ADP-induced Platelet Aggregation Research Articles

Demand Ischemia due to Gastrointestinal Bleeding Secondary to Ticagrelor

Ticagrelor, an orally consumed antiplatelet medication, acts as an antagonist on the P2Y12 receptor preventing adenosine diphosphate-induced platelet aggregation. Gastrointestinal bleeding triggered by ticagrelor can lead to demand ischemia, as the demand for oxygen by the tissues surpasses the oxygen supply available following acute blood loss. A male patient in his 80s had his antiplatelet medication switched from clopidogrel to ticagrelor following a cardiac stent. He presented to the emergency department a few months later due to black stools and fatigue. The patient had a critically elevated initial troponin level of 780 ng/L which increased to 892 ng/L two hours later. Hemoglobin was low at 8.5 g/dL. The patient suspended usage of ticagrelor and switched back to clopidogrel. Three weeks later, he had an endoscopy, which demonstrated no active bleeding sources.

Can CYP2C19 genotyping improve antiplatelet therapy efficacy in real-life practice? Recent advances.

Clopidogrel remains the most widely used P2Y₁₂ receptor inhibitor worldwide and is often used in combination with aspirin for secondary prevention in patients with arterial disease. The drug is associated with a wide response variability with one on three patients exhibiting little or no inhibition of adenosine diphosphate-induced platelet aggregation. It is a prodrug that is mainly metabolized by hepatic cytochrome P450 (CYP) 2C19. Patients who carry a CYP2C19 loss-of-function (LoF) allele have reduced metabolism of clopidogrel that is associated with reduced platelet inhibition compared to non-carriers that is associated with increased risk for thrombotic event occurrences, particularly, stent thrombosis. The United States Food and Drug Administration (US FDA) issued a black box warning in the clopidogrel label highlighting the importance of presence of CYP2C19 LOF allele during the insufficient metabolism of clopidogrel and availability of other potent P2Y₁₂ inhibitor for the treatment in CYP2C19 poor metabolizers. Clinical trials have conclusively demonstrated greater anti-ischemic benefits of prasugrel/ticagrelor in the treatment of patients carrying the CYP2C19 LoF allele. However, uniform use of these more potent P2Y₁₂ inhibitors has been associated with greater bleeding and cost, and lower adherence. The latter information provides a strong rationale for personalizing P2Y₁₂ inhibitor therapy based on the laboratory determination of CYP2C19 genotype. However, cardiologists have been slow to take up pharmacogenetic testing possibly due to lack of provider and patient education, clear cardiology guidelines and, and lack of positive results from adequately sized randomized clinical trials. However, current evidence strongly supports genotyping of patients who are candidates for clopidogrel. Physicians should strongly consider performing genetic tests to identify LoF carriers and treat these patients with more pharmacodynamically predictable P2Y₁₂ inhibitors than clopidogrel.

Differential Effect of Omega-3 Fatty Acids on Platelet Inhibition by Antiplatelet Drugs In Vitro.

The omega-3 polyunsaturated fatty acids (PUFAs) Docosahexaenoic acid (DHA) and Eicosapentaenoic acid (EPA) exert multiple cardioprotective effects, influencing inflammation, platelet activation, endothelial function and lipid metabolism, besides their well-established triglyceride lowering properties. It is not uncommon for omega-3 PUFAs to be prescribed for hypertriglyceridemia, alongside antiplatelet therapy in cardiovascular disease (CVD) patients. In this regard, we studied the effect of EPA and DHA, in combination with antiplatelet drugs, in platelet aggregation and P-selectin and αIIbβ3 membrane expression. The antiplatelet drugs aspirin and triflusal, inhibitors of cyclooxygenase-1 (COX-1); ticagrelor, an inhibitor of the receptor P2Y12; vorapaxar, an inhibitor of the PAR-1 receptor, were combined with DHA or EPA and evaluated against in vitro platelet aggregation induced by agonists arachidonic acid (AA), adenosine diphosphate (ADP) and TRAP-6. We further investigated procaspase-activating compound 1 (PAC-1) binding and P-selectin membrane expression in platelets stimulated with ADP and TRAP-6. Both DHA and EPA displayed a dose-dependent inhibitory effect on platelet aggregation induced by AA, ADP and TRAP-6. In platelet aggregation induced by AA, DHA significantly improved acetylsalicylic acid (ASA) and triflusal's inhibitory activity, while EPA enhanced the inhibitory effect of ASA. In combination with EPA, ASA and ticagrelor expressed an increased inhibitory effect towards ADP-induced platelet activation. Both fatty acids could not improve the inhibitory effect of vorapaxar on AA- and ADP-induced platelet aggregation. In the presence of EPA, all antiplatelet drugs displayed a stronger inhibitory effect towards TRAP-6-induced platelet activation. Both omega-3 PUFAs inhibited the membrane expression of αIIbβ3, though they had no effect on P-selectin expression induced by ADP or TRAP-6. The antiplatelet drugs exhibited heterogeneity regarding their effect on P-selectin and αIIbβ3 membrane expression, while both omega-3 PUFAs inhibited the membrane expression of αIIbβ3, though had no effect on P-selectin expression induced by ADP or TRAP-6. The combinatory effect of DHA and EPA with the antiplatelet drugs did not result in enhanced inhibitory activity compared to the sum of the individual effects of each component.

