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.

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.

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.

Role of circadian clock in the chronoefficacy and chronotoxicity of clopidogrel.

BACKGROUND AND PURPOSE: The role of circadian locomotor output cycles kaput (CLOCK) in regulating drug chronoefficacy and chronotoxicity remains elusive. Here, we aimed to uncover the impact of CLOCK and dosing time on clopidogrel efficacy and toxicity. EXPERIMENTAL APPROACH: The antiplatelet effect, toxicity and pharmacokinetics experiments were conducted with Clock-/- mice and wild-type mice, after gavage administration of clopidogrel at different circadian time points. The expression levels of drug-metabolizing enzymes were determined by quantitative polymerase chain reaction (qPCR) and western blotting. Transcriptional gene regulation was investigated using luciferase reporter and chromatin immunoprecipitation assays. KEY RESULTS: The antiplatelet effect and toxicity of clopidogrel in wild-type mice showed a dosing time-dependent variation. Clock ablation reduced the antiplatelet effect of clopidogrel, but increased clopidogrel-induced hepatotoxicity, with attenuated rhythms of clopidogrel active metabolite (Clop-AM) and clopidogrel, respectively. We found that Clock regulated the diurnal variation of Clop-AM formation by modulating the rhythmic expression of CYP1A2 and CYP3A1, and altered clopidogrel chronopharmacokinetics by regulation of CES1D expression. Mechanistic studies revealed that CLOCK activated Cyp1a2 and Ces1d transcription by directly binding to the enhancer box (E-box) elements in their promoters, and promoted Cyp3a11 transcription through enhancing the transactivation activity of albumin D-site-binding protein (DBP) and thyrotroph embryonic factor (TEF). CONCLUSIONS AND IMPLICATIONS: CLOCK regulates the diurnal rhythmicity in clopidogrel efficacy and toxicity through regulation of CYP1A2, CYP3A11 and CES1D expression. These findings may contribute to optimizing dosing schedules for clopidogrel and may deepen understanding of the circadian clock and chronopharmacology.

Clopidogrel Administration Impairs Post-Stroke Learning and Memory Recovery in Mice.

Clopidogrel, which is one of the most prescribed antiplatelet medications in the world, is given to stroke survivors for the prevention of secondary cardiovascular events. Clopidogrel exerts its antiplatelet activity via antagonism of the P2Y12 receptor (P2RY12). Although not widely known or considered during the initial clinical trials for clopidogrel, P2RY12 is also expressed on microglia, which are the brain's immune cells, where the receptor facilitates chemotactic migration toward sites of cellular damage. If microglial P2RY12 is blocked, microglia lose the ability to migrate to damaged sites and carry out essential repair processes. We aimed to investigate whether administering clopidogrel to mice post-stroke was associated with (i) impaired motor skills and cognitive recovery; (ii) physiological changes, such as survival rate and body weight; (iii) changes in the neurovascular unit, including blood vessels, microglia, and neurons; and (iv) changes in immune cells. Photothrombotic stroke (or sham surgery) was induced in adult male mice. From 24 h post-stroke, mice were treated daily for 14 days with either clopidogrel or a control. Cognitive performance (memory and learning) was assessed using a mouse touchscreen platform (paired associated learning task), while motor impairment was assessed using the cylinder task for paw asymmetry. On day 15, the mice were euthanized and their brains were collected for immunohistochemistry analysis. Clopidogrel administration significantly impaired learning and memory recovery, reduced mouse survival rates, and reduced body weight post-stroke. Furthermore, clopidogrel significantly increased vascular leakage, significantly increased the number and appearance of microglia, and significantly reduced the number of T cells within the peri-infarct region post-stroke. These data suggest that clopidogrel hampers cognitive performance post-stroke. This effect is potentially mediated by an increase in vascular permeability post-stroke, providing a pathway for clopidogrel to access the central nervous system, and thus, interfere in repair and recovery processes.

Validation of storage and shipping of feline blood samples for analysis on the Platelet Function Analyzer-200 for determining the effect of clopidogrel.

BACKGROUND: The Platelet function analyzer-200 (PFA-200) can determine the effect of clopidogrel in cats, but analysis traditionally must be performed at point-of-care (POC). The ability to ship samples of blood to a laboratory would allow widespread access. OBJECTIVES: We aimed to validate the shipping of blood samples for PFA-200 analysis in cats to determine the effect of clopidogrel. METHODS: Twenty healthy cats and 10 cats receiving clopidogrel were recruited. Blood was collected from cats and aliquoted into two samples, one was analyzed at POC within 2 hours using the PFA-200, and the other was packaged and transported to a location 4 km away, stored, and transported back to the lab for analysis the following day. RESULTS: Median closure times (CTs) with the collagen/adenosine diphosphate (COL/ADP) cartridge in healthy cats were 51.5 seconds (POC) and 78.8 seconds (shipped), which were significantly different (P < 0.001), and for cats on clopidogrel, median CTs were 147.5 seconds (POC) and 190 seconds (shipped), which were not significantly different (P = 0.131). Median CTs with the P2Y cartridge in healthy cats were 50.5 seconds (POC) and 64.9 seconds (shipped), which were significantly different (P = 0.03), and in cats receiving clopidogrel, median CTs were 300 seconds (POC) and 300 seconds (shipped) which were not significantly different (P = 1.000). Reference intervals for CTs differed for COL/ADP at POC (19.8-89.7 seconds) and shipped (50.9-161.6 seconds) and for P2Y at POC (35.5-118.8 seconds) and shipped (35.1-108.9 seconds). Receiver operating characteristics showed similar areas under the curve (AUCROCs) regarding the effect of clopidogrel for COL/ADP at POC (0.994 seconds) and shipped (0.932) and for P2Y at POC (0.904 seconds) and shipped (0.975 seconds). When classifying for the presence of clopidogrel effects, Cohen's Kappa was 0.62 for COL/ADP and 1.00 for P2Y. CONCLUSIONS: Shipping blood samples for PFA analysis are feasible with similar performance to POC analyses for determining the effect of clopidogrel in cats.

