Search

Targeted therapies are becoming more common. In targeted therapy development, suppose its companion diagnostic test divides patients into a marker-positive subgroup and its complementary marker-negative subgroup. To find the right patient population for the therapy to target, inference on efficacy in the marker-positive and marker-negative subgroups as well as efficacy in the overall mixture population are all of interest. Depending on the type of clinical endpoints, inference on mixture population can be nontrivial and commonly used efficacy measures may not be suitable for a mixture population. Correlations among estimates of efficacy in the marker-positive, marker-negative, and overall mixture population play a crucial role in using an earlier phase study to inform on the design of a confirmatory study (e.g., determination of sample size). This article first shows that when the clinical endpoint is binary (such as respond or not), odds ratio is inappropriate as an efficacy measure in this setting, but relative response (RR) is appropriate. We show a safe way of calculating estimated correlations is to consider mixing subgroup response probabilities within each treatment arm first, and then derive the joint distribution of RR estimates. We also show, if one calculates RR within each subgroup first, how wrong the correlations can be if the Delta method derivation fails to take randomness of estimating the mixing coefficient into account., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Long-term evidence shows an increased risk of cardiovascular events in the morning hours and recent studies in aspirin-treated patients have shown increased platelet reactivity at the end of the dosing interval. Similar pharmacodynamic analyses of adenosine-diphosphate (ADP) receptor inhibitors are scarce. We therefore investigated changes in clopidogrel-dependent platelet function and activation over 24 h and whether enhanced platelet turnover might explain diurnal variability of platelet function and activation. Twenty-one patients after acute coronary syndromes (ACS) on maintenance doses of clopidogrel (75 mg) and aspirin (100 mg) Once per day (OD) were included. Blood was collected at five time points in 24 h. Platelet function and activation was analyzed by vasodilator-stimulated phosphoprotein-phosphorylation (VASP-P), Verify Now, multiple electrode aggregometry (MEA), and platelet PAC-1 and P-selectin (P-sel) expression. Additionally, platelet count, mean platelet volume (MPV), and reticulated platelet fraction (RPF) were analyzed. There was significant diurnal variability of clopidogrel effects as documented with VASP-P, Verify Now, and PAC-1 and P-sel (all p < 0.05), whereas MEA did not differ over 24 h. Neither MPV nor RPF varied significantly over 24 h. In patients with high RPF, platelet function and activation was significantly higher in all assays, compared to patients with low RPF (all p < 0.05). However, the changes over time in low versus high RPF groups were similar. ADP-dependent platelet function and activation recovers significantly at the end of the 24-h dosing interval in patients with ACS on a maintenance dose of clopidogrel and aspirin. Although platelet function and activation is increased in patients with higher RPF, platelet turnover might not explain the observed diurnal variability.

Acute coronary syndrome (ACS) has become a vital disease with high mortality worldwide. A combined antiplatelet therapy (aspirin and a P2Y 12 antagonist) is commonly used to prevent re-infarction in ACS patients who have undergone percutaneous coronary intervention (PCI). Clopidogrel, a P2Y 12 antagonist, plays an important role in the inhibition of platelet aggregation (IPA). However, it is a pro-drug requiring biotransformation by cytochrome P450 (CYP450). The aim of this study is to unravel the effect of clopidogrel-associated genetic variants on inhibition of platelet activity and clinical outcomes in ACS patients. In our study, a total of 196 patients with metabolic gene polymorphism of clopidogrel were enrolled, and their antiplatelet effect as well as their cardiovascular events were collected. Approximately 2 mL of venous blood samples were used for genotype detection and another 4 mL were collected for platelet reactivity with thrombelastography. The primary clinical end-point was defined as a combination of cardiovascular mortality and revascularization for targeted vascular lesion. Based on the results of IPA, the prevalence of high on-treatment platelet reactivity (HPR) was 17.3% and the majority of patients (82.7%) obtained normal on-treatment platelet reactivity (NPR). The HPR group had significantly higher body mass index (BMI) and lower arachidonic acid (AA) induced IPA (P < 0.05). Therapy including Glycoprotein (GP) IIb/IIIa antagonist increased IPA (P < 0.05). ADP-induced IPA effect was lower with the presence of CYP2C19*2, *3 and paraoxonase (PON)1 Q192R loss-of-function (LOF) alleles, respectively (P < 0.05). Multivariate logistic regression analysis demonstrated that aspirin resistance (AA-induced IPA < 50%) had a greater risk of the occurrence of major adverse cardiovascular events (MACE) (OR = 3.817; 95% CI: 1.672-8.700; P = 0.002). CYP2C19*2 LOF alleles were associated with high risk of MACE in 1-year post PCI operations (OR = 2.571; 95% CI: 1.143-5.780; P = 0.030). For the ACS patients, the presence of CYP2C19*2 and PON1 Q192R LOF alleles were the major drivers of HPR., (© 2018 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).)

Background: Elevated mean platelet volume (MPV) and immature platelet fraction (IPF) are predictive for vascular risk. Both can be associated with residual platelet reactivity. We aimed to explore associations among platelet characteristics and responder status in stroke patients on clopidogrel., Methods: Blood samples from 46 patients and 15 healthy subjects were analyzed for platelet count, MPV, IPF, large cell ratio (LCR) and high-fluorsecent immature platelet fraction (H-IPF). As a novelty, not only whole blood, but upper and lower half blood samples after 1-hour gravity sedimentation were analyzed. Platelet aggregometry was used for the whole blood and separated samples to explore area under the curve (AUC) in patients and controls., Results: The AUC of the whole blood showed significant differences compared to the upper and lower samples separated after 1-hour sedimentation in patients and controls (p < 0.001 and p = 0.005 respectively). Remarkably, AUC measured in the upper samples in 59% of patients on clopidogrel were exceeding the therapeutic range suggesting that ascending platelets exert aggregation in the presence of ADP. This observation was associated with increased MPV and LCR in the upper samples (both p = 0.04). Patients on clopidogrel were characterized as responders and non-responders and the percentage of H-IPF was significantly higher among non-responders compared to controls in the upper samples (p = 0.04)., Conclusions: The modified platelet function test may help to stratify patients with high residual platelet reactivity.

Background: Carotid stenting stimulates intimal proliferation through platelet and stem cell activation., Objective: The aim of this study is to evaluate whether the administration before or after carotid stenting of clopidogrel loading dose may play a role on circulating endothelial progenitor cells, stromal cell-derived factor-1α (SDF-1α) and neointimal hyperplasia., Methods: We recruited 13 patients (aged 74.52±7.23) with indication of carotid revascularization and in therapy with salicylic acid and statin. We blindly randomized them in two groups: pre-carotid angioplasty with stent (Pre-CAS group) receiving 300 mg of clopidogrel before stenting, and post-carotid angioplasty with stent (Post-CAS group) receiving 300 mg after stenting. At the admission, we valued endothelial progenitor cells, SDF-1α and prospectively we repeated blood samples and measured intima-media thickness to estimate neointimal hyperplasia on the stent at 3, 6 and 12 months., Results: In the days following the CAS, we found a lower, statistically not significant, trend of endothelial progenitor cells in Pre-CAS group. The SDF-1α concentration tended to be lower at baseline in the pre-CAS group than in the post-CAS group and it did not show an increase in the observed time. On the contrary, in the Post-CAS group we observed a peak at six hours with a significant reduction (p < 0.001) at one day after stenting.The intima-media thickness was significantly lower in the Pre-CAS group than the Post-CAS group both at six months and 12 months after stenting., Conclusions: Pre-stenting clopidogrel loading dose leaded to short-time modification of endothelial progenitor cells and platelets and to long-term a minor neointimal hyperplasia.

