR and low platelet reactivity (LPR) were defined as ADP-induced aggregation ≥ 46 U and ≤ 18 U, respectively. TRAP-6-induced aggregation reference was 94-156 U. The primary outcome was time to all-cause death, myocardial infarction, or stroke at 6 months. The secondary outcome was time to non-major clinically relevant bleedings or major bleedings. 159 patients were enrolled between May 2020 and May 2021. The median age was 78 years (interquartile range 72-82) and 111 (70%) were male. Median ADP- and TRAP-induced aggregation were 12 (6-17) and 49 (35-68) U, respectively. 147 (93%) patients had a low overall aggregability. HPR was detected in 2 patients (1%) and 125 (79%) had LPR. ADP-induced aggregation did not significantly associate with the primary outcome (r = 0.081, p = 0.309) but correlated inversely with bleeding risk (r = - 0.201, p = 0.011). HPR status as assessed by MEA among patients with AF after PCI was rare and overall aggregability was low. Conventional cut-off values for HPR might be inappropriate for these patients. ADP-induced aggregation might be helpful to identify patients at risk for bleeding.

A novel machine learning model to predict high on-treatment platelet reactivity on clopidogrel in Asian patients after percutaneous coronary intervention.

BACKGROUND: Various genetic and nongenetic variables influence the high on-treatment platelet reactivity (HTPR) in patients taking clopidogrel. AIM: This study aimed to develop a novel machine learning (ML) model to predict HTPR in Chinese patients after percutaneous coronary intervention (PCI). METHOD: This cohort study collected information on 507 patients taking clopidogrel. Data were randomly divided into a training set (90%) and a testing set (10%). Nine candidate Machine learning (ML) models and multiple logistic regression (LR) analysis were developed on the training set. Their performance was assessed according to the area under the receiver operating characteristic curve, precision, recall, F1 score, and accuracy on the test set. Model interpretations were generated using importance scores by transforming model variables into scaled features and representing in radar plots. Finally, we established a prediction platform for the prediction of HTPR. RESULTS: A total of 461 patients (HTPR rate: 19.52%) were enrolled in building the prediction model for HTPR. The XGBoost model had an optimized performance, with an AUC of 0.82, a precision of 0.80, a recall of 0.44, an F1 score of 0.57, and an accuracy of 0.87, which was superior to those of LR. Furthermore, the XGBoost method identified 7 main predictive variables. To facilitate the application of the model, we established an XGBoost prediction platform consisting of 7 variables and all variables for the HTPR prediction. CONCLUSION: A ML-based approach, such as XGBoost, showed optimum performance and might help predict HTPR on clopidogrel after PCI and guide clinical decision-making. Further validated studies will strengthen this finding.

Transcriptome-wide map of N6-methyladenosine (m6A) profiling in coronary artery disease (CAD) with clopidogrel resistance.

BACKGROUND: Clopidogrel resistance profoundly increases the risk of major cardiovascular events in coronary artery disease (CAD) patients. Here, we comprehensively analyse global m6A modification alterations in clopidogrel-resistant (CR) and non-CR patients. METHODS: After RNA isolation, the RNA transcriptome expression (lncRNA, circRNA, and mRNA) was analysed via RNA-seq, and m6A peaks were identified by MeRIP-seq. The altered m6A methylation sites on mRNAs, lncRNAs, and circRNAs were identified, and then, GO and KEGG pathway analyses were performed. Through joint analysis with RNA-seq and MeRIP-seq data, differentially expressed mRNAs harbouring differentially methylated sites were identified. The changes in m6A regulator levels and the abundance of differentially methylated sites were measured by RT-PCR. The identification of m6A-modified RNAs was verified by m6A-IP-qPCR. RESULTS: The expression of 2919 hypermethylated and 2519 hypomethylated mRNAs, 192 hypermethylated and 391 hypomethylated lncRNAs, and 375 hypermethylated and 546 hypomethylated circRNAs was shown to be altered in CR patients. The m6A peaks related to CR indicated lower mark density at the CDS region. Functional enrichment analysis revealed that inflammatory pathways and insulin signalling pathways might be involved in the pathological processes underlying CR. The expression of mRNAs (ST5, KDM6B, GLB1L2, and LSM14B), lncRNAs (MSTRG.13776.1 and ENST00000627981.1), and circRNAs (hsa_circ_0070675_CBC1, hsa-circRNA13011-5_CBC1, and hsa-circRNA6406-3_CBC1) was upregulated in CR patients, while the expression of mRNAs (RPS16 and CREG1), lncRNAs (MSTRG.9215.1), and circRNAs (hsa_circ_0082972_CBC1) was downregulated in CR patients. Moreover, m6A regulators (FTO, YTHDF3, and WTAP) were also differentially expressed. An additional combined analysis of gene expression and m6A peaks revealed that the expression of mRNAs (such as ST5, LYPD2, and RPS16 mRNAs) was significantly altered in the CR patients. CONCLUSION: The expression of m6A regulators, the RNA transcriptome, and the m6A landscape was altered in CR patients. These findings reveal epitranscriptomic regulation in CR patients, which might be novel therapeutic targets in future.