The metabolic activation of and platelet response to vicagrel vary with P-glycoprotein deficiency, rather than P-glycoprotein inhibition, in mice

This study aimed to determine changes in the hydrolysis of vicagrel, a substrate drug of arylacetamide deacetylase (Aadac) and carboxylesterase 2 (Ces2), in P-glycoprotein (P-gp)-deficient or P-gp-inhibited mice and to elucidate the mechanisms involved. Male wild-type (WT) and P-gp knock-out (KO) mice were used to investigate the systemic exposure of vicagrel thiol active metabolite H4 and platelet response to vicagrel, and the mRNA and protein expression levels of intestinal Aadac and Ces2. Moreover, WT mice were administered vicagrel alone or in combination with elacridar (a potent P-gp inhibitor) to determine drug-drug interactions. Compared with WT mice, P-gp KO mice exhibited significant increases in the systemic exposure of H4, the protein expression levels of intestinal Aadac and Ces2, and inhibition of ADP-induced platelet aggregation by vicagrel. Further, the H4 exposure was positively correlated with intestinal Aadac protein expression levels but did not vary with short-term inhibition of P-gp efflux activity by elacridar. P-gp-deficient mice, rather than elacridar-treated mice, exhibited significant upregulation of intestinal Aadac and Ces2 and thus, enhanced metabolic activation of and platelet response to vicagrel, suggesting that the metabolic activation of vicagrel may vary with P-gp deficiency, not P-gp inhibition, in mice.

Effect of Ticagrelor Versus Clopidogrel After Off-Pump Coronary Artery Bypass Grafting on Postoperative Atrial Fibrillation: A Cohort Study.

This study aimed to explore the effect of a P2Y12 inhibitor regimen on the occurrence of postoperative atrial fibrillation (POAF) after off-pump coronary artery bypass graft surgery in carriers with the cytochrome P450 family 2 subfamily C member19 loss-of-function allele. From May 2019 to November 2023, patients containing the cytochrome P450 family 2 subfamily C member19*2 or *3 allele undergoing elective first-time off-pump coronary artery bypass graft surgery including aspirin 100 mg/d and ticagrelor 180 mg/d (AT group; n=95) versus clopidogrel 75 mg/d (aspirin and clopidogrel group; n=95) were prospectively followed. The primary end point was the cumulative incidence of POAF in a week. The secondary end points were POAF burden, platelet aggregability, systemic immune-inflammation index and heart rate variability. The incidence of POAF was 21.1% in the AT group versus 41.1% in the aspirin and clopidogrel group (hazard ratio, 0.46 [95% CI, 0.27-0.76]; P=0.003). POAF burden, ADP-induced platelet aggregation and systemic immune-inflammation index was notably lower in the AT group than the aspirin and clopidogrel group. Heart rate variability data showed an increase in both high-frequency and SD of normal-to-normal RR intervals in the AT group with a decreased low-frequency/high-frequency ratio, suggesting that the sympathetic/parasympathetic activation was balanced. In patients carrying the cytochrome P450 family 2 subfamily C member19 loss-of-function allele, an AT regimen after off-pump coronary artery bypass grafting was associated with a lower incidence of POAF, paralleled by lower atrial fibrillation burden, ADP-induced platelet aggregation, lower systemic immune-inflammation index reaction, and a balanced automatic nerve system compared with an aspirin and clopidogrel regimen. Inhibiting the systemic immune-inflammation response and sustaining automatic nerve balance may underlie the therapeutic effect of POAF by a potent antiplatelet combination.

Effects of batroxobin on the antithrombotic system in patients with cerebral venous thrombosis: Clues to mechanisms.

More evidence supports the benefits of batroxobin combined with anticoagulation in correcting acute cerebral venous thrombosis (CVT). The dynamic fluctuations of peripheral blood platelets, fibrinolysis, and coagulation biomarkers during this therapy were analyzed. We investigated batroxobin's effects on the antithrombotic system under two regimens. The pretreatment group included patients on anticoagulants for at least 1 week before starting batroxobin. The simultaneous treatment group began both treatments upon admission. The control group received only anticoagulation. Batroxobin was given on alternate days at doses of 10BU, 5BU, and 5BU, totaling three doses. Anticoagulation was continuous. Baseline data were T0; the next day after each batroxobin dose was T1, T2, and T3. Data from these four time points was analyzed. The time-point paired sample T-test results of the pretreatment group [n = 60; mean age (SD), 43.3(16.5); 38 (63.35%) women] showed that batroxobin significantly inhibited ADP-induced platelet aggregation rate (T1-T0: p = 0.015; T2-T0: p = 0.025; T3-T0: p = 0.013), decreased fibrinogen level (T1-T0: p < 0.001; T2-T0: p < 0.001; T3-T0: p < 0.001), and increased D-dimer (T1-T0:p < 0.001; T2-T0: p < 0.001; T3-T0: p < 0.001), TT (T1-T0:p = 0.046; T2-T0: p = 0.003; T3-T0: p < 0.001), and APTT (T1-T0:p = 0.021; T2-T0: p = 0.012; T3-T0: p = 0.026). Compared to the control group, the simultaneous treatment group showed significantly higher TT (T2: p = 0.002; T3: p = 0.004) and D-dimer (T1: p < 0.001; T2: p < 0.001; T3: p < 0.001) values, while fibrinogen (T2: p < 0.001; T3: p < 0.001) levels were significantly lower. Using batroxobin can alleviate the amplitude of changes in coagulation indicators other than TT caused by anticoagulants. The above conclusions are consistent with the results of repeated measurement data analysis. Batroxobin can significantly inhibit ADP-induced platelet aggregation rate, increase D-dimer, decrease fibrinogen, and prolong TT and APTT in the presence of anticoagulant agents. Using batroxobin can reduce the amplitude of changes in coagulation indicators caused by anticoagulants. These results reveal the potential mechanism of batroxobin combined with anticoagulation in the safe and effective treatment of CVT.