The effect of CYP2C19 gene polymorphism on clinical outcomes of patients with coronary artery disease (CAD) treated with clopidogrel remains controversial. Ethnicity has been proposed to influence clopidogrel response following stent implantation in CAD patients with different CYP2C19 genotypes. Furthermore, Asian populations are reported to have a relatively greater prevalence of CYP2C19 loss-of-function (LOF) alleles. We aimed to evaluate the impact of CYP2C19 gene polymorphism on clinical outcomes in Asian populations who underwent percutaneous coronary interventions (PCI) and received clopidogrel therapy. We conducted a comprehensive search in PubMed, EMBASE, and Cochrane Library from their inceptions to January 20, 2017. Studies that reported clopidogrel therapy information, clinically relevant outcomes (adverse cardiovascular events, stent thrombosis and bleeding), and CYP2C19 genotypes among Asian populations were included. The primary endpoint was major adverse cardiovascular events (MACE), defined as a composite of cardiovascular death and myocardial infarction. The safety endpoint was any kind of bleeding. We retrieved 20 studies of 15056 patients reporting 1301 cardiovascular events. The primary analysis showed at least one CYP2C19 LOF allele (*2 and/or *3) carriers were at an increased risk of MACE compared with non-carriers (10.58% vs. 6.07%, OR: 1.99, 95% CI: 1.64 to 2.42, p < .001). Stent thrombosis (ST) was also more frequent in LOF allele carriers (2.22% vs. 0.44%, OR: 4.77, 95% CI: 2.84 to 8.01, p < .001). Inversely, the risk of bleeding was lower in LOF allele carriers (OR: 0.66, 95% CI: 0.46 to 0.96, p < .001). Subgroup analysis was performed to assess differences by high (600 mg) or routine (300 mg) loading dose of clopidogrel and by different nationalities. The risk of MACE in LOF allele carriers remained significantly higher even in high loading dose group (high loading dose: OR 1.72, 95% CI: 1.37 to 2.16, and routine loading dose: OR 2.22, 95% CI: 1.68 to 2.94, p for subgroup heterogeneity = 0.16). Subgroup analysis between three nationalities of China, Korea, and Japan demonstrated that the risk of MACE among Chinese LOF allele carriers was the greatest (OR: 2.28; 95% CI:1.91 to 2.73). In conclusion, among Asian populations with CAD undergoing stent implantation, CYP2C19 LOF allele carriers are at greater risk of adverse cardiovascular events and lower risk of bleeding compared with non-carriers. Genetic testing may be helpful for clinicians to personalize antiplatelet therapy especially in Asian population.

Dual antiplatelet therapy (DAPT) with aspirin and a P2Y 12 inhibitor is a mainstay of the prevention of stent thrombosis following percutaneous coronary intervention (PCI). In the 2015 European guidelines for the management of acute coronary syndrome (ACS), prasugrel (PRA) and ticagrelor (TICA) combined with aspirin are recommended as first-line therapy. Clopidogrel (CLO) is recommended as an alternative medication for patients with contradictions to these new drugs. This single-center study analyzed the platelet function of 809 ACS patients undergoing PCI and treatment with DAPT. The platelet response to ADP was determined using Multiplate® analyzer at a median of 3 days after PCI in 254 patients treated with PRA (loading dose [LD] 60 mg, 10 mg qd), 162 patients receiving TICA (LD 180 mg, D 90 mg bid), and 393 CLO-treated patients (LD 600 mg, 75 mg qd). An aggregation >468 arbitrary units (AU)*min was defined as "high on-treatment platelet reactivity" (HPR), <188 AU*min as "low on-treatment platelet reactivity" (LPR). Platelet response in PRA-treated patients was lower compared to CLO or TICA (median; interquartile range: PRA 220 [163-275] AU*min vs. CLO 268 [186-387] AU*min, p < 0.001 vs. TICA 245 [190-320] AU*min, p = 0.001). Only 1.6% of PRA patients were stratified as HPR and 34.6% as LPR, while in the TICA group 1.9% fulfilled the criteria of HPR and 24.1% criteria of LPR. Sixteen percent of CLO patients were stratified as HPR and 26.2% as LPR. In a real-world cohort of ACS patients following PCI, PRA results in more potent inhibition of platelet function compared to CLO and TICA. TICA achieves a consistent antiplatelet effect with reduced rates of HPR and LPR in relation to CLO.

Clopidogrel is the cornerstone antiplatelet used in the treatment and prevention of thrombotic events. Some studies examined the effect of CYP2C19 polymorphism and nongenetic factors on clopidogrel response in the Middle East and North Africa (MENA) region. However, the consistency among these studies is yet unknown. This study aims to estimate the prevalence of CYP2C19 genetic variants in MENA region and to evaluate the effect of these variants as well as the nongenetic factors on clopidogrel responsiveness. A systematic literature search was performed to identify relevant articles. Only observational studies were included. A total of 20 studies in 8 different populations were included. The CYP2C19*2 variant is the most prevalent loss-of-function (LOF) allele in the MENA region (1.7%-35%). The frequency of CYP2C19*17 ranged from 5.3% to 26.9%. Of the 9 studies, 6 found an association between carriers of at least 1 LOF allele and clopidogrel resistance. Older age, high body mass index, females, and the use of calcium channel blockers were associated with clopidogrel resistance as well. Association between the CYP2C19*2 allele and clopidogrel resistance is common among MENA populations. Future studies should focus on having larger sample sizes to detect other minor variant alleles and their effect on bleeding and cardiovascular outcomes.

Objective: Oral antiplatelet drugs are a key to modern pharmacotherapy in cardiovascular atherothrombotic diseases. Clopidogrel (CLO) constitutes the main preventive treatment of atherothrombosis. However, a considerable inter-individual variation in CLO response has been documented, resulting in suboptimal therapy and an increased risk of recurrent adverse effects in some patients. The enzyme CYP2C19 has been reported to be the CYP isoform that activates CLO to its active metabolite. Several single nucleotide polymorphisms in the CYP2C19 gene have been identified as strong predictors of CLO-impaired pharmacological response. At least 16 variants have been associated with changes in CYP2C19 activity., Materials and Methods: The following research was composed of a total of 102 subjects with high cardiovascular risk in the northeast of Mexico, with a maintenance dose of 75 mg of CLO per day. The platelet reactivity was measured with VerifyNow P2Y12 assay, while the presence of CYP2C19*2 was identified by real-time polymerase chain reaction., Results: Patients were categorized by CYP2C19 metabolizer status based on *2 genotypes using the common consensus star allele nomenclature as normal metabolizer (G/G), intermediate metabolizer (G/A), and poor metabolizer (A/A), respectively. The phenotype frequency for CYP2C19*2 was 74.5% (G/G), 21.6% (G/A), and 3.9% (A/A). The subjects with the A allele presented ≥235 P2Y12 reaction unit levels, classifying them how poor metabolizer. The prevalence of reduced CLO effectiveness was associated with the presence of CYP2C19*2 polymorphism among Mexican patients., Conclusion: The presence of the CYP2C19*2 allele is related to resistance to the antiplatelet effect of CLO (p = 0.003)., (Copyright: © 2019 Permanyer.)

Aims: P2Y12 antagonists are the standard in antiplatelet therapy but their potential effects on functional myocardial recovery and cardioprotection post-myocardial infarction (MI) are unknown. We investigated in a preclinical model of MI whether ticagrelor and clopidogrel differently affect cardiac repair post-MI., Methods and Results: Pigs either received: (i) clopidogrel (600 mg; 75 mg/qd); (ii) ticagrelor (180 mg; 90 mg/bid); and (iii) placebo control. MI was induced by mid-left anterior descending coronary artery balloon occlusion (60 min) and animals received the maintenance doses for the following 42 days. Serial cardiac magnetic resonance was performed at Day 3 and Day 42 for the assessment of global and regional cardiac parameters. We determined cardiac AMP-activated protein kinase (AMPK), Akt/PKB, aquaporin-4, vascular density, and fibrosis. In comparison to controls, both P2Y12 antagonists limited infarct expansion at Day 3, although ticagrelor induced a further 5% reduction (P < 0.05 vs. clopidogrel) whereas oedema was only reduced by ticagrelor (≈23% P < 0.05). Scar size decreased at Day 42 in ticagrelor-treated pigs vs. controls but not in clopidogrel-treated pigs. Left ventricular ejection fraction was higher 3 days post-MI in ticagrelor-treated pigs and persisted up to Day 42 (P < 0.05 vs. post-MI). Regional analysis revealed that control and clopidogrel-treated pigs had severe and extensive wall motion abnormalities in the jeopardized myocardium and a reduced myocardial viability that was not as evident in ticagrelor-treated pigs (χ2P < 0.05 vs. ticagrelor). Only ticagrelor enhanced myocardial AMPK and Akt/PKB activation and reduced aquaporin-4 levels (P < 0.05 vs. control and clopidogrel). No differences were observed in vessel density and fibrosis markers among groups., Conclusions: Ticagrelor is more efficient than clopidogrel in attenuating myocardial structural and functional alterations post-MI and in improving cardiac healing. These benefits are associated with persistent AMPK and Akt/PKB activation.

