Evaluation of clopidogrel, hypercoagulability, and platelet count in dogs undergoing splenectomy for splenic masses.

Dogs that had splenectomy are predisposed to fatal thrombotic conditions, and thrombocytosis is a risk factor for post-splenectomy hypercoagulability. However, in veterinary medicine, there are no specific therapeutic approaches for managing this hypercoagulability. This study aimed to determine the preventive effect of clopidogrel on post-operative hypercoagulability during the first 2 weeks post-splenectomy in dogs with splenic masses. This study included 12 dogs that had splenectomy. Seven dogs received no treatment (group A), and five were treated with clopidogrel (group B). Clopidogrel was loaded at 10 mg/kg on day 2 and continued at 2 mg/kg until day 14. Blood samples were collected on the day of surgery and 2, 7, and 14 days after splenectomy in both groups. In group B, thromboelastography (TEG) was performed on the same days. In group A, there was significant elevation of platelet counts on days 7 (p = 0.007) and 14 (p = 0.001) compared to day 0. In group B, the platelet counts were significantly elevated on day 7 (p = 0.032) but no significant difference was found on day 14 compared to day 0. Platelet counts on day 14 were significantly higher in group A than in group B (p = 0.03). The lower platelet counts were correlated with alterations in TEG parameters, and no significant differences were found in the K and α-angle values at all postoperative assessment points compared to day 0. Our study suggests that clopidogrel may reduce post-operative thrombocytosis and hypercoagulability in dogs that undergo splenectomy for splenic masses.

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

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

Evaluating anthelmintic, anti-platelet, and anti-coagulant activities, and identifying the bioactive phytochemicals of Amaranthus blitum L.

BACKGROUND: Highlighting affordable alternative crops that are rich in bioactive phytoconstituents is essential for advancing nutrition and ensuring food security. Amaranthus blitum L. (AB) stands out as one such crop with a traditional history of being used to treat intestinal disorders, roundworm infections, and hemorrhage. This study aimed to evaluate the anthelmintic and hematologic activities across various extracts of AB and investigate the phytoconstituents responsible for these activities. METHODS: In vitro anthelmintic activity against Trichinella spiralis was evaluated in terms of larval viability reduction. The anti-platelet activities were assessed based on the inhibitory effect against induced platelet aggregation. Further, effects on the extrinsic pathway, the intrinsic pathway, and the ultimate common stage of blood coagulation, were monitored through measuring blood coagulation parameters: prothrombin time (PT), activated partial thromboplastin time (aPTT), and thrombin time (TT), respectively. The structures of isolated compounds were elucidated by spectroscopic analysis. RESULTS: Interestingly, a previously undescribed compound (19), N-(cis-p-coumaroyl)-ʟ-tryptophan, was isolated and identified along with 21 known compounds. Significant in vitro larvicidal activities were demonstrated by the investigated AB extracts at 1 mg/mL. Among tested compounds, compound 18 (rutin) displayed the highest larvicidal activity. Moreover, compounds 19 and 20 (N-(trans-p-coumaroyl)-ʟ-tryptophan) induced complete larval death within 48 h. The crude extract exhibited the minimal platelet aggregation of 43.42 ± 11.69%, compared with 76.22 ± 14.34% in the control plasma. Additionally, the crude extract and two compounds 19 and 20 significantly inhibited the extrinsic coagulation pathway. CONCLUSIONS: These findings extend awareness about the nutritional value of AB as a food, with thrombosis-preventing capabilities and introducing a promising source for new anthelmintic and anticoagulant agents.

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

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

Antiplatelet Effects of Flavonoid Aglycones Are Mediated by Activation of Cyclic Nucleotide-Dependent Protein Kinases.