Prospective Trial on the Pharmacokinetics of Clopidogrel in Hemodialysis Patients

IntroductionPatients undergoing hemodialysis (HDP) exhibit extensive cardiovascular risk. The widely prescribed anti-platelet agent clopidogrel is metabolically activated by cytochrome enzymes, which may be impaired by uremia and chronic low-grade inflammation, typically present in HDP. We conducted a prospective multicenter study to investigate the pharmacokinetics and pharmacodynamics of clopidogrel in HDP and healthy volunteers (HV). MethodsWe enrolled HDP receiving long-term clopidogrel (75mg) and pantoprazole treatment (40 mg). HV received a loading dose of 300 mg clopidogrel, followed by 75 mg once daily. Pantoprazole, a substrate and probe drug of CYP2C19, was administered intravenously (40 mg). Plasma concentrations were quantified by mass spectrometry. Pharmacokinetics were calculated, and a population pharmacokinetic model was developed. The primary endpoint was the maximum concentration of clopidogrel’s active metabolite (Cmax_CAM). Platelet aggregation was measured using ADP-induced whole-blood aggregometry. ResultsSeventeen HDP and 16 HV were included. Cmax_CAM was significantly lower in HDP compared to HV (median [IQR] 12.2 [4.6-23.4] vs. 24.7 [17.8-36.5] ng/mL, p=0.02). The Cmax ratio of CAM to prodrug was 8.5-fold lower in HDP, and an 82.7% reduced clopidogrel clearance, including CAM formation, was found using population pharmacokinetic modeling. ADP-induced platelet aggregation at 120 min was significantly higher in HDP than in HV (median [IQR]: 26 [14-43] vs. 12 [11-18] U, p=0.004. Pantoprazole terminal half-life was ∼1.7-fold higher in HDPs compared to HVs. ConclusionOur data demonstrate an altered metabolism of clopidogrel in HDP in the context of lower CYP2C19 activity, with potential implications for other substances metabolized by this enzyme.

Clopidogrel Resistance in Ischemic Stroke Patients.

Stroke remained the second leading cause of death globally in 2019. The antiplatelet drug clopidogrel is used to keep blood clots from forming in people who have experienced a stroke. Although most people find clopidogrel to be safe and beneficial, there is inevitably a range in how each patient responds.The review covers about Clopidogrel resistance in stroke patients, their risk factors and the methods to identify it. Clopidogrel resistance is characterized as the drug's inability to prevent the target enzyme from acting. The prognosis of patients with ischemic stroke and the responsiveness to clopidogrel are significantly impacted by the various genetic polymorphism CYP2C19 genotypes. The two primary mutant alleles, CYP2C19 *2 and CYP2C19 *3, have been found to be the most prevalent genotypes. Better mRS scores six months after treatment showed a higher response rate in patients without these CYP2C19 variant alleles. Other factors are drug-drug interaction (proton pump inhibitors), demographics (age, sex, social history), comorbid conditions, etc. Blood samples for testing platelet reactivity were drawn one month after discharge from a peripheral blood sample. Several methods are used to identify the clopidogrel resistance. Some of them are ADP-Induced platelet aggregation, Platelet Reactivity Index VASP, Verify Now Assay, TEG Analyzer, Plasma microRNA-223. Drugs that are not prodrugs and whose metabolism is not dependent upon CYP2C19 can be selected as a superior alternative in case of CR. Ticagrelor is one such effective substitute. Proton pump inhibitors and clopidogrel should only be used concurrently in patients with reliable clinical indications.

Clinical and Molecular Characteristics of Megakaryocytes in Myelodysplastic Syndrome.