Filamin-binding LIM protein 1 (FBLIM1) is related to regulation of inflammatory responses, such as chronic recurrent multifocal osteomyelitis; however, the relevance of FBLIM1 in oral squamous cell carcinoma (OSCC) is unknown. The aim of the current study was to elucidate the possible role of FBLIM1 in the carcinogenesis of OSCC. We analyzed FBLIM1 expression using quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), immunoblot analysis, and immunohistochemistry. The expression levels of FBLIM1 were up-regulated significantly (P < 0.05) in OSCC-derived cell lines and primary OSCCs specimens compared with normal counterparts. FBLIM1 expression also was correlated with the primary tumoral size (P < 0.05) and vascular invasion (P < 0.05). We then assessed tumoral progression after treatment with FBLIM1 siRNA and clopidogrel, an antiplatelet agent. Similar to the FBLIM1 knockdown effect, clopidogrel-treated cells had attenuated functions of proliferation, migration, and invasiveness. Interestingly, clopidogrel treatment led to down-regulation of epidermal growth factor receptor (EGFR) and FBLIM1. These findings identify FBLIM1 as a putative therapeutic target by using clopidogrel for inhibiting over activation of EGFR signaling to prevent OSCC malignancy., (© 2018 Wiley Periodicals, Inc.)

Background Platelets play a role in promoting inflammatory responses under several disease conditions. Platelets are activated in hypertensive patients. However, the mechanisms responsible for platelet-mediating vascular inflammation are unknown. The present study investigated the role of platelets in promoting vascular inflammation following angiotensin II (Ang II ) stimulation, and the efficacy of antiplatelet intervention. Methods and Results Within a mouse model of Ang II infusion (490 ng/kg per min), we measured the portion of P-selectin-positive platelets and platelet-monocyte (P-M) binding in blood samples, and platelet accumulation and P-M binding in vessels under Ang II stimulation at days 1, 3, and 7. We tested the efficacy of clopidogrel (15 mg/kg per day, followed by 5 mg/kg per day) on Ang II -induced platelet activation, P-M binding, vascular platelet accumulation, as well as vascular inflammation and remodeling at day 7 or 14. Clopidogrel reduced platelet vascular deposition (28.7±2.4% versus 18.3±2.9%), suppressed inflammatory cell infiltration (3.6±0.8×10 4 /vessel versus 2.3±1.2×10 4 /vessel) and oxidative stress, and attenuated vascular remodeling and dysfunction (55.0±5.5% versus 84.0±6.0%) following Ang II stimulation at day 7 or 14. Clopidogrel suppressed Ang II -induced P-M binding both at circulating (13.4±3.3% versus 5.9±2.7%) and regional (33.4±4.3% versus 11.9±2.7%) levels. Conclusions Platelets play a critical role in vascular inflammation under Ang II stimulation, with a marked promotion of P-M binding as an important mechanism. Clopidogrel prevented vascular inflammation in Ang II -infused mice.

Present study was to evaluate whether switching ticagrelor to clopidogrel would impact platelet reactivity and cardiovascular outcomes in acute coronary syndrome (ACS) patients after percutaneous coronary intervention (PCI).A total of 202 ACS patients after PCI were enrolled and prescribed ticagrelor. Before discharge, 138 (68%) patients were switched to clopidogrel. Peripheral blood was obtained before switching and at 48 hours after switching to measure platelet reactivity. Patients were followed for 30 days to evaluate cardiovascular events.Compared to ticagrelor group, patients in clopidogrel group were more likely to be male (69.6% vs 65.6%), smokers (34.1% vs 31.3%) and had higher prevalence of hypertension (75.4% vs 71.9%). The frequency of right coronary artery lesion was significantly higher in ticagrelor group (34.4% vs 30.4%). There were no significant differences in baseline platelet reactivity (37.6 ± 5.2% vs 38.4 ± 4.9%). Forty-eight hours after switching to clopidogrel, platelet reactivity in clopidogrel group was significantly higher (46.3 ± 5.6% vs 38.1 ± 5.0%, P <.05). Patients in clopidogrel group had significantly higher incidence of cardiovascular events (3.6% vs 1.6%, P <.05). However, after further adjusted for platelet reactivity at 48 hours of switching, clopidogrel switching was not significantly associated with composite outcomes, with hazard ratio 1.08 (95% confidence interval 0.98-1.21, P = .063), indicating that platelet reactivity was a critical mediator between antiplatelet drug switching and cardiovascular outcomes.ACS patients after PCI treatment, early switching ticagrelor to clopidogrel results in increased platelet reactivity and higher incidence of short-term cardiovascular events.

P2Y 12 receptor antagonists are a class of drugs that act on platelet P2Y 12 receptors and inhibit adenosine diphosphate-induced platelet aggregation. As a thienopyridine antiplatelet agent which is approved by the US Food and Drug Administration for the treatment of cardiovascular diseases, currently, clopidogrel was once considered to be the most safe and effective antiplatelet drug in the P2Y 12 receptor antagonists, however, it has become increasingly clear that clopidogrel does not satisfactorily inhibit the platelets of approximately one-third of patients. This is in part due to clopidogrel is a prodrug and reliance on multiple cytochrome P450 enzymes for conversion into its active metabolite. Prasugrel and ticagrelor reduces the risk of adverse cardiovascular events compared to clopidogrel in acute coronary syndromes patients, however, the cardiovascular benefit of both drugs is counter-balanced by increased rates of spontaneous bleeding. Unlike clopidogrel, which is a prodrug, cangrelor is an active drug not requiring metabolic conversion, despite fewer bleeding events during cardiac surgery, cangrelor carries the risk of potential autoimmune reactions manifesting as breathlessness. DV-127 was synthesized by using three generations of thienopyridine P2Y 12 receptor antagonists as research models, using high resolution mass spectrometry, selective deuteration, and 2,7-position replacement groups technologies in order to maximize cardiovascular benefit while minimizing adverse effects on hemostasis. Our results show that although the dose of DV-127 is greatly reduced, it can achieve similar anticoagulant and antiplatelet effects as clopidogrel, and DV-127 can more strongly inhibit the release of α-granules even though the inhibitory effect of dense granules is similar to clopidogrel., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background and Objectives: Prasugrel and clopidogrel are inhibitors of the ADP-P 2 Y 12 platelet receptor used in acute coronary syndrome patients. They require bioactivation via isoenzymes such as cytochrome P450 (CYP) 3A4, CYP2C19 and CYP2B6. Ritonavir and cobicistat are potent CYP3A inhibitors, prescribed as pharmacokinetic (PK) enhancers in the treatment of human immunodeficiency virus (HIV) infection., Methods: In this study, the impact of boosted antiretroviral therapies (ARTs) on the PK of clopidogrel and prasugrel active metabolites (AMs), and on the efficacy of prasugrel and clopidogrel, were evaluated in a randomized crossover clinical trial., Results: A significantly lower exposure to clopidogrel AM [3.2-fold lower area under the concentration-time curve (AUC) and maximum plasma concentration (C max )] and prasugrel AM (2.1-fold and 1.7-fold lower AUC and C max ) were demonstrated in HIV-infected patients treated with boosted ARTs compared with healthy controls; however, a differential impact was observed on platelet inhibition between clopidogrel and prasugrel. Clopidogrel 300 mg induced adequate (although modest) platelet inhibition in all healthy subjects, while platelet inhibition was insufficient in 44% of HIV patients. On the contrary, prasugrel 60 mg induced a potent platelet inhibition in both healthy and HIV-infected subjects., Conclusion: Prasugrel appears to remain an adequate antiplatelet agent in HIV-infected patients and could be preferred to clopidogrel in this context, regardless of the metabolic interaction and inhibition of its bioactivation pathways.

Hydrogen sulfide has emerged as a critical endogenous signaling transmitter and a potentially versatile therapeutic agent. The key challenges in this field include the lack of approved hydrogen sulfide-releasing probes for in human exploration and the lack of controllable hydrogen sulfide promoieties that can be flexibly installed for therapeutics development. Here we report the identification of the widely used antithrombotic drug clopidogrel as a clinical hydrogen sulfide donor. Clopidogrel is metabolized in patients to form a circulating metabolite that contains a thioenol substructure, which is found to undergo spontaneous degradation to release hydrogen sulfide. Model studies demonstrate that thioenol derivatives are a class of controllable promoieties that can be conveniently installed on a minimal structure of ketone with an α-hydrogen. These results can provide chemical tools for advancing hydrogen sulfide biomedical research as well as developing hydrogen sulfide-releasing drugs.