Non-Random Enrichment of Single-Nucleotide Polymorphisms Associated with Clopidogrel Resistance within Risk Loci Linked to the Severity of Underlying Cardiovascular Diseases: The Role of Admixture.

Cardiovascular disease (CVD) is one of the leading causes of death in Puerto Rico, where clopidogrel is commonly prescribed to prevent ischemic events. Genetic contributors to both a poor clopidogrel response and the severity of CVD have been identified mainly in Europeans. However, the non-random enrichment of single-nucleotide polymorphisms (SNPs) associated with clopidogrel resistance within risk loci linked to underlying CVDs, and the role of admixture, have yet to be tested. This study aimed to assess the possible interaction between genetic biomarkers linked to CVDs and those associated with clopidogrel resistance among admixed Caribbean Hispanics. We identified 50 SNPs significantly associated with CVDs in previous genome-wide association studies (GWASs). These SNPs were combined with another ten SNPs related to clopidogrel resistance in Caribbean Hispanics. We developed Python scripts to determine whether SNPs related to CVDs are in close proximity to those associated with the clopidogrel response. The average and individual local ancestry (LAI) within each locus were inferred, and 60 random SNPs with their corresponding LAIs were generated for enrichment estimation purposes. Our results showed no CVD-linked SNPs in close proximity to those associated with the clopidogrel response among Caribbean Hispanics. Consequently, no genetic loci with a dual predictive role for the risk of CVD severity and clopidogrel resistance were found in this population. Native American ancestry was the most enriched within the risk loci linked to CVDs in this population. The non-random enrichment of disease susceptibility loci with drug-response SNPs is a new frontier in Precision Medicine that needs further attention.

LUNG PROTECTIVE EFFECTS OF CLOPIDOGREL IN POLYMICROBIAL SEPSIS.

OBJECTIVE: Aim: The goal of this experiment was to examine if Clopidogrel might protect the lungs during sepsis by modulating the inflammatory and oxidative stress markers. PATIENTS AND METHODS: Materials and Methods: Twenty-four adult male Swiss-albino mice aged 8-12 weeks, with a weighing of 20-30 g, were randomized into 4 equal groups (n=6): sham (Laparotomy without cecal ligation and puncture [CLP]), CLP (laparotomy plus CLP), vehicle (DMSO 1 hour prior to CLP), Clopidogrel (50 mg/g IP 1 hour before to CLP). ELISA was used to assess Lung tissue levels of pro-inflammatory and oxidative stress markers. RESULTS: Results: F2 isoprostane levels were significantly higher in the sepsis group (p<0.05) in comparison with sham group, while Clopidogrel was considerably lower (p<0.05) in the inflammatory and oxidative stress markers in comparison to sepsis group. Histologically, all mice in the sepsis group had considerable (p=0.05) lung tissue damage, but Clopidogrel considerably decreased lung tissue injury (p=0.05). CONCLUSION: Conclusion: Clopidogrel was found to reduce lung tissue cytokine concentrations (IL-1, TNF a, IL-6, F2 isoprostane, GPR 17, MIF) in male mice during CLP-induced polymicrobial sepsis by modulation of pro-inflammatory and oxidative stress cascade signaling pathways, to the best of our abilities, no study has looked at the effect of Clopidogrel on MIF levels.