Objective Myelodysplastic syndrome (MDS) is a malignant clonal disorder of hematopoietic stem cells which is characterized by morphologic dysplasia. However, the pathological characteristics of megakaryocytes (MKs) in MDS patients with gene mutation are not well established. Methods Bone marrow MK specimens from 104 patients with primary MDS were evaluated, and all patients were distributed into two groups according to gene mutation associated with functional MKs. The morphologic and cellular characteristics of MKs and platelets were recorded and compared. Results The more frequently mutated genes in MDS patients were TUBB1 (11.54%), VWF (8.65%), NBEAL2 (5.77%), and the most common point mutation was TUBB1 p.(R307H) and p.(Q43P). Patients with MK mutation showed a decrease in adenosine diphosphate-induced platelet aggregation, high proportion of CD34 + CD61 + MKs (10.00 vs. 4.00%, p = 0.012), and short overall survival (33.15 vs. 40.50 months, p = 0.013). Further, patients with a higher percent of CD34 + CD61 + MKs (≧20.00%) had lower platelet counts (36.00 × 10 9 /L vs. 88.50 × 10 9 /L, p = 0.015) and more profound emperipolesis ( p = 0.001). By analyzing RNA-sequencing of MKs, differentially expressed mRNA was involved in physiological processes including platelet function and platelet activation, especially for MDS patients with high percent of CD34 + CD61 + MKs. The high levels of expression of CD62P, CXCL10, and S100A9 mRNA, shown by RNA sequencing, were validated by PCR assay. Conclusion High proportion of CD34 + CD61 + MKs was a poor prognostic factor in MDS patients with MK mutation. CD62P, CXCL10, and S100A9 may be the potential targets to evaluate the molecular link between gene defects and platelet function.

Clinical prediction factors of nonchronic total occlusion lesion progression in patients with unstable angina receiving percutaneous coronary intervention for chronic total occlusion lesions

BackgroundIn this study, we investigated clinical prediction factors of nonchronic total occlusion lesion (NCTOL) progression in patients who underwent percutaneous coronary intervention (PCI) for chronic total occlusion (CTO) lesions. MethodsIn total, 450 patients with unstable angina (mean age = 57.1 ± 9.2 years) who underwent PCI for CTO lesions between January 2016 and December 2018 at Beijing Anzhen Hospital were enrolled in this study. A clinical and angiographic follow-up examination was performed 12 months postoperatively. The patients were divided into NCTOL progression (145 cases) and control (305 cases) groups based on the outcome of the 12-month angiographic follow-up. The clinical and angiographic features of the participants were analyzed. ResultsThe adenosine diphosphate-induced platelet aggregation (ADP-IPA) rate and levels of lipoprotein (a) (Lp(a)) in the NCTOL progression group were significantly higher than those in the control group (51.89 ± 14.81 vs. 39.63 ± 17.12, P < 0.01; 0.22 ± 0.26 vs. 0.14 ± 0.18, P < 0.05, respectively). Logistic regression showed that the ADP-IPA rate (odds ratio = 1.047, 95 % confidence interval: 1.014–1.082, P = 0.005) and Lp(a) (odds ratio = 11.972, 95 % confidence interval: 1.230–116.570, P = 0.033) were independent predictors of NCTOL progression. Partial correlation analysis demonstrated that the ADP-IPA rate was positively correlated with NCTOL progression (r = 0. 351, P < 0.001). Receiver operating characteristic curve showed that the boundary point of the ADP-IPA rate to predict NCTOL progression was 30 % (sensitivity, 86.2 %; specificity, 68.9 %). ConclusionsNCTOL progression is an important cause of recurrent PCI in patients with coronary artery disease after PCI for CTO lesions. The ADP-IPA rate is a useful predictor for NCTOL progression in patients with unstable angina who undergo PCI for CTO lesions.

Inhibition of mitochondrial calcium transporters alters adp-induced platelet responses.

ADP-stimulated elevation of cytosolic Ca2+ is an important effector mechanism for platelet activation. The rapidly elevating cytosolic Ca2+ is also transported to mitochondrial matrix via Mitochondrial Ca2+ Uniporter (MCU) and extruded via Na+/Ca2+/Li+ Exchanger (NCLX). However, the exact contribution of MCU and NCLX in ADP-mediated platelet responses remains incompletely understood. The present study aimed to elucidate the role of mitochondrial Ca2+ transport in ADP-stimulated platelet responses by inhibition of MCU and NCLX with mitoxantrone (MTX) and CGP37157 (CGP), respectively. As these inhibitory strategies are reported to cause distinct effects on matrix Ca2+ concentration, we hypothesized to observe opposite impact of MTX and CGP on ADP-induced platelet responses. Platelet aggregation profiling was performed by microplate-based spectrophotometery while p-selectin externalization and integrin αIIbβ3 activation were analyzed by fluorescent immunolabeling using flow cytometery. Our results confirmed the expression of both MCU and NCLX mRNAs with relatively low abundance of NCLX in human platelets. In line with our hypothesis, MTX caused a dose-dependent inhibition of ADP-induced platelet aggregation without displaying any cytotoxicity. Likewise, ADP-induced p-selectin externalization and integrin αIIbβ3 activation was also significantly attenuated in MTX-treated platelets. Concordantly, inhibition of NCLX with CGP yielded an accelerated ADP-stimulated platelet aggregation which was associated with an elevation of p-selectin surface expression and αIIbβ3 activation. Together, these findings uncover a vital and hitherto poorly characterized role of mitochondrial Ca2+ transporters in ADP-induced platelet activation.