Background: Adenosine-5'-diphosphate (ADP) can influence intrarenal vascular tone and tubular transport, partly through activation of purine P2Y12 receptors (P2Y12-R), but their actual in vivo role in regulation of renal circulation and excretion remains unclear., Methods: The effects of intravenous ADP infusions of 2-8mg/kg/hour were examined in anesthetized Wistar rats that were untreated or chronically pretreated with clopidogrel, 20mg/kg/24hours, a selective P2Y12-R antagonist. Renal blood flow (transonic probe) and perfusion of the superficial cortex and medulla (laser-Doppler fluxes) were measured, together with urine osmolality (U osm ), diuresis (V), total solute (U osm V), sodium (U Na V) and potassium (U K V) excretion., Results: ADP induced a gradual, dose-dependent 15% decrease of mean arterial pressure, a sustained increase of renal blood flow and a 25% decrease in renal vascular resistance. Clopidogrel pretreatment attenuated the mean arterial pressure decrease, and did not significantly alter renal blood flow or renal vascular resistance. Renal medullary perfusion was not affected by ADP whereas U osm decreased from 1,080 ± 125 to 685 ± 75 mosmol/kg H 2 0. There were also substantial significant decreases in U osm V, U Na V and U K V; all these changes were attenuated or abolished by clopidogrel pretreatment. Two-weeks' clopidogrel treatment decreased V while U osm U osm V and U Na V increased, most distinctly after 7 days. Acute clopidogrel infusion modestly decreased mean arterial pressure and significantly increased outer- and decreased inner-medullary perfusion., Conclusions: Our functional studies show that ADP can cause systemic and renal vasodilation and a decrease in mean arterial pressure, an action at least partly mediated by P2Y12 receptors. We confirmed that these receptors exert tonic action to reduce tubular water reabsorption and urine concentration., (Copyright © 2018 Southern Society for Clinical Investigation. Published by Elsevier Inc. All rights reserved.)

CYP2C19 genotype influences clopidogrel response but only accounts for a small part of the variability in platelet reactivity. Recently, exome sequencing identified a variant of the gene encoding B4GALT2 as a potential candidate implicated in on-treatment platelet reactivity. Carriers of the B4GALT2 c.909C>T variant have lower platelet reactivity indicating that B4GALT2 could influence clopidogrel sensitivity and could expose to the risk of bleeding events. We undertook this observational retrospective study to determine if B4GALT2 c.909C>T influences P2RY12-specific vasodilator-stimulated phosphoprotein phosphorylation and agonist-induced platelet aggregation in a nonselected cohort of 174 patients under clopidogrel-based antiplatelet therapy. Our results indicate that in individuals under dual antiplatelet therapy, B4GALT2 c.909C>T might be an independent genetic predictor of on-treatment platelet reactivity.

Pharmacotherapy with agents that inhibit platelet function has proven to be effective in the treatment of acute coronary syndrome. Proper re-endothelization after angioplasty prevents adverse cardiovascular events. Therefore, in this in vitro study we examined how antiplatelet P2Y 12 receptor blockers can affect endothelial cells' angiogenic properties. Endothelial cells were exposed to ticagrelor, prasugrel and clopidogrel in their highest concentrations obtained in serum after the treatment with loading and clinical doses. Further, the viability, apoptosis, and necrosis were tested and the following angiogenic properties such as proliferation, migration, invasiveness, tube formation, wound healing and the production of angiogenic mediators (bFGF, PDGF, MMP-2, Ang-2, TIMP-1). The results of this study showed that P2Y 12 receptor blockers in the tested concentrations are safe for endothelial cells. They neither induced necrosis or apoptosis nor changed the endothelial cell viability, migration, invasiveness, tube formation, wound healing, the production of VEGF or its receptors. However, they reduced cell proliferation. It was shown that out of these three drugs, ticagrelor in its loading concentration had the most potent angiogenic property. It reduced cell proliferation and changed the production of angiogenic (bFGF, MMP-2) and angiostatic mediators (Ang-2). In conclusion, P2Y 12 receptor blockers in the concentrations obtained in the serum during standard therapy reduced endothelial cell proliferation. Despite this slight antimitogenic effect, they did not change endothelial cell tube formation or wound healing. Out of the three tested drugs, ticagrelor had the most potent angiogenic effect in vitro, but not strong enough to disturb tube formation and wound healing.

Antiplatelet therapy is considered as a standard procedure against atherosclerotic cardiovscular disease but this therapy has limited effect if resistance to acetylsalicylic acid or clopidogrel is present. Important factors associated with resistance are gender; or inflammation possibly associated with membrane microparticles (MP). It was decided to challenge the hypothesis that differential responses to dual antiplatelet therapy are conditioned by gender and/or proinflammatory status. The study involved 160 patients with stable coronary heart disease (118 men, 42 women) aged 65.2 ± 7.8 years. Patients were treated long-term with acetylsalicylic acid (ASA); plus clopidogrel starting 6 days before percutaneous coronary intervention (both 75 mg/day). Response was evaluated using platelet aggregation with either arachidonic acid (the ASPI test; predominantly for ASA response) or adenosine diphosphate (the ADP test; predominantly for clopidogrel response). MP levels were measured as follows: total (MP-total); with TF expression (MP-TF); or platelet-derived microparticles (PDMP), as well as proinflammatory parameters: C-reactive protein (CRP), leukocytes (WBC) and platelet numbers (PLT). Analysis of platelet-aggregation levels with regard to gender revealed higher aggregation in women: with resistance to ASA (ASPI test: P = 0.0383, ADP test: P = 0.0027); resistance to clopidogrel (ASPI test: P = 0.0003; ADP test: P = 0.0566) and with sensitivity to both drugs with the ADP test (P = 0.0190). In women relative to men, regardless of response, significantly higher CRP (P = 0.0012), WBC (P = 0.0244) and PLT numbers (P = 0.0001) were found. In contrast, in men significantly higher concentrations of MP-TF (P = 0.0286) and triglycerides (P = 0.0296) were found in the clopidogrel-resistant group. We conclude that women have an inferior response to dual antiplatelet therapy relative to men, possibly associated with higher platelet reactivity (especially when measured with the ADP test), with a more accentuated proinflammatory status. In contrast, among the factors supporting the resistance in men can be an elevated concentration of MP-TF which, together with the coexistence of hypertriglyceridemia, may constitute an important mechanism of resistance to clopidogrel.

Formation of thrombosis is mainly associated with dysfunctions of endothelial cells. NaoXinTong capsule (NXT), a traditional Chinese medicine, has been demonstrated multiple protective effects on vascular systems. However, it is unknown the effect of NXT on thrombosis. In this study, we determined whether NXT can inhibit carrageenan-induced thrombosis and the underlying mechanisms. Two days after carrageenan injection, severe thrombi were found in blood vessels of mouse tail and liver. By contrast, thrombi were substantially reduced by NXT treatment, and the reduction was associated with reduced serum tumor necrosis factor α and P-selectin levels. In vitro, NXT reduced lipopolysaccharide-activated adhesion of THP-1 monocytes to human umbilical vein endothelial cells (HUVECs) by inhibiting expression of adhesion molecules and interleukin 6, and reducing production of mitochondrial superoxide that is related to activation of antioxidant enzymes expression. NXT also reduced oxidized low-density lipoprotein-activated adhesion of platelets to HUVECs. In addition, NXT protected HUVECs against clopidogrel-induced cell death by inhibiting expression of tumor necrosis factor-like cytokine 1A and activating expression of vascular endothelial growth factor α. Taken together, our study indicates the potential application of NXT in antithrombosis by multiple antithrombotic functions.

Background: Dual-antiplatelet therapy (DAPT) is a standard strategy in acute coronary heart disease; however, it confers a considerable bleeding risk. Single-antiplatelet therapy (SAPT) inhibits haemostatic system activation ex vivo to a similar extent as DAPT. Extracellular vesicles (EV) are procoagulant and contribute to haemostatic system activation. We aimed to investigate the effect of DAPT compared with SAPT on EV., Methods: In a randomized, double-blind, placebo-controlled trial, 44 healthy volunteers received DAPT (clopidogrel + aspirin) or SAPT (clopidogrel + placebo) for 7 days. Blood was obtained from a standardized microvascular injury and through venipuncture at baseline (BL) and at 2 h, 24 h, and 8 days after treatment initiation. The number, origin, and surface expression of EV were assessed using flow cytometry. Data are given as median (quartiles). Non-parametric tests were used to evaluate the short-term (BL vs 2 h) and long-term differences (2 h to 8 days), as well as the differences between treatment groups., Results: There was no difference either in the short-term effects on the number (×10 3  mL -1 ) of EV in microvascular blood between DAPT [BL: 1433 (653; 3184) vs 2 h: 862 (545; 2026), p = 0.39] and SAPT [(BL: 614 (552; 1402) vs 2 h: 1079 (781; 1538), p = 0.75)] or in the long-term effects. DAPT and SAPT did not exhibit differential short-term effects on the number and proportion (36% and 27% vs 55% and 36%) of platelet-derived EV. DAPT and SAPT resulted in a significant short-term increase in phosphatidylserine expression in microvascular blood. The effects of DAPT and SAPT on EV in venous blood were similar to those in microvascular blood., Conclusion: DAPT and SAPT have comparable effects on the amount, origin, and surface characteristics of EV., (Copyright © 2018. Published by Elsevier Ltd.)