Platelet function analyzer-200 closure curve analysis and assessment of flow-obstructed samples.

BACKGROUND: The Platelet function analyzer-200 can determine the effect of clopidogrel in cats. Flow obstruction is an error that causes uninterpretable results. Closure curves and parameters initial flow rate (IF) and total volume (TV) are displayed by the PFA-200 and may allow interpretation of results in cases of flow obstruction. The primary hemostasis components (PHC) are calculated values that normalize these parameters. OBJECTIVES: To determine if closure curves and research parameters allow detecting the effect of clopidogrel in cases of flow obstruction. METHODS: A review of closure curves identified those with flow obstruction and paired analysis that did not. Non-flow-obstructed curves were used to categorize curves with respect to clopidogrel effects. IF, TV, PHC(1), and PHC(2) were evaluated to determine if these could be used to categorize if a sample exhibited the effects of clopidogrel. Curves were visually analyzed, and characteristics identified that were more common with or without the effect of clopidogrel. Visual analysis of curves was performed by blinded observers to determine if a visual analysis was able to predict the effect of clopidogrel. RESULTS: Analysis of parameters was able to predict closure or non-closure in flow-obstructed curves. TV, PHC(1), and PHC(2) had area under the curve of the receiver operating characteristics of 0.79, 0.79, and 0.87. Visual curve analysis was unable to predict closure, with an average accuracy of only 55%, among three reviewers. Agreement between reviewers was poor (Fleiss' Kappa 0.06). CONCLUSIONS: Visual curve analysis was unable to determine the effect of clopidogrel in flow-obstructed samples. Numerical parameters were able to detect the effect of clopidogrel with a high degree of accuracy in flow-obstructed samples.

On-treatment platelet reactivity through the thromboxane A2 or P2Y12 platelet receptor pathways is not affected by pelacarsen.

BACKGROUND: Pelacarsen decreases plasma levels of lipoprotein(a) [Lp(a)] and oxidized phospholipids (OxPL). It was previously reported that pelacarsen does not affect the platelet count. We now report the effect of pelacarsen on on-treatment platelet reactivity. METHODS: Subjects with established cardiovascular disease and screening Lp(a) levels ≥60 mg per deciliter (~ ≥150 nmol/L) were randomized to receive pelacarsen (20, 40, or 60 mg every 4 weeks; 20 mg every 2 weeks; or 20 mg every week), or placebo for 6-12 months. Aspirin Reaction Units (ARU) and P2Y12 Reaction Units (PRU) were measured at baseline and the primary analysis timepoint (PAT) at 6 months. RESULTS: Of the 286 subjects randomized, 275 had either an ARU or PRU test, 159 (57.8%) were on aspirin alone and 94 (34.2%) subjects were on dual anti-platelet therapy. As expected, the baseline ARU and PRU were suppressed in subjects on aspirin or on dual anti-platelet therapy, respectively. There were no significant differences in baseline ARU in the aspirin groups or in PRU in the dual anti-platelet groups. At the PAT there were no statistically significant differences in ARU in subjects on aspirin or PRU in subjects on dual anti-platelet therapy among any of the pelacarsen groups compared to the pooled placebo group (p > 0.05 for all comparisons). CONCLUSION: Pelacarsen does not modify on-treatment platelet reactivity through the thromboxane A2 or P2Y12 platelet receptor pathways.

Effects of clopidogrel bisulfate on B16-F10 cells and tumor development in a murine model of melanoma.

Metastatic melanoma is a very aggressive skin cancer. Platelets are constituents of the tumor microenvironment and, when activated, contribute to cancer progression, especially metastasis and inflammation. P2Y12 is an adenosine diphosphate receptor that triggers platelet activation. Inhibition of P2Y12 by clopidogrel bisulfate (CB) decreases platelet activation, which is also controlled by the extracellular concentration and the metabolism of purines by purinergic enzymes. We evaluated the effects of CB on the viability and proliferation of cultured B16-F10 cells. We also used a metastatic melanoma model with C57BL-6 mice to evaluate cancer development and purine metabolism modulation in platelets. B16-F10 cells were administered intraperitoneally to the mice. Two days later, the animals underwent a 12-day treatment with CB (30 mg/kg by gavage). We have found that CB reduced cell viability and proliferation in B16-F10 culture in 72 h at concentrations above 30 µm. In vivo, CB decreased tumor nodule counts and lactate dehydrogenase levels and increased platelet purine metabolism. Our results showed that CB has significant effects on melanoma progression.