Role of &lt;i&gt;ITGB3&lt;/i&gt;, &lt;i&gt;GP1B1&lt;/i&gt;, and &lt;i&gt;ITGA2&lt;/i&gt; gene polymorphisms in platelet dysfunction in patients with COVID-19-associated lung damage

The aim of the work. To investigate platelet aggregation, polymorphism in the genes that ensure its implementation, and the association between these indicators in patients with COVID-19-associated lung damage, depending on the severity of the clinical course. Methodology. The study involved 75 patients with COVID-19, which, depending on the severity of lung involvement, were divided into two groups: patients with damage of up to 50 % of the lung parenchyma (n = 48) and with damage of more than 50 % (n = 27) respectively. The control group consisted of healthy people (n = 24), comparable in sex and age. In all individuals, the number of platelets, platelet aggregation induced by ADP, collagen and ristomycin were studied; polymorphisms rs6065 in the GP1BA gene, rs1126643 in the ITGA2 gene, and rs5918 in the ITGB3 gene were determined by polymerase chain reaction. The analysis of the obtained data was executed using the IBM SPSS Statistics v. 23 (IMB Corp., USA). Results and discussion. In patients with moderate and severe COVID-19-associated lung damage, platelet aggregation induced by ADP, collagen, and ristomycin accelerated; in severe cases, the number of platelets decreased. The frequency of variants of the rs6065 polymorphism did not change, the frequency of occurrence of the T/C genotype of the rs5918 polymorphism increased; with moderate severity, the frequency of occurrence of the C/T and T/T genotypes of the rs1126643 polymorphism increased; with severe lung damage, the frequency of occurrence of the mutant C/C genotype polymorphism rs5918 increased. In moderate lung damage, the presence of the mutant T/T polymorphism rs1126643 accelerated collagen-induced platelet aggregation; in severe cases, the presence of mutant C/C and heterozygous variant C/T polymorphism rs5918 accelerated ADP-induced platelet aggregation. There was no effect of the rs6065 polymorphism on platelet aggregation. The data obtained indicate the possible role of genetic predisposition in the activation of platelet aggregation in patients with COVID-19-associated lung damage.

Phytochemical characterization and phospholipase A2 inhibitory effect of Vitex negundo L. root extracts

Ethnopharmacological relevanceSnake bites are a critical health issue in many parts of the world particularly in Asian countries lacking efficient health facilities in rural areas. Cobra is the most common snake type in Asia and is responsible for a large number of mortalities particularly in rural areas. Plants are usually considered the most effective and easy-to-approach treatment for snake bites in rural areas of various countries. Vitex negundo L. is an important medicinal plant traditionally used to treat snake bite envenomation in many countries of Asia. Aim of the studyFrom literature survey of plants traditionally used in the treatment of snake bites in Asian countries including India, Pakistan and Sri Lanka, roots of V. negundo were selected for the present study. Anti-snake venom potential of its roots was assessed through various in vitro assays targeting the phospholipase A2 enzyme. Materials and methodsV. negundo roots were sequentially extracted in different organic solvents to get fractions and in methanol to get total extract. The extracts were evaluated for phospholipase A2 (PLA2) inhibitory potential through inhibition of venom-induced hemolysis, ADP-induced platelet aggregation, PLA2-induced fatty acid hydrolysis and anticoagulant effect of cobra venom. Antioxidant power was determined using DPPH and superoxide radical scavenging assays. GC-MS and HPLC analysis was performed for the total methanol extract. ResultsStrong PLA2 inhibitory effect was observed for all the extracts. The ethyl acetate, acetone and methanol fractions significantly inhibited toxic effects of cobra venom under in vitro conditions. Radical scavenging potential of these fractions was also significantly high as compared to non-polar fractions in both DPPH and superoxide scavenging assays. Phytochemical analysis indicated high phenolic and flavonoid contents in these fractions. GC-MS and HPLC analysis of total methanol extract confirmed the presence of bis(2-ethylhexyl) phthalate, phenol, o-Guaiacol, palmitic acid-methyl ester, methyl stearate, quercetin and kaempferol in the plant. ConclusionThe study concluded that the roots of V. negundo, particularly their polar extracts, have strong PLA2 inhibitory effect against cobra venom confirming their traditional use to manage snake bites. The roots of this plant can be further studied for isolation of plant-based antisera.

GDF-15 Inhibits ADP-Induced Human Platelet Aggregation through the GFRAL/RET Signaling Complex.