Flavonoid aglycones are secondary plant metabolites that exhibit a broad spectrum of pharmacological activities, including anti-inflammatory, antioxidant, anticancer, and antiplatelet effects. However, the precise molecular mechanisms underlying their inhibitory effect on platelet activation remain poorly understood. In this study, we applied flow cytometry to analyze the effects of six flavonoid aglycones (luteolin, myricetin, quercetin, eriodictyol, kaempferol, and apigenin) on platelet activation, phosphatidylserine externalization, formation of reactive oxygen species, and intracellular esterase activity. We found that these compounds significantly inhibit thrombin-induced platelet activation and decrease formation of reactive oxygen species in activated platelets. The tested aglycones did not affect platelet viability, apoptosis induction, or procoagulant platelet formation. Notably, luteolin, myricetin, quercetin, and apigenin increased thrombin-induced thromboxane synthase activity, which was analyzed by a spectrofluorimetric method. Our results obtained from Western blot analysis and liquid chromatography-tandem mass spectrometry demonstrated that the antiplatelet properties of the studied phytochemicals are mediated by activation of cyclic nucleotide-dependent signaling pathways. Specifically, we established by using Förster resonance energy transfer that the molecular mechanisms are, at least partly, associated with the inhibition of phosphodiesterases 2 and/or 5. These findings underscore the therapeutic potential of flavonoid aglycones for clinical application as antiplatelet agents.

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.

Correlations among different platelet aggregation pathways in a group of healthy volunteers.

Platelet aggregation is a complicated process mediated by different signaling pathways. As the process is highly complex and apparently redundant, the relationships between these pathways are not yet fully known. The aim of this project was to study the interconnections among seven different aggregation pathways in a group of 53 generally healthy volunteers aged 20 to 66 years. Platelet aggregation was induced with thrombin receptor activating peptide 6 (TRAP), arachidonic acid (AA), platelet activating factor 16 (PAF), ADP, collagen, thromboxane A2 analogue U46619 or ristocetin (platelet agglutination) ex vivo in fasting blood samples according to standardized timetable protocol. Additionally, some samples were pre-treated with known clinically used antiplatelet drugs (vorapaxar, ticagrelor or acetylsalicylic acid (ASA)). Significant correlations among all used inducers were detected (Pearson correlation coefficients (rP): 0.3 to 0.85). Of all the triggers, AA showed to be the best predictor of the response to other inducers with rP ranging from 0.66 to 0.85. Interestingly, the antiplatelet response to ticagrelor strongly predicted the response to unrelated drug vorapaxar (rP = 0.71). Our results indicate that a response to one inducer can predict the response for other triggers or even to an antiplatelet drug. These data are useful for future testing but should be also confirmed in patients.

What is the context?• Platelet activation is a complicated process with multiple signaling cascades involved.• A total of seven common platelet triggers (ADP, collagen, TRAP-6, PAF, arachidonic acid/AA/, ristocetin and U46619) were tested.• The process is dependent on many factors including sex, age, concomitant disease(s), pharmacotherapy.What is new?• There were significant correlations between all tested aggregatory cascades.• AA has the highest rate of response predictability in our heterogeneous generally healthy volunteer group.• There was no correlation between impedance aggregometry in whole blood and turbidimetric measurement with platelet-rich plasma.What is the impact?• The effect of antiplatelet drugs can be assessed from the reaction to different trigger(s) at least in this group of healthy patients.• Future studies must test these relationships in patients with different diseases.

Linking triphenylphosphonium cation to a bicyclic hydroquinone improves their antiplatelet effect via the regulation of mitochondrial function.

Platelets are the critical target for preventing and treating pathological thrombus formation. However, despite current antiplatelet therapy, cardiovascular mortality remains high, and cardiovascular events continue in prescribed patients. In this study, first results were obtained with ortho-carbonyl hydroquinones as antiplatelet agents; we found that linking triphenylphosphonium cation to a bicyclic ortho-carbonyl hydroquinone moiety by a short alkyl chain significantly improved their antiplatelet effect by affecting the mitochondrial functioning. The mechanism of action involves uncoupling OXPHOS, which leads to an increase in mitochondrial ROS production and a decrease in the mitochondrial membrane potential and OCR. This alteration disrupts the energy production by mitochondrial function necessary for the platelet activation process. These effects are responsive to the complete structure of the compounds and not to isolated parts of the compounds tested. The results obtained in this research can be used as the basis for developing new antiplatelet agents that target mitochondria.

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.

Immature platelets and platelet reactivity in patients with acute ST-segment Elevation Myocardial Infarction using whole blood flow cytometry with SYTO-13 staining.