Differential inhibition of platelet function by cilostazol in combination with clopidogrel.

PURPOSE: To assess the antiplatelet effect of cilostazol clinically, we compared the effects of cilostazol in combination with clopidogrel on various platelet function tests. METHODS: We recruited patients with ischemic stroke at high risk of recurrence who were treated with clopidogrel alone within 180 days after stroke onset. Subjects underwent baseline platelet function tests, and were then randomly assigned to receive dual antiplatelet therapy (DAPT) comprising clopidogrel and cilostazol or clopidogrel monotherapy (SAPT). After 6 months, platelet function was measured again and compared to that at baseline in each group, and the rate of change was compared between groups. RESULTS: Thirty-four patients were enrolled, but 4 patients were excluded for various reasons. In total, 30 subjects (13 in DAPT and 17 in SAPT group) were analyzed. Adenosine diphosphate- and collagen-induced aggregation, VerifyNow P2Y12 reaction units, vasodilator-stimulated phosphoprotein (platelet reactivity index: PRI) and plasma p-selectin concentration were significantly lower (P = 0.004, 0.042, 0.049, 0.003 and 0.006 respectively), while VerifyNow % inhibition was significantly higher at 6 months compared to baseline (P = 0.003) in the DAPT group only. Comparison of the rate of change in each parameter from baseline to 6 months showed that while PRI decreased at a greater rate (P = 0.012), VerifyNow % inhibition increased at a greater rate (P = 0.003) in the DAPT group than the SAPT group. CONCLUSIONS: The inhibitory effects of adjunctive cilostazol added to clopidogrel on platelet function differed by type of platelet function test. VerifyNow % inhibition and PRI were more inhibited than the other platelet function tests. TRIAL REGISTRATION: CSPS.com substudy in TWMU (UMIN000026672), registered on April 1, 2017. This study was performed as a substudy of CSPS.com (UMIN000012180, registered on October 31, 2013) and was retrospectively registered.

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

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

Investigation of Platelet Function Analyzer 200 platelet function measurements in healthy cats and cats receiving clopidogrel.

The Platelet Function Analyzer 200 (PFA-200; Siemens) is an in vitro substitute for in vivo bleeding time that is designed to investigate platelet function in a more physiologic manner than traditional aggregometry. The analyzer reports a closure time (CT) as a marker of platelet function, and may also report the calculated platelet function measurement primary hemostasis components, PHC1 and PHC2. These incorporate the measured total volume (TV) of blood aspirated and the initial flow rate (IF). We determined, for the COL/ADP and P2Y cartridges, the median total volume (TVmedian), and RIs for CT, IF, TV, PHC1, and PHC2, and investigated the sensitivity and specificity of those parameters at the determined interpretation thresholds in determination of the clopidogrel effect. Healthy client-owned cats were recruited prospectively to determine RIs for CT, IF, TV, PHC1, and PHC2. Healthy blood-donor cats and cats on clopidogrel therapy were included retrospectively to determine test performance. In 20 healthy cats, RIs for COL/ADP were CT (19.5-87.2 s), IF (199-278 µL/min), TV (199-332 µL), PHC1 (94-106%), and PHC2 (52-148%); and for P2Y, CT (4.2-94.3 s), IF (112-208 µL/min), TV (151-294 µL), PHC1 (35-178%), and PHC2 (90-109%). CVs were calculated for all of these values. Specificity for detection of the clopidogrel effect was calculated from a group of healthy blood donors, and sensitivity for detection of the clopidogrel effect from a group of cats with known clopidogrel effect. Sensitivity and specificity were, for COL/ADP: CT (83.3%, 66.6%), IF (41.4%, 83.3%), TV (83.3%, 100%), PHC1 (100%, 100%) and PHC2 (100%, 83.3%); and for P2Y: CT (100%, 94.4%), IF (30%, 44.4%), TV (100%, 94.4%), PHC1 (100%, 100%), and PHC2 (100%, 97.7%). These PFA-200 values may be beneficial in the determination of platelet function in cats.