Growth differentiation factor-15 (GDF-15) is proposed to be strongly associated with several cardiovascular diseases, such as heart failure and atherosclerosis. Moreover, some recent studies have reported an association between GDF-15 and platelet activation. In this study, we isolated peripheral blood platelets from healthy volunteers and evaluated the effect of GDF-15 on adenosine diphosphate (ADP)-induced platelet activation using the platelet aggregation assay. Subsequently, we detected the expression of GDF-15-related receptors on platelets, including the epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), human epidermal growth factor receptor 3 (HER3), transforming growth factor-beta receptor I (TGF-βRI), transforming growth factor-beta receptor II (TGF-βRII), glial-cell-line-derived neurotrophic factor family receptor α-like (GFRAL), and those rearranged during transfection (RET). Then, we screened for GDF-15 receptors using the GDF-15-related receptor microarray comprising these recombinant proteins. We also performed the immunoprecipitation assay to investigate the interaction between GDF-15 and the receptors on platelets. For the further exploration of signaling pathways, we investigated the effects of GDF-15 on the extracellular signal-regulated kinase (ERK), protein kinase B (AKT), and Janus kinase 2 (JAK2) pathways. We also investigated the effects of GDF-15 on the ERK and AKT pathways and platelet aggregation in the presence or absence of RET agonists or inhibition. Our study revealed that GDF-15 can dose-independently inhibit ADP-induced human platelet aggregation and that the binding partner of GDF-15 on platelets is GFRAL. We also found that GDF-15 inhibits ADP-induced AKT and ERK activation in platelets. Meanwhile, our results revealed that the inhibitory effects of GDF-15 can be mediated by the GFRAL/RET complex. These findings reveal the novel inhibitory mechanism of ADP-induced platelet activation by GDF-15.

Low ambient temperature exposure increases the risk of ischemic stroke by promoting platelet activation

BackgroundAccumulating epidemiological evidence suggests the association between low ambient temperature exposure and the risk of ischemic stroke, but the underlying mechanisms remain unclear. ObjectiveGiven the crucial role of platelet activation and thrombosis in ischemic stroke, this study aims to investigate the effect of ambient temperature on platelet activation through multi-center clinical data in Tianjin as well as animal experiments. MethodsFrom 2018 to 2020, nearly 3000 ischemic stroke patients from three stroke centers in Tianjin were included in the analysis, among them the ADP induced platelet aggregation rate was available. Meteorological data from the same period had also been collected. After controlling for confounding factors, the generalized additive mixed model (GAMM) was used to evaluate the correlation between environmental temperature and platelet aggregation rate. In further animal experiments, platelet function assessments were conducted on mice from the cold exposure group and the normal temperature group, including platelet aggregation, spreading, and clot retraction. Additionally, tail bleeding and mesentery thrombosis were also tested to monitor hemostasis and thrombosis in vivo. ResultA nonlinear “S” shaped relationship between outdoor temperature and platelet aggregation was found. Each 1 °C decrease of mean temperature was associated with an increase of 7.77 % (95 % CI: 2.06 % - 13.48 %) in platelet aggregation. The ambient temperature is not related to other platelet parameters. Subgroup analysis found that males, people aged ≥65 years, and hypertensive individuals are more susceptible to temperature changes. Furthermore, animal experiments demonstrated that the increased CIRBP levels and subsequent activation of p-AKT/p-ERK may be one of the reasons for cold exposure induced platelets activation. ConclusionBoth clinical data and basic research support that low ambient temperature exposure has the potential to increase platelet activation. These results provide a basis for understanding the potential mechanism of temperature variations on the pathogenesis of cerebrovascular diseases.

Soluble urokinase plasminogen activator receptor, platelet aggregation, and carotid plaque thickness in diabetes: A cross-sectional analysis

BackgroundSoluble urokinase plasminogen activator receptor (suPAR) is an inflammatory biomarker strongly linked with cardiovascular disease in diabetes. By investigating its association with platelet aggregation levels and carotid plaque thickness, we can potentially improve the characterization of cardiovascular pathophysiology in type 1 (T1D) and type 2 diabetes (T2D). MethodsSuPAR was measured post-hoc in plasma collected in two cross-sectional studies including 1) individuals with T1D and 2) individuals with T2D without known cardiovascular disease. Platelet aggregation was analyzed employing impedance aggregometry using the agonists arachidonic acid (ASPI), adenosine diphosphate (ADP), and thrombin receptor activating peptide-6 (TRAP). Carotid plaque thickness was assessed bilaterally, and the thickest plaque (cPTmax) was analyzed. Correlations and associations of suPAR with platelet aggregation and cPTmax were assessed with Pearson's R and linear regressions adjusted for sex, age, C-reactive protein, estimated glomerular filtration rate, and platelet levels (only for aggregation analysis). ASPI analyses were only performed in participant not receiving aspirin treatment. Standardized estimates with 95 % confidence intervals are presented. ResultsTwo-hundred eighty-three with T1D (mean age 55 years, 44 % women) and 493 individuals with T2D (mean age 65 years, 33 % women) were included. SuPAR levels were positively correlated to platelet aggregation in both T1D (ADP: R = 0.18, p = 0.003; ASPI R = 0.13, p = 0.026) and T2D (ADP R = 0.14, p = 0.002; ASPI R = 0.14, p = 0.017). In adjusted linear regressions higher SuPAR levels were associated with higher ADP induced platelet aggregation in both T1D (26 (8, 44) AU/min, p = 0.004) and T2D (22 (3, 42) AU/min, p = 0.025), while associations to the other agonists were not conclusive. Higher suPAR levels were associated with thicker cPTmax in T1D (0.19 (0.07, 0.32) mm, p = 0.003), but not in T2D (p = 0.94). ConclusionHigher levels of suPAR, an inflammatory generalized disease biomarker, were associated with increased platelet aggregation and thicker cPTmax, in type 1 diabetes. Similar associations with platelet aggregation were found in type 2 diabetes, but not for carotid plaque thickness. These results suggest distinct cardiovascular pathomechanisms in T1D and T2D, although prospective studies are needed to confirm these findings.