BACKGROUND: Reduced effect of antiplatelet therapy has been reported in patients with ST-segment elevation myocardial infarction (STEMI). Multiple factors may concur to explain this, including increased amount of highly reactive immature platelets. OBJECTIVES: To investigate the association between immature platelets and reactivity determined with multicolour flow cytometry using the SYTO-13 dye in STEMI patients. METHODS: We conducted an observational study of 59 patients with acute STEMI. Blood samples were obtained within 24 h after admission and after loading doses of dual antiplatelet therapy. For comparison, samples were obtained from 50 healthy individuals. Immature platelets and platelet reactivity were investigated using multicolour flow cytometry including the SYTO-13 dye that binds to platelet RNA and thus provides a method for subdividing platelets into immature and mature platelets. Additionally, we assessed platelet aggregation, serum-thromboxane B2 levels and standard immature platelet markers. RESULTS: Immature platelets were more reactive than mature platelets in both STEMI patients and healthy individuals (p-values < 0.05). STEMI patients had lower platelet aggregation and thromboxane B2 levels than healthy individuals. We found a positive association between automatically determined immature platelet markers and CD63 expression on activated platelets (Spearman's rho: 0.27 to 0.58, p-values < 0.05). CONCLUSIONS: Our study shows that immature platelets identified with a multicolour flow cytometric method using the SYTO-13 dye are more reactive than mature platelets in patients with acute STEMI and in healthy individuals. The presence of immature platelets may be important for the overall platelet reactivity, which may have implications for the effect of antiplatelet therapy.

WY-14643, a novel antiplatelet and antithrombotic agent targeting the GPIbα receptor.

BACKGROUND AND PURPOSE: Hypolipidemia and platelet activation play key roles in atherosclerotic diseases. Pirinixic acid (WY-14643) was originally developed as a lipid-lowering drug. Here we focused on its antiplatelet and antithrombotic abilities and the underlying mechanism. EXPERIMENTAL APPROACH: The effects of WY-14643 on platelet aggregation was measured using a lumi-aggregometer. Clot retraction and spreading on fibrinogen were also assayed. PPARα-/- platelets were used to identify the target of WY-14643. The interaction between WY-14643 and glycoprotein Ibα (GPIbα) was detected using cellular thermal shift assay (CETSA), surface plasmon resonance (SPR) spectroscopy and molecular docking. GPIbα downstream signaling was examined by Western blot. The antithrombotic effect was investigated using mouse mesenteric arteriole thrombosis model. Mouse tail bleeding model was used to study its effect on bleeding side effects. KEY RESULTS: WY-14643 concentration-dependently inhibits human washed platelet aggregation, clot retraction, and spreading. Significantly, WY-14643 inhibits thrombin-induced activation of human washed platelets with an IC50 of 7.026 µM. The antiplatelet effect of WY-14643 is mainly dependent of GPIbα. CESTA, SPR and molecular docking results indicate that WY-14643 directly interacts with GPIbα and acts as a GPIbα antagonist. WY-14643 also inhibits phosphorylation of PLCγ2, Akt, p38, and Erk1/2 induced by thrombin. Noteworthily, 20 mg/kg oral administration of WY-14643 inhibits FeCl3-induced thrombosis of mesenteric arteries in mice similarly to clopidogrel without increasing bleeding. CONCLUSION AND IMPLICATIONS: WY-14643 is not only a PPARα agonist with lipid-lowering effect, but also an antiplatelet agent as a GPIbα antagonist. It may have more significant therapeutic advantages than current antiplatelet agents for the treatment of atherosclerotic thrombosis, which have lipid-lowering effects without bleeding side effects.

Evaluating thrombosis risk and patient-specific treatment strategy using an atherothrombosis-on-chip model.