Objective: The aims of this study were to determine whether the detection of preoperative clopidogrel resistance in patients undergoing cardiac surgery while using clopidogrel could play a guiding role in the prediction of postoperative excessive bleeding, transfusion requirements, and risks and to provide clinically significant data., Methods: Two hundred and twenty-two patients [median age: 59.4 (38-83) years; 38 females] undergoing emergency and elective coronary artery bypass graft (CABG) surgeries in our clinic were evaluated prospectively. Patients with multiple systemic diseases, other than diabetes mellitus (DM) and hypertension (HT), were excluded. Patients receiving clopidogrel were also evaluated for clopidogrel resistance and grouped according to the results of this test. Assessments of platelet functions were performed by multiplate impedance aggregometry method and adenosine diphosphate test., Results: The use of postoperative fresh blood replacement and platelet transfusion was higher in patients receiving clopidogrel than in those not receiving it (P=0.001, P=0.018). DM, HT, myocardial infarction, and the number of presentation to the emergency room were significantly higher in patients receiving clopidogrel than in those not receiving it (P<0.05). No significant difference was determined between patients with and without clopidogrel resistance regarding the amount of bleeding during and after surgery, erythrocyte suspension and fresh-frozen plasma transfusion rates, preoperative troponin values, ejection fraction values, and length of hospital stays (P>0.05)., Conclusion: We think that resistance studies in patients receiving clopidogrel before cardiac surgery are not efficient to predict bleeding and bleeding-related complications in patients undergoing emergency and elective CABG surgeries.

Background: Hepatitis B virus (HBV) infection has been reported to down-regulate the expression of CYP2C19 gene, which may decrease the bioactivation of clopidogrel into active metabolites. We aimed to evaluate the impact of HBV infection on platelet response to clopidogrel in patients undergoing coronary stent implantation., Methods: A total of 1805 patients who had received coronary stent implantation and taken aspirin 100 mg in combination with clopidogrel 75 mg daily ≥5 days were consecutively recruited. The serologic identifications for HBV, platelet aggregations in response to arachidonic acid (PL AA ) and adenosine diphosphate (PL ADP ), as well as ABCB1, CYP2C19, CYP3A5, PON1 and P2RY12 genotypes were determined. Clopidogrel low response (CLR) was defined as PL ADP  > 40%., Results: Among the recruited subjects, 102 patients showed hepatitis B surface antigen (HBsAg) positive and 1703 patients negative. PL ADP was significantly higher in HBsAg positive group than that in HBsAg negative group [38 (24-48) % vs. 29 (20-39) %, p < 0.001] while the difference of PL AA was not statistically significant (p = 0.329). The incidence of CLR was significantly higher in HBsAg positive group compared with that in HBsAg negative group (43.1% vs. 23.4%, p < 0.001). After adjusted for CYP2C19 genotype and known risk factors, HBsAg positive patients exhibited a significantly higher risk of CLR (adjusted odds ratio: 2.81, 95% confidence interval: 1.73 to 4.58, p < 0.001)., Conclusions: HBV infection is an independent risk factor of CLR, in addition to CYP2C19 gene mutations. (Pharmacogenetic and Pharmacokinetic Study of Clopidogrel; NCT01968499)., (Copyright © 2018. Published by Elsevier Ltd.)

Purpose: Some studies in the white population have shown that carriers of at least 1 loss-of-function allele in the gene that encodes the cytochrome P-450 2C19 isozyme (CYP2C19) have lower levels of the clopidogrel active metabolite (CAM) and a reduced antiplatelet effect of clopidogrel. However, data are limited regarding the association between CYP2C19 genetic variants and exposure to CAM and on the pharmacodynamic properties of CAM in the Chinese population. Data from the white population cannot be extrapolated to the Chinese population because of the marked interethnic differences in CYP2C19 variants. This study was aimed to investigate the influence of CYP2C19 genetic polymorphisms on the pharmacokinetic properties of CAM and the antiplatelet effect of clopidogrel in healthy Chinese volunteers, and to provide evidence for the role of a CYP2C19 genotyping test in predicting the antiplatelet effect of clopidogrel in the Chinese population., Methods: Twenty healthy subjects received a single 300-mg dose of clopidogrel and were assigned to 1 of 3 groups according to CYP2C19 genotype: CYP2C19 *1/*1 (normal metabolizers [NM]; n = 8), CYP2C19 *1/*2 or *3 (intermediate metabolizers [IM]; n = 10) and CYP2C19 *2/*2 or *3 and *3/*3 (poor metabolizers [PM]; n = 2). Blood samples were collected at baseline and at 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 12, and 24 hours after administration. The plasma concentrations of clopidogrel and CAM were analyzed by LC-MS/MS, and adenosine diphosphate-induced platelet aggregation was measured by light-transmittance aggregometry., Findings: There were no significant differences in C max and AUC 0-t of clopidogrel prodrug in the NM group compared with the IM and PM groups. The mean CAM C max value was significantly higher in the NM group than in IM and PM groups (45.39 [12.57] vs 29.15 [7.92] ng/mL [P = 0.003] and 19.55 [2.19] ng/mL [P = 0.004], respectively). The mean CAM AUC 0-t value was significantly higher in the NM group than in the IM and PM groups (61.05 [21.63] vs 37.67 [11.01] ng · h/mL [P = 0.007] and 27.08 [2.72] ng · h/mL [P = 0.016]). The NM group exhibited a significantly higher percentage of inhibition of platelet aggregation than did the IM or PM group (P = 0.001). The correlations between the pharmacokinetic properties (C max , AUC 0-t) of CAM and the pharmacodynamic data (maximal and inhibition of platelet aggregation) were significant (both, Pearson r > 0.5 and P < 0.01)., Implication: In these healthy Chinese subjects, carriers of CYP2C19 loss-of-function allele(s) had significantly reduced exposure of CAM and decreased levels of inhibition of platelet aggregation with clopidogrel; these genotypes therefore might be a determinant for the formation of CAM and its antiplatelet effects. Study identifier: ChiCTR-OCH-14004382., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Objective To explore the inhibitory effect of aspirin and clopidogrel on platelet adhesion and aggregation behaviors under the physiological flow condition using microfluidic chip technology for health volunteers. Methods Peripheral venous blood samples collected from twelve randomly recruited health volunteers were treated with 20 μmol/L acetylsalicylic acid,50 μmol/L 2-methlthioadenosine-5'-monophosphate triethylammonium salt,and their combination,respectively,with untreated blood samples being control group. The blood samples were flowed through a microchannel modified with type I collagen protein at a physiological relevant shear rate of 1000 s -1 for 300 s,while the fluorescent images of platelet aggregations were dynamic captured using a microscope. Based on the images,the platelet coverage rates were calculated and used as quantitative parameters for evaluating platelet adhesion and aggregation behaviors. Results Under a flow condition of 1000 s -1 shear rate,an expected in vivo-like platelet adhesion and aggregation behaviors were observed at the surfaces of collagen proteins for control blood samples. Aspirin alone or clopidogrel alone suppressed platelet adhesion and aggregation at the later period of flow(200-300 s),while the combination of aspirin and clopidogrel reduced the adhesion numbers of platelets at the earlier stage of flow(≤150 s) and compromised the stability of platelet aggregation at the later period of flow(200-300 s). The combination showed synergistic effect in inhibiting platelet aggregation. Furthermore,such inhibitory effect was heterogeneous among 12 volunteers. Conclusion This simple microfluidic technology can offer a new technical platform for analyzing the inhibitory effect of antiplatelet drugs.