Synergistic inhibitory effects of clopidogrel and rivaroxaban on platelet function and platelet-dependent thrombin generation in cats.

BACKGROUND: Dual antithrombotic treatment (DAT) with clopidogrel and rivaroxaban sometimes is prescribed to cats with hypertrophic cardiomyopathy at risk of thromboembolism. To date, no studies have evaluated their combined effects on platelet function. OBJECTIVES/HYPOTHESIS: Evaluate the safety of DAT in healthy cats and compare, ex vivo, platelet-dependent thrombin generation and agonist-induced platelet activation and aggregation in cats treated with clopidogrel, rivaroxaban, or DAT. We hypothesized that DAT would safely modulate agonist-induced platelet activation and aggregation more effectively than single agent treatment. ANIMALS: Nine apparently healthy 1-year-old cats selected from a research colony. METHODS: Unblinded, nonrandomized ex vivo cross-over study. All cats received 7 days of rivaroxaban (0.6 ± 0.1 mg/kg PO), clopidogrel (4.7 ± 0.8 mg/kg PO), or DAT with defined washout periods between treatments. Before and after each treatment, adenosine diphosphate (ADP)- and thrombin-induced platelet P-selectin expression was evaluated using flow cytometry to assess platelet activation. Platelet-dependent thrombin generation was measured by fluorescence assay. Platelet aggregation was assessed using whole blood impedance platelet aggregometry. RESULTS: No cats exhibited adverse effects. Of the 3 treatments, only DAT significantly decreased the number of activated platelets (P = .002), modulated platelet activation in response to thrombin (P = .01), dampened thrombin generation potential (P = .01), and delayed maximum reaction velocity (P = .004) in thrombin generation. Like clopidogrel, DAT inhibited ADP-mediated platelet aggregation. However, rivaroxaban alone resulted in increased aggregation and activation in response to ADP. CONCLUSION AND CLINICAL IMPORTANCE: Treatment combining clopidogrel and rivaroxaban (DAT) safely decreases platelet activation, platelet response to agonists, and thrombin generation in feline platelets more effectively than monotherapy with either clopidogrel or rivaroxaban.

Decreased platelet miR-199a-5p level might lead to high on-clopidogrel platelet reactivity in patients with coronary artery disease.

Clopidogrel combined with aspirin is widely used in coronary artery disease (CAD) patients, while some patients exhibit high platelet activity when receiving the combined treatment. Current environmental and genetic factors could only explain part of the variability in clopidogrel efficacy. Human platelets harbor abundant miRNAs which might affect clopidogrel efficacy by regulating the expression of key proteins in the clopidogrel antiplatelet signaling pathway. This study aimed to investigate the association between platelet miRNA levels and clopidogrel efficacy. Here we recruited 508 CAD patients who underwent clopidogrel antiplatelet therapy and determined the platelet reactivity index (PRI) to evaluate antiplatelet reactivity responses to clopidogrel. Subsequently, 22 patients with extreme clopidogrel response were selected for platelet small RNA sequencing. Another 41 CAD patients taking clopidogrel were collected to verify the differentially expressed candidate miRNAs. We found the metabolic types of the CYP2C19 enzyme (based on CYP2C19 * 2 and * 3 polymorphisms) could significantly affect the PRI of CAD patients with or without percutaneous coronary intervention (PCI) in Chinese. A total of 43 miRNAs were differentially expressed in the platelets from the 22 extreme clopidogrel response samples, and 109 miRNAs were differentially expressed in the 13 CYP2C19 extensive metabolizers with extreme clopidogrel response. Platelet miR-199a-5p levels were correlated negatively with PRI after clopidogrel therapy. Studies in cultured cells revealed that miR-199a-5p inhibited the expression of VASP, a key effector protein downstream of the P2Y12 receptor. In conclusion, we found the expression of VASP could be inhibited by miR-199a-5p, and decreased platelet miR-199a-5p was associated with high on-clopidogrel platelet reactivity in CAD patients.

What is the context?● Clopidogrel combined with aspirin is widely used in coronary artery disease (CAD) patients, while some patients exhibit high platelet activity when receiving the combined treatment.● Current environmental and genetic factors could only explain part of the variability in clopidogrel efficacy.● Human platelets harbor abundant miRNAs which might affect clopidogrel efficacy by regulating the expression of key proteins in the clopidogrel antiplatelet signaling pathway.What is new?● We found that decreased platelet miR-199a-5p level was associated with high on-clopidogrel platelet reactivity.● Overexpression of miR-199a-5p significantly down-regulated the expression of VASP protein in cultured cells.What is the impact?● The current study provided new insights into the exploration of interindividual variability in clopidogrel response from the perspective of miR-199a-5p and VASP interaction.