Platelets play an essential role in thrombotic processes. Recent studies suggest a direct link between increased plasma glucose, lipids, and inflammatory cytokines with platelet activation and aggregation, resulting in an increased risk of atherothrombotic events in cardiovascular patients. Antiplatelet therapies are commonly used for the primary prevention of atherosclerosis. Transitioning from a population-based strategy to patient-specific care requires a better understanding of the risks and advantages of antiplatelet therapy for individuals. This proof-of-concept study evaluates the potential to assess an individual's risk of forming atherothrombosis using a dual-channel microfluidic model emulating multiple atherogenic factors in vitro, including high glucose, high cholesterol, and inflammatory cytokines along with stenosis vessel geometry. The model shows precise sensitivity toward increased plasma glucose, cholesterol, and tumour necrosis factor-alpha (TNF-α)-treated groups in thrombus formation. An in vivo-like dose-dependent increment in platelet aggregation is observed in different treated groups, benefiting the evaluation of thrombosis risk in the individual condition. Moreover, the model could help decide the effective dosing of aspirin in multi-factorial complexities. In the high glucose-treated group, a 50 µM dose of aspirin could significantly reduce platelet aggregation, while a 100 µM dose of aspirin was required to reduce platelet aggregation in the glucose-TNF-α-treated group, which proves the model's potentiality as a tailored tool for customised therapy.

Clopidogrel induced reduction in neutrophil count: An overlooked beneficial effect?

Complement-stimulated neutrophils are able to adhere to the endothelium and damage endothelial cells both in vitro and in vivo. These blood cells participate in the early stages, growth and complications of atherosclerotic plaques. Recent findings, based on mendelian randomization analysis, support the concept that high neutrophil counts are a causal risk factor for ischemic heart disease and myocardial infarction . Clopidogrel decreases leukocyte count and inflammatory markers in patients with acute coronary syndromes; this off-target effect, which is independent of the antiplatelet action, may help explaining secondary prevention data showing a superiority of clopidogrel over aspirin in reducing new cardiovascular events.

Glycoursodeoxycholic Acid Alleviates Arterial Thrombosis via Suppressing Diacylglycerol Kinases Activity in Platelet.

BACKGROUND: Glycoursodeoxycholic acid (GUDCA) has been acknowledged for its ability to regulate lipid homeostasis and provide benefits for various metabolic disorders. However, the impact of GUDCA on arterial thrombotic events remains unexplored. The objective of this study is to examine the effects of GUDCA on thrombogenesis and elucidate its underlying mechanisms. METHODS: Plasma samples from patients with arterial thrombotic events and diet-induced obese mice were collected to determine the GUDCA concentrations using mass spectrometry. Multiple in vivo murine thrombosis models and in vitro platelet functional assays were conducted to comprehensively evaluate the antithrombotic effects of GUDCA. Moreover, lipidomic analysis was performed to identify the alterations of intraplatelet lipid components following GUDCA treatment. RESULTS: Plasma GUDCA level was significantly decreased in patients with arterial thrombotic events and negatively correlated with thrombotic propensity in diet-induced obese mice. GUDCA exhibited prominent suppressing effects on platelet reactivity as evidenced by the attenuation of platelet activation, secretion, aggregation, spreading, and retraction (P<0.05). In vivo, GUDCA administration robustly alleviated thrombogenesis (P<0.05) without affecting hemostasis. Mechanistically, GUDCA inhibited DGK (diacylglycerol kinase) activity, leading to the downregulation of the phosphatidic acid-mediated signaling pathway. Conversely, phosphatidic acid supplementation was sufficient to abolish the antithrombotic effects of GUDCA. More importantly, long-term oral administration of GUDCA normalized the enhanced DGK activity, thereby remarkably alleviating the platelet hyperreactivity as well as the heightened thrombotic tendency in diet-induced obese mice (P<0.05). CONCLUSIONS: Our study implicated that GUDCA reduces platelet hyperreactivity and improves thrombotic propensity by inhibiting DGKs activity, which is a potentially effective prophylactic approach and promising therapeutic agent for arterial thrombotic events.

Determination of clopidogrel effect in cats using point-of-care Plateletworks ADP and shipped samples for PFA-200 analysis in a clinical practice setting.