Cerebral infarction causes severe morbidity and mortality. Most patients with cerebral infarction should take antiplatelet drugs daily, so the effects of those drugs on the regeneration of the brain need to be investigated. Aspirin and clopidogrel are the most widely used antiplatelet drugs for the prevention of ischemic stroke. We investigated the effects of aspirin and clopidogrel on neural stem cells (NSCs). NSCs were dissociated from fetal rat cortex and cultured with basic fibroblast growth factor and N2 medium. To measure the effects of aspirin and clopidogrel on NSCs, NSCs were treated with several concentrations of aspirin, clopidogrel bisulfate, and clopidogrel resinate for 24 h. After the treatment, we measured cell viability by cell counting kit-8, MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay, trypan blue staining, flow cytometry, and lactate dehydrogenase assay. To evaluate their effects on NSC proliferation, we performed BrdU cell proliferation assay and colony-forming unit assay. We compared the intracellular protein level in the NSCs treated with aspirin and two types of clopidogrel, by proteomics analysis. Various viability tests showed that clopidogrel resinate and clopidogrel bisulfate did not affect the viability and proliferation of NSCs whereas aspirin decreased them even at low concentrations which are clinically relevant. Moreover, through the proteomics, it was confirmed that the toxicity of aspirin to NSCs might be associated with the alteration of several intracellular proteins. Taken together, these results suggest that clopidogrel resinate and clopidogrel bisulfate are safe but aspirin could be toxic to NSCs. Therefore, when these antiplatelet agents are prescribed over the long-term, the finding that aspirin could be toxic to NSCs should be considered.

Essentials Procoagulant platelets can be detected using GSAO in human whole blood. Stable coronary artery disease is associated with a heightened procoagulant platelet response. Agonist-induced procoagulant platelet response is not inhibited by aspirin alone. Collagen plus thrombin induced procoagulant platelet response is partially resistant to clopidogrel., Summary: Background Procoagulant platelets are a subset of highly activated platelets with a critical role in thrombin generation. Evaluation of their clinical utility in thrombotic disorders, such as coronary artery disease (CAD), has been thwarted by the lack of a sensitive and specific whole blood assay. Objectives We developed a novel assay, utilizing the cell death marker, GSAO [(4-(N-(S-glutathionylacetyl)amino)phenylarsonous acid], and the platelet activation marker, P-selectin, to identify procoagulant platelets in whole blood by flow cytometry. Patients/Methods Using this assay, we characterized the procoagulant platelet population in healthy controls and a cohort of patients undergoing elective coronary angiography. Results In patients with CAD, compared with patients without CAD, there was a heightened procoagulant platelet response to thrombin (25.2% vs. 12.2%), adenosine diphosphate (ADP) (7.8% vs. 2.7%) and thrombin plus collagen (27.2% vs. 18.3%). The heightened procoagulant platelet potential in CAD patients was not associated with other markers of platelet function, including aggregation, dense granule release and activation of α 2b β 3 integrin. Although dual antiplatelet therapy (DAPT) was associated with partial suppression of procoagulant platelets, this inhibitory effect on a patient level could not be predicted by aggregation response to ADP and was not fully suppressed by clopidogrel. Conclusions We report for the first time that procoagulant platelets can be efficiently detected in a few microliters of whole blood using the cell death marker, GSAO, and the platelet activation marker, P-selectin. A heightened procoagulant platelet response may provide insight into the thrombotic risk of CAD and help identify a novel target for antiplatelet therapies in CAD., (© 2018 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis.)

Background: Variations in several clopidogrel-pharmacogenes have been linked to clopidogrel response variability and clinical outcomes. We aimed to determine the frequency distribution of major polymorphisms on CYP2C19 , PON1 , ABCB1 and P2RY12 pharmacogenes in Puerto Ricans. Methods : This was a cross-sectional, population-based study of 200 unrelated "Guthrie" cards specimens from newborns registered in the Puerto Rican newborn screening program (PRNSP) between 2004 and 2014. Taqman ® SNP assay techniques were used for genotyping. Results: Minor allele frequencies (MAF) were 46% for PON1 (rs662), 41% for ABCB1 (rs1045642), 14% for CYP2C19 *17, 13% for CYP2C19 *2, 12% for P2RY12 -H2 and 0.3% for CYP2C19 *4. No carriers of the CYP2C19 *3 variants were detected. All alleles and genotype proportions were found to be in Hardy⁻Weinberg equilibrium (HWE). Overall, there were no significant differences between MAFs of these variants in Puerto Ricans and the general population ( n = 453) of the 1000 Genome project, except when comparisons to each individual parental group were performed (i.e., Africans, Europeans and East-Asians; p < 0.05). As expected, the prevalence of these markers in Puerto Ricans most resembled those in the 181 subjects from reference populations of the Americas. Conclusions: These prevalence data provide a necessary groundwork for future clinical studies of clopidogrel pharmacogenetics in Caribbean Hispanics.

Background: The CYP2C19 nonfunctional genotype reduces clopidogrel effectiveness after percutaneous coronary intervention (PCI). Following clinical implementation of CYP2C19 genotyping at University Florida (UF) Health Shands Hospital in 2012, where genotype results are available approximately 3 days after PCI, testing was expanded to UF Health Jacksonville in 2016 utilizing a rapid genotyping approach. We describe metrics with this latter implementation., Methods: Patients at UF Health Jacksonville undergoing left heart catheterization with intent to undergo PCI were targeted for genotyping using the Spartan RX™ system. Testing metrics and provider acceptance of testing and response to genotype results were examined, as was antiplatelet therapy over the 6 months following genotyping., Results: In the first year, 931 patients, including 392/505 (78%) total patients undergoing PCI, were genotyped. The median genotype test turnaround time was 96 min. Genotype results were available for 388 (99%) PCI patients prior to discharge. Of 336 genotyped PCI patients alive at discharge and not enrolled in an antiplatelet therapy trial, 1/6 (17%) poor metabolizers (PMs, with two nonfunctional alleles), 38/93 (41%) intermediate metabolizers (IMs, with one nonfunctional allele), and 119/237 (50%) patients without a nonfunctional allele were prescribed clopidogrel (p = 0.110). Clopidogrel use was higher among non-ACS versus ACS patients (78.6% vs. 42.2%, p < 0.001). Six months later, among patients with follow-up data, clopidogrel was prescribed in 0/4 (0%) PMs, 33/65 (51%) IMs, and 115/182 (63%) patients without a nonfunctional allele (p = 0.008 across groups; p = 0.020 for PMs versus those without a nonfunctional allele)., Conclusion: These data demonstrate that rapid genotyping is clinically feasible at a high volume cardiac catheterization facility and allows informed chronic antiplatelet prescribing, with lower clopidogrel use in PMs at 6 months. Trial registration ClinicalTrials.gov Identifier: NCT02724319; registered March 31, 2016; https://www.clinicaltrials.gov/ct2/show/NCT02724319?term=angiolillo&rank=7.

Essentials Inflammation plays a key role in the development of colorectal cancer. Understanding mechanisms of cancer initiation might reveal new anticancer preventive strategy. Hyperactive platelets promote tumor formation by fostering immune evasion of cancer. Platelet inhibition by clopidogrel prevents carcinogenesis by restoring antitumor immunity., Summary: Background Clinical and experimental evidence support a role for inflammation in the development of colorectal cancer, although the mechanisms are not fully understood. Beyond thrombosis and hemostasis, platelets are key actors in inflammation; they have also been shown to be involved in cancer. However, whether platelets participate in the link between inflammation and cancer is unknown. Objective To investigate the contribution of platelets and platelet-derived proteins to inflammation-elicited colorectal tumor development. Methods We used a clinically relevant mouse model of colitis-associated cancer. Platelet secretion and platelet reactivity to thrombin were assessed at each stage of carcinogenesis. We conducted an unbiased proteomic analysis of releasates of platelets isolated at the pretumoral stage to identify soluble factors that might act on tumor development. Plasma levels of the identified proteins were measured during the course of carcinogenesis. We then treated the mice with clopidogrel to efficiently inhibit platelet release reaction. Results At the pretumoral stage, hyperactive platelets constituted a major source of circulating protumoral serum amyloid A (SAA) proteins. Clopidogrel prevented the early elevation of the plasma SAA protein level, decreased colitis severity, and delayed the formation of dysplastic lesions and adenocarcinoma. Platelet inhibition hindered the expansion and function of immunosuppressive myeloid cells, as well as their infiltration into tumors, but increased the number of tissue CD8 + T cells. Platelets and releasates of platelets from mice with cancer were both able to polarize myeloid cells towards an immunosuppressive phenotype. Conclusions Thus, platelets promote the initiation of colitis-associated cancer by enhancing myeloid cell-dependent immunosuppression. Antiplatelet agents may help to prevent inflammation-elicited carcinogenesis by restoring antitumor immunity., (© 2018 International Society on Thrombosis and Haemostasis.)