Effect of evolocumab on platelet function in patients with acute coronary syndromes. An analysis of the randomized, double-blind, placebo-controlled EVOPACS Trial

Abstract Background Proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors substantially lower levels of low-density lipoprotein cholesterol (LDL-C) and favourably impact clinical outcomes in patients with acute coronary syndromes (ACS). Although PCSK9 inhibitors have been suggested in ex vivo studies to suppress platelet activation, the impact of evolocumab on platelet function in ACS patients receiving potent antiplatelet therapy remains largely unknown. Methods The EVOPACS trial was a randomized, double-blind, placebo-controlled trial assessing the use of evolocumab initiated in the acute phase of ACS. Patients presenting with ST-elevation myocardial infarction (STEMI) or Non-ST-elevation ACS (NSTE-ACS) were randomly allocated in a 1:1 ratio to either evolocumab 420mg SC or matching placebo at baseline (during hospitalization for index ACS event) and after 4 weeks. All patients received atorvastatin 40mg daily. Dual antiplatelet therapy consisting of aspirin and a potent P2Y12 inhibitor was initiated according to STEMI / NSTE-ACS guidelines, unless a less potent treatment was indicated (e.g. due to concomitant use of oral anticoagulation). Platelet function was measured at baseline (during index hospitalization) and after 8 weeks, using the Multiplate analyser. The main outcome for this analysis was the change from baseline to 8 weeks in adenosine diphosphate (ADP)-induced platelet aggregation. Results Serial platelet function measurements were available in 260 of all 308 enrolled patients, 124 in the evolocumab and 136 in the placebo group. Mean age was 60.2±11.0 years and 83% were men. Baseline clinical characteristics were well balanced between the two treatment groups. At discharge, the proportion of patients treated with ticagrelor, prasugrel, and clopidogrel was 73%, 11%, and 9% respectively, without differences between groups. Evolocumab significantly reduced LDL-C compared to placebo at 8 weeks (difference in mean percentage change -40.75%, 95% CI -45.39 to -36.11; p&amp;lt;0.001). Change in ADP-induced platelet aggregation between baseline and 8-week follow-up was -5.6±23.5 U (from 24.1±19.8 to 29.7±25.6 U) in the evolocumab group vs. -1.7±198.7 U (from 23.0±14.7 U to 24.7±19.1 U) in the placebo group (p=0.13). Similar results were found in a subgroup of 189 patients (86 in the evolocumab and 103 in the placebo group) treated with ticagrelor at discharge, showing a change in ADP-induced platelet aggregation of -2.7±19.5 U with evolocumab vs. -2.4±19.9 U with placebo (p=0.80). Conclusions In this randomized trial of ACS patients largely treated with a potent P2Y12 inhibitor, evolocumab initiated during index hospitalization on top of high-intensity statin therapy had no measurable effect on ADP-induced platelet aggregation. These findings suggest that the early therapeutic benefit of PCSK9 inhibitors in ACS patients is likely related to plaque regression/stabilization rather than changes in platelet activity.

In vitro comparison of platelet aggregation between zalunfiban and selatogrel in healthy donors

Abstract Background Two subcutaneously injected antiplatelet drugs, zalunfiban and selatogrel, are under development for prehospital administration for STEMI and (N)STEMI, respectively. Zalunfiban is a glycoprotein IIb/IIIa inhibitor that produces high-grade inhibition of platelet aggregation induced by ADP or a thrombin receptor activating peptide (TRAP) within 15 minutes. Selatogrel is a P2Y12 inhibitor that produces high-grade inhibition of platelet aggregation initiated by ADP within 15 minutes. We have studied the relative potencies of the drugs at levels achievable in vivo in inhibiting platelet aggregation. Since trisodium citrate (TSC) has been reported to enhance the antiplatelet effect of zalunfiban and inhibit the antiplatelet effect off selatogrel, aggregation studies were performed with both blood anticoagulated with TSC and the non-chelating anticoagulant PPACK. Whereas both ADP and thrombin are present at the site of vascular injury within seconds, we tested the effects of both ADP and TRAP. Purpose To compare directly the effects of zalunfiban and selatogrel on ADP- and TRAP-induced platelet aggregation. Methods Light transmission platelet aggregometry was performed within 4 hours of blood collection (BioData PAP-8E). Whole blood from healthy donors not taking aspirin (N=10) was anticoagulated with 3.2% TSC and 100 μM PPACK. Platelet-rich plasma (PRP) was prepared and adjusted to a platelet count of 250,000/µl. Three clinically relevant incremental concentrations of zalunfiban and selatogrel were added to PRP and platelet aggregation was initiated with 20 μM ADP or 20 μM TRAP. Tracings were recorded for at least 10 minutes after agonist addition. Platelet aggregation was quantified based on the instrument’s measurement of the primary slope (PrSl) of aggregation, which is a measure of the change in light transmission per unit time sustained over at least a 15 second period. Results In TSC-anticoagulated PRP, zalunfiban was more effective in inhibiting the PrSl of both ADP- and TRAP-induced platelet aggregation at all drug concentrations [Figure 1, panel A and B]. In PPACK-anticoagulated PRP, selatogrel showed greater inhibition of ADP-induced platelet aggregation at the lowest concentration tested, similar inhibition at the intermediate concentration, and slightly less inhibition at the highest doses. In sharp contrast, with TRAP-induced aggregation, zalunfiban demonstrated dose-related inhibition, reaching greater than 80% inhibition at the highest dose, whereas selatogrel produced less than 20% inhibition at all three doses. Conclusion Selatogrel is a potent inhibitor of ADP-induced platelet aggregation, but has low impact on TRAP-induced platelet aggregation, whereas zalunfiban is a potent inhibitor of both ADP and TRAP-induced platelet aggregation. The difference between the drugs needs to be considered in light of the known generation of thrombin at the site of vascular injury within seconds.Comparison of Platelet Inhibition