OBJECTIVES: Clopidogrel is the recommended first-line antithrombotic in cats for a variety of conditions; however, it is ineffective in 15-20% of cats. The determination of clopidogrel effectiveness with platelet function assays has historically been limited to specialty centers; however, recent work has suggested that in-hospital or shipped analyses of samples may be feasible. The aim of the present study was to investigate the utility of an in-house analysis and shipping of blood samples collected in primary practices for the determination of clopidogrel effectiveness. METHODS: Citrated blood samples were collected from cats receiving clopidogrel therapy by veterinarians in clinical practices across Canada, a median of 304.4 km from the reference laboratory (range 8-4425). Samples were analyzed in-house using Plateletworks ADP and shipped for remote analysis using PFA-200 P2Y and COL/ADP cartridges. RESULTS: A total of 30 samples were collected from 25 cats. Of these, the percentage of samples analyzable for the presence or absence of the clopidogrel effect was 86% for Plateletworks ADP, 90% for PFA-200 P2Y and 87% for PFA-200 COL/ADP. There was no significant difference in the number of samples unable to be analyzed by each modality (P = 0.689) due to flow obstruction or other sample characteristics. The prevalence of absence of clopidogrel effectiveness on platelet function assays was 8% with the PFA-200 COL/ADP assay, 25% with the PFA-200 P2Y assay and 30% with the Plateletworks ADP assay. CONCLUSIONS AND RELEVANCE: The results of this study confirm that samples of feline blood can be collected in clinical practices and shipped to a reference laboratory for PFA-200 analysis with a high rate of success, comparable to point-of-care analysis.

Investigation of smoking on the antiplatelet response to clopidogrel: Unravelling the smoker's paradox.

The intricate relationship between smoking and the effects of the antiplatelet drug clopidogrel has been termed the "smoker's paradox". This paradox details the enhanced efficacy of clopidogrel in smokers compared to non-smokers. This review begins with an exploration of the proposed mechanisms of the smoker's paradox, particularly drawing attention to the induction of cytochrome P450 (CYP) isoenzymes via tobacco smoke, specifically the enzymes CYP1A2 and CYP2C19. Moreover, an investigation of the effects of genetic variability on the smoker's paradox was undertaken from both clinical and molecular perspectives, delving into the effects of ethnicity and genetic polymorphisms. The intriguing role of CYP1A2 genotypes and the response to clopidogrel in smoking and non-smoking populations was examined conferring insight into the individuality rather than universality of the smoker's paradox. CYP1A2 induction is hypothesised to elucidate the potency of smoking in exerting a counteracting effect in those taking clopidogrel who possess CYP2C19 loss of function polymorphisms. Furthermore, we assess the comparative efficacies of clopidogrel and other antiplatelet agents, namely prasugrel and ticagrelor. Studies indicated that prasugrel and ticagrelor provided a more consistent effect and further reduced platelet reactivity compared to clopidogrel within both smoking and non-smoking populations. Personalised dosing was another focus of the review considering patient comorbidities, genetic makeup, and smoking status with the objective of improving the antiplatelet response of those taking clopidogrel. In summation, this review provides insight into multiple areas of research concerning clopidogrel and the smoker's paradox taking into account proposed mechanisms, genetics, other antiplatelet agents, and personalised dosing.

REDV-Functionalized Recombinant Spider Silk for Next-Generation Coronary Artery Stent Coatings: Hemocompatible, Drug-Eluting, and Endothelial Cell-Specific Materials.

Coronary artery stents are life-saving devices, and millions of these devices are implanted annually to treat coronary heart disease. The current gold standard in treatment is drug-eluting stents, which are coated with a biodegradable polymer layer that elutes antiproliferative drugs to prevent restenosis due to neointimal hyperplasia. Stenting is commonly paired with systemic antiplatelet therapy to prevent stent thrombosis. Despite their clinical success, current stents have significant limitations including inducing local inflammation that drives hyperplasia; a lack of hemocompatibility that promotes thrombosis, increasing need for antiplatelet therapy; and limited endothelialization, which is a critical step in the healing process. In this research, we designed a novel material for use as a next-generation coating for drug-eluting stents that addresses the limitations described above. Specifically, we developed a recombinant spider silk material that is functionalized with an REDV cell-adhesive ligand, a peptide motif that promotes specific adhesion of endothelial cells in the cardiovascular environment. We illustrated that this REDV-modified spider silk variant [eADF4(C16)-REDV] is an endothelial-cell-specific material that can promote the formation of a near-confluent endothelium. We additionally performed hemocompatibility assays using human whole blood and demonstrated that spider silk materials exhibit excellent hemocompatibility under both static and flow conditions. Furthermore, we showed that the material displayed slow enzyme-mediated degradation. Finally, we illustrated the ability to load and release the clinically relevant drug everolimus from recombinant spider silk coatings in a quantity and at a rate similar to that of commercial devices. These results support the use of REDV-functionalized recombinant spider silk as a coating for drug-eluting stents.