Pharmacological inhibition of platelet collagen interaction is a promising therapeutic strategy to treat intra-vascular thrombosis. S007-867 is a novel synthetic inhibitor of collagen-induced platelet aggregation. It has shown better antithrombotic protection than aspirin and clopidogrel with minimal bleeding tendency in mice. The present study is aimed to systematically investigate the antithrombotic efficacy of S007-867 in comparison to aspirin and clopidogrel in vivo and to delineate its mechanism of action in vitro. Aspirin, clopidogrel, and S007-867 significantly reduced thrombus weight in arterio-venous (AV) shunt model in rats. In mice, following ferric chloride induced thrombosis in either carotid or mesenteric artery; S007-867 significantly prolonged the vessel occlusion time (1.2-fold) and maintained a sustained blood flow velocity for >30 min. Comparatively, clopidogrel showed significant prolongation in TTO (1.3-fold) while aspirin remained ineffective. Both S007-867 and aspirin did not alter bleeding time in either kidney or spleen injury models, and thus maintained hemostasis, while clopidogrel showed significant increase in spleen bleeding time (1.7-fold). The coagulation parameters namely thrombin time, prothrombin time or activated partial thromboplastin time remained unaffected even at high concentration of S007-867 (300 µM), thus implying its antithrombotic effect to be primarily platelet mediated. S007-867 significantly inhibited collagen-mediated platelet adhesion and aggregation in mice ex-vivo. Moreover, when blood was perfused over a highly thrombogenic combination of collagen mimicking peptides like CRP-GFOGER-VWF-III, S007-867 significantly reduced total thrombus volume or ZV 50 (53.4 ± 5.7%). Mechanistically, S007-867 (10-300 μM) inhibited collagen-induced ATP release, thromboxane A 2 (TxA 2 ) generation, intra-platelet [Ca +2 ] flux and global tyrosine phosphorylation including PLCγ2. Collectively the present study highlights that S007-867 is a novel synthetic inhibitor of collagen induced platelet activation, that effectively maintains blood flow velocity and delays vascular occlusion. It inhibits thrombogenesis without compromising hemostasis. Therefore, S007-867 may be further developed for the treatment of thrombotic disorders in clinical settings., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Prasugrel has been shown to provide more potency and less variability than clopidogrel, but its potential temporal variability has not been described.Methods and Results:We conducted a prospective open-label study, evaluating platelet reactivity overtime in acute coronary syndrome (ACS) patients on aspirin and clopidogrel (n=60) or prasugrel (n=61), after a percutaneous coronary intervention (PCI). Blood samples were taken at discharge and at 3 and 6 months. Platelet function tests included VerifyNow (VN-P2Y12), and Multiplate Aggregometry (MEA). By means of VN-P2Y12, prasugrel patients displayed significantly (P<0.001) higher platelet inhibition than clopidogrel patients over time, although there were not significant differences using MEA. Prasugrel patients showed higher platelet inhibition at baseline than at 3 months (59.3±8.1 vs. 105.0±49.2; P<0.001), without significant change at 6 months (107.9±72.0; P=0.919 vs. 3 months). Clopidogrel patients showed a similar trend (160.1±65.1, 184.8±62.7 and 185.0±53.3; baseline vs. 3 months P=0.060; 3 months vs. 6 months P=0.974). High platelet reactivity (HPR) was shown in 16.3% prasugrel patients, with no patient consistently remaining in HPR over time. HPR was detected in 36.6% of the clopidogrel patients, being consistently observed in 15.0% of them. Low platelet reactivity (LPR) was detected in 60.5% prasugrel and 9.8% clopidogrel patients., Conclusions: Prasugrel patients showed less temporal variation than patients on clopidogrel in terms of HPR. In contrast, higher variability in LPR was detected in prasugrel patients for up to 6 months' follow-up.

This review discusses the response variability to acetylsalicylic acid (ASA) and particularly to clopidogrel, and their relation to adverse recurrent ischaemic events in patients with arterial diseases. The higher rate of ASA resistance reported in the literature may be mainly due to the cyclooxygenase-1 non-specific assays, non-compliance, and underdosing. Clopidogrel response variability and non-responsiveness are established concepts. Moreover, high platelet reactivity (HPR) to adenosine diphosphate during clopidogrel therapy is now a known risk factor for recurrent ischaemic events in high-risk percutaneous coronary intervention/acute coronary syndrome patients. Variable active metabolite generation is the primary explanation for clopidogrel response variability and non-responsivenes. Variable levels of active metabolite generation following clopidogrel administration could be mainly explained by functional variability in hepatic cytochrome (CYP)P450 isoenzyme activity that is influenced by drug-drug interactions and single nucleotide polymorphisms of specific genes encoding CYP450 isoenzymes. Treatment with more potent P2Y12 receptor blockers, such as prasugrel and ticagrelor are credible alternative strategies to overcome HPR during clopidogrel therapy.

This study investigates the efficiency and predictive value of light-transmission aggregometry (LTA), vasodilator-stimulated phosphoprotein (VASP) and VerifyNow for ischemia in patients undergoing percutaneous coronary intervention (PCI). Studies that used LTA, VASP or VerifyNow to predict ischemia were included, and their quality and efficiency were analyzed using Review Manager 5.3. The sensitivity and specificity of subgroup studies based on the outcome, cut-off value, and follow-up days were calculated and the summary ROC (sROC) curves were compared after having been fitted. Thirty-one studies including a total of 17,314 participants were analyzed. LTA, VASP and VerifyNow presented a considerable efficiency in predicting ischemic clinical events. In the subgroup analysis, the sensitivities of LTA, VASP and VerifyNow in predicting cardiac death, all-cause death, myocardial infarction, stent thrombosis, stroke, and revascularization were 0.40/0.63/0.62, 0.47/0.56/0.39, 0.40/0.48/0.60, 0.44/0.58/0.70, 0.29/not applicable/0.60 and 0.44/0.57/0.37, respectively and the specificities of LTA, VASP, and VerifyNow were 0.85/0.48/0.63, 0.73/0.52/0.63, 0.74/0.55/0.64, 0.75/0.47/0.61, 0.72/not applicable/ 0.61, and 0.70/0.47/0.67, respectively. LTA showed a higher sensitivity in predicting the outcomes over six months than those within six months, while VerifyNow prediction sensitivity was found to be higher within six months. Meanwhile, VerifyNow showed no statistically significant higher AUC of sROC in comparison to LTA and VASP in predicting ischemic events in patients undergoing clopidogrel treatment. The cut-off values of LTA, VASP and VerifyNow were suggested to be 56%, 50% and 235 respectively according to our study., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Purpose: This study aimed to explore the effects of ticagrelor (a P2Y12 receptor inhibitor) on interleukin (IL)-17 and myeloperoxidase (MPO) expression in coronary thrombus as well as on the coronary blood flow in ST-segment elevation myocardial infarction (STEMI) patients following percutaneous coronary intervention (PCI)., Methods: Forty STEMI patients who were admitted to the First Affiliated Hospital of Harbin Medical University between August 1, 2014 and December 30, 2014 were enrolled in this study according to a set inclusion criteria. They were randomized to ticagrelor and clopidogrel groups and treated with 180 mg ticagrelor and 600 mg clopidogrel before PCI, respectively. Intracoronary thrombus aspiration was performed by a physician during PCI. Immunohistochemistry and Western blot analysis were carried out to detect the expression of IL-17 and MPO in the thrombus. Corrected thrombolysis in myocardial infarction frame count (CTFC) was used to evaluate blood flow after PCI., Results: Immunohistochemistry results showed that the average positive staining area percentage of IL-17 and MPO in the clopidogrel group was significantly higher than that in the ticagrelor group. Western blot analysis also showed similar results for IL-17 (clopidogrel 0.71 ± 0.036, ticagrelor 0.50 ± 0.56) and MPO (clopidogrel 0.50 ± 0.040; ticagrelor 0.38 ± 0.06). CTFC was lower in the ticagrelor group than that in the clopidogrel group (P < 0.05)., Conclusions: Ticagrelor is more effective than clopidogrel in reducing inflammation thrombosis and improving postprocedural PCI blood flow in STEMI patients. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

Carboxylesterase 1 (CES1) hydrolyzes the prodrug clopidogrel to an inactive carboxylic acid metabolite. The effects of CES1 S75N (rs2307240,C>T) on clopidogrel response among 851 acute coronary syndrome patients who came from the north, central and south of China were studied. The occurrence ratios of each endpoint in the CC group were significantly higher than in the CT + TT group for cerebrovascular events (14% vs 4.8%, p < 0.001, OR = 0.31), acute myocardial infarction (15.1% vs 6.1%, p < 0.001, OR = 0.37) and unstable angina (62.8% vs 37.7%, p < 0.001, OR = 0.36). The results showed that there was a significant association between CES1 S75N (rs2307240) and the outcome of clopidogrel therapy. Moreover, the frequency of the T allele of rs2307240 in acute coronary syndrome patients (MAF = 0.22) was more than four times higher than that in the general public (MAF = 0.05).