Designing energy transfer-based color-tunable, information-encrypting, luminous hydro-gels, and latent fingerprint detection systems using BaLa2ZnO5:Tb3+, Bi3+ nanophosphors

Synthesizing cost-effective blue-green emitting BaLa2ZnO5:Tb3+, Bi3+ nanophosphors (BLZO:Tb3+, Bi3+ NPs) through an eco-friendly urea-aided combustion method. Thorough examination of phase purity, structure, surface characteristics, and luminescent properties of the resulting products reveals a robust blue-green emission linked to the 5D4 → 7F5 transition of Tb3+ under 365 nm UV excitation, as well as evident energy transfer from Bi3+ to Tb3+ within the co-doped BLZO, discussed in detail. Optimal doping concentrations are determined to be 5 mol% for Tb3+ and 0.3 mol% for Bi3+ in BLZO phosphor, with non-radiative transition mechanisms explaining concentration quenching. BLZO:Tb3+ NPs emits green light, while co-doping with Bi3+ shifted to the blue-green region. The optimized phosphor is employed for latent fingerprint (LFP) development on porous, semi-porous, and non-porous surfaces, with clear visualization under 365 nm UV light. The generated fingerprints (FPs) display level I–III ridge features with exceptional sensitivity, selectivity, and contrast. The luminescent hydro-gels exhibit outstanding photo-stability, chemical stability, and long-term durability, making them ideal for anti-counterfeiting (AC) applications. Furthermore, BLZO:Tb3+, Bi3+ NPs demonstrate the ability to mitigate oxidative stress and thrombosis, evidenced by their DPPH scavenging activity and their capacity to normalize stress indicators in Red Blood Cells (RBC) exposed to NaNO2-induced oxidative stress. These NPs also reduce ADP-induced platelet aggregation. Collectively, these results highlight the effective utilization of the optimized phosphor in display devices, advanced forensics, anti-counterfeiting measures, and biomedical applications.

Abstract 14010: Synergistic Influence of Rivaroxaban on Platelets, Inflammation and Coagulation Biomarkers in Patients With CAD and/or PAD on Aspirin Therapy

Background: Cardiovascular benefits of 2.5 mg twice daily rivaroxaban plus aspirin therapy (RIV+ASA) has been demonstrated in patients with arterial diseases. Hypothesis: RIV+ASA is associated with reduced platelet activation and plasma inflammation and coagulation activation markers in patients with CAD and/or PAD who were on ASA. Methods: In this open-label biomarker study, patients on 81 mg/day ASA were randomized to continue ASA or RIV+ASA for 12 weeks. We assessed ADP-, α-thrombin-, and tissue factor-induced platelet aggregation (PA) using conventional aggregometry, platelet-fibrin clot strength (PFCS) by INTEM (intrinsic pathway activator) and EXTEM (extrinsic pathway activator) using thromboelastometry, shear-induced PA by platelet function analyzer-100, D-dimer and fibrinogen using coagulation analyzer, hs-CRP and interleukin-6 using ELISA method at baseline, and four weeks and 12 weeks post-randomization. Results: Data was available in 9 patients with RIV + ASA and ten patients with ASA-only therapy. Most patients were male, Caucasians, obese and older. There were no differences in baseline demographics, medications, and laboratory values between groups, except patients in RIV + ASA group had higher white blood cell counts (p=0.028) and lower baseline 2uM ADP-induced PA (p=0.03). There were no differences in other laboratory measurements between baseline and post-randomization time points within the group or at 4- and 12-week time points between groups, except D-dimer values were significantly lower at 12 weeks (p=0.038) in the RIV + ASA vs. ASA-only group. No significant adverse events were observed. Conclusions: Twelve weeks of rivaroxaban plus aspirin vs. aspirin was associated with similar levels of platelet aggregation, platelet fibrin clot strength, fibrinogen and inflammation markers, and lower d-dimer levels.