The study of platelet aggregation using a microtiter plate reader ‒ methodological considerations.

Optical aggregometry by 96-well plate assay, the microplate method, is a fast, efficient, and readily available method for measuring the pharmacological effects of antiplatelet drugs. Even though recent years have witnessed growing interest in adopting the microplate method for widespread use, it remains in the shadow of the standard light transmission aggregometry (LTA). Regardless of the method used, the results of platelet aggregation depend on a variety of factors and often vary among laboratories worldwide. While several methodological papers have examined the microplate method, no standards have been established, most likely because the approach is not used as a diagnostic tool. Currently, the microplate method is recommended by researchers to be used in conjunction with LTA or as an adjunct to LTA. This raises the question of whether an optimal protocol exists for microplate aggregometry, and what are the key considerations in a good experimental protocol for obtaining reliable results? This article attempts to address these questions by summarizing the knowledge accumulated in this field over the last three decades.

Impact of very low dose rivaroxaban in addition to dual antiplatelet therapy on endogenous fibrinolysis in acute coronary syndrome: The VaLiDate-R study.

BACKGROUND: Impaired endogenous fibrinolysis is adverse cardiovascular risk factor in acute coronary syndrome (ACS) patients. Addition of very low dose rivaroxaban (VLDR) to dual antiplatelet therapy (DAPT) reduces cardiovascular events but increases bleeding. OBJECTIVE: We aimed to assess whether addition of VLDR to DAPT can enhance endogenous fibrinolysis. METHODS: In a prospective, open-label trial, we assessed endogenous fibrinolysis in whole blood, in 549 patients with ACS using the Global Thrombosis Test (GTT) and Thromboelastography (TEG). Patients (n = 180) who demonstrated impaired endogenous fibrinolysis (lysis time [LT] >2000s with the GTT) were randomised 1:1:1 to (i) clopidogrel 75 mg daily; (ii) clopidogrel 75 mg daily plus rivaroxaban 2.5 mg twice daily; or (iii) ticagrelor 90 mg twice daily, for 30 days, in addition to aspirin. Fibrinolytic status was assessed at 0, 2, 4 and 8 weeks. The primary outcome was the change in LT from admission to week 4. We also measured thrombotic occlusion time (OT) at high shear, and rivaroxaban level. RESULTS: There was no difference between the groups with respect to LT or clot lysis with TEG, and no change in these parameters compared to baseline during study drug allocation. In the rivaroxaban plus clopidogrel group, OT was prolonged compared to the other groups, although rivaroxaban levels were low, suggesting non-compliance. CONCLUSION: Addition of rivaroxaban 2.5 mg twice daily to DAPT does not affect endogenous fibrinolysis of thrombus formed at either high or low shear. Further studies are needed to determine whether higher doses of rivaroxaban can favourably modulate fibrinolysis. CONDENSED ABSTRACT: Impaired endogenous fibrinolysis is a strong risk factor in ACS. We aimed to assess whether adding very low dose rivaroxaban (VLDR) to DAPT can enhance fibrinolysis. Fibrin and clot lysis were assessed in whole blood. ACS patients with impaired fibrinolysis were randomised 1:1:1 to clopidogrel 75 mg daily; clopidogrel 75 mg plus VLDR; or ticagrelor 90 mg twice daily, in addition to aspirin. At 30-days, there was no difference in lysis time between the groups, nor change from baseline. VLDR does not improve fibrinolysis at high or low shear. Further studies are needed to determine whether alternative antithrombotic regimens can enhance endogenous fibrinolysis.