Objective To observe the effect of clopidogrel on plasma protein binding rates of gin- senoside Rg1. Methods Concentrations of ginsenoside Rg1 were measured in fetal bovine serum and phosphate buffered solution (PBS). Samples were randomly divided into ginsenoside Rg1 groups (low: 0.4; middle: 1. 0; high 5. 0 mg/L, respectively) and clopidogrel combined ginsenoside Rg1 groups (low: 0. 4 mg/L +2. 0 mg/L clopidogrel; middle: 1. 0 mg/L +2. 0 mg/L clopidogrel; high: 5. 0 mg/L +2. 0 mg/L clo- pidogrel). The effect of clopidogrel on plasma protein binding rates of ginsenoside Rgl was observed u- sing equilibrium dialysis. Then 3-dimensional structure of bovine serum albumin (BSA) was constructed using homology modeling. On this basis, binding effect of small compounds (ginsenoside Rg1 and clopi- dogrel) and BSA was observed using molecular docking method. Results The serum protein binding rate was 11. 2% ±2. 1% in the low dose ginsenoside Rg1 group, 13. 4% ±2. 2% in the middle dose ginsenoside Rgl group, and 14. 6% ±1. 4% in the high dose ginsenoside Rg1 group, respectively. It was 6. 5% ±2. 3% in the clopidogrel combined low dose ginsenoside Rg1 group, 9. 2% ±1. 5% in the clopi- dogrel combined middle dose ginsenoside Rg1 group, 12.1% ± 1. 7% in the clopidogrel combined high dose ginsenoside Rg1 group, respectively. They were lower in clopidogrel combined ginsenoside Rg1 groups than in ginsenoside Rg1 groups with statistical difference (P <0. 05). Results of molecular doc- king showed that competitive binding effect existed between compounds (ginsenoside Rg1 and clopi- dogrel) and BSA. Conclusion Results of equilibrium dialysis and molecular docking comprehensively in- dicated clopidogrel had effect on plasma protein binding rate of ginsenoside Rg1.

Objective To observe correlation between CYP2C19 *2/CYP2C19 *3 gene polymorphism with clopidogrel resistance and distribution of Chinese medicine ( CM) syndrome in acute coronary syndrome (ACS) population. Methods Peripheral blood was collected from 229 ACS patients from June 2014 to March 2015. DNAs were extracted, amplified, and sequenced. Correlations between CYP2C19 *2/CYP2Cl9 *3 gene polymorphisms and clopidogrel resistance/distribution of CM syndrome were analyzed. Gene frequency and allele frequency were tested using gene counting and one-sample K-S test. Correlation between gene types and distribution of CM syndrome was tested by Pearson corre- lation test. Results (1) The CYP2C19 *2 polymorphism distribution: CYP2C19 *2(A/A) (mutant homozygous) 12 cases (5. 2%) ; CYP2C19 * 2 ( G/A ) ( mutant heterozygote ) 93 cases (40. 6%), and CYP2C19 *2 (G/G) (normal homozygous) 124 cases (54. 2%). The mutant allele frequency was 0. 255. (2) The CYP2C19 *3 polymorphism distribution: CYP2C19 *3 (A/A) 0 case (0) ; CYP2C19 *3 (G/A) 26 cases (11. 4%), and CYP2C19 *3 (G/G) 203 cases (88. 6%). The mutant allele frequency was 0. 056. (3) Correlation between CYP2C19 gene polymorphism and clopidogrel resistance: Clopidogrel resistance was more liable to occur in mutant homozygous than in mutant heterozygote and normal homozygous (R =0. 30, P <0. 01). Clopidogrel resistance was more liable to occur in mutant heterozygote than in normal homozygous (R =0. 34, P <0. 01). (4) Among the 229 patients, the CM syndrome distribution were distributed as follows. Blockage of Xin vessels syndrome (BXVS, 33 cases, 14. 41%) ; qi deficiency blood stasis syndrome (QDBSS, 51 cases, 22. 27%) ; qi stagnation blood stasis syndrome (QSBSS, 92 cases, 40.18%) ; phlegm obstructing Xin vessel syndrome (POXVS, 17 cases, 7. 42%) ; yin-cold coag- ulation syndrome (YCCS, 8 cases, 3. 49%) ; qi-yin deficiency syndrome (QYDS, 13 cases, 5.68%) ; Xin-Shen yin deficiency syndrome (XSYDS, 5 cases, 2.18%), yang and qi deficiency syndrome (YQDS, 10 cases, 4. 37%). (5) CYP2C19 *2 gene type was significantly correlated with syndrome typing of CM (R =0. 26, P <0. 01). Mutant homozygous and most mutant heterozygote patients were syndrome typed as QDBSS. Conclusions The polymorphism of CYP2C19 was closely correlated with clopidogrel resist- ance in 229 ACS patients. Its occurrence rate was correlated with CYP2C19 *2/CYP2C19 *3 gene muta- tion frequency. Blood stasis syndrome ( QSBSS, QDBSS, BXVS) were main syndromes of ACS. Be- sides, QSBSS was obviously higher than the rest syndrome types. The polymorphism of CYP2C19 * 2 was correlated with syndrome typing of CM. CYP2C19 *2 gene defect mostly existed in QSBSS.

Plumbagin (PLB) isolated from Plumbago zeylanica L (Plumbaginaceae) was evaluated for the suppressive effect and mechanism on ADP induced rat platelet aggregation. Adult male SD rats were randomly divided into control group, clopidogrel group, PLB 25mg/kg group and PLB 50mg/kg group. Clopidogrel (13.5mg/kg per day) and PLB (25 and 50mg/kg per day) were orally given to experimental rats by gavage for seven consecutive days. The antiplatelet properties were assessed by measuring the ADP-induced platelet aggregation rate (Agg max ). The level of cAMP in platelets before aggregation was determined by ELISA. The protein expression of pAkt, Akt, pPLC β3 and PLC β3 in platelets was measured by western blot. Our data indicated that PLB (25 and 50mg/kg) significantly inhibited ADP-induced rat platelet aggregation as well as clopidogrel (13.5mg/kg) in a dose dependent manner compared with the control group. PLB (25 and 50mg/kg) remarkably reduced the ADP-induced PLC β3 phosphorylation but not Akt in platelets as compared with the control group. The present study suggests that PLB exerts a suppressive effect on ADP-induced rat platelet aggregation, at least in part, through P2Y 1 -PLC signaling pathway.

Objective: To elucidate the degree of genetic polymorphisms CYP2C19 (CYP2C19*2, CYP2C19*3) of key drug metabolizing enzymes on the antiplatelet effect of clopidogrel response in patients with acute ischemic stroke from Saudi Arabia., Methods: A case-control study carried out at Neurology Clinics at Asser Central Hospital, Abha, Kingdom of Saudi Arabia from October 2015 to January 2016 and included 25 stroke patients responding to clopidogrel therapy and 25 stroke patients non responding to clopidogrel monotherapy. After obtaining their informed consent, the blood samples were collected and genotyped for CYP2C19 polymorphisms by the polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP Method). Allele frequencies were derived from genotypic data and platelet aggregation was measured using multiple electrode aggregometry on the multiplate analyser. Chi Square tests, p-values, odds ratio (OR) and corresponding confidence intervals were calculated for each polymorphism., Results: The CYP2C19*2 (681G>A) and CYP2C19*3 (636 G>A) polymorphism were seen to be in Hardy-Weinberg equilibrium and showed significant allelic and genotypic association between responders and non-responders to clopidogrel (p<0.01). The CYP2C19*2: allelic chi-square=21.49, p=0.000036, OR=5.52 (2.42-12.83); Genotypic Chi-square=10.27, p=0.001, OR=7.88 (1.78-9.73). The CYP2C19*3: Allelic chi-square=11.66, p=0.0006, OR=3.45 (1.57-7.70); genotypic chi-square=4.37, p=0.036, OR=3.69 (0.90-5.81). The variant allele (homozygous and homozygous Mutant) showed significant influence on platelet inhibition and the antiplatelet effect of clopidogrel in ischemic stroke., Conclusion: Our findings provide certain evidence on the genetic effect of CYP2C19 on clopidogrel responsiveness in stroke patients from Saudi Arabia.