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.

Association of CYP2C19 genotypes with postoperative atrial fibrillation after coronary artery bypass surgery.

This cohort study aims to assess the connection between cytochrome P450 family 2 subfamily C member 19 (CYP2C19) genotyping, platelet aggregability following oral clopidogrel administration, and the occurrence of postoperative atrial fibrillation (POAF) after off-pump coronary artery bypass graft (CABG) surgery. From May 2017 to November 2022, a total of 258 patients undergoing elective first-time CABG surgery, receiving 100 mg/day oral aspirin and 75 mg/day oral clopidogrel postoperatively, was included for analysis. These patients were categorized based on CYP2C19 genotyping. Platelet aggregability was assessed serially using multiple-electrode aggregometry before CABG, 1 and 5 days after the procedure, and before discharge. The incidences of POAF were compared using the log-rank test for cumulative risk. CYP2C19 genotyping led to categorization into CYP2C19*1*1 (WT group, n = 123) and CYP2C19*2 or *3 (LOF group, n = 135). Baseline characteristics and operative data showed no significant differences between the two groups. The incidence of POAF after CABG was 42.2% in the LOF group, contrasting with 22.8% in the WT group (hazard risk [HR]: 2.061; 95% confidence interval [CI]: 1.347, 3.153; p = 0.0013). Adenosine diphosphate-stimulated platelet aggregation was notably higher in the LOF group compared to the WT group 5 days after CABG (30.4% ± 6.5% vs. 17.9% ± 4.1%, p < 0.001), remaining a similar higher level at hospital discharge (25.6% ± 6.1% vs. 12.2% ± 3.5%, p < 0.001). The presence of CYP2C19 LOF was linked to a higher incidence of POAF and relatively elevated platelet aggregation after CABG surgery under the same oral clopidogrel regimen.

Safety, Tolerability, and Pharmacodynamics of AZD3366 (Optimized Human CD39L3 Apyrase) Alone and in Combination With Ticagrelor and Acetylsalicylic Acid: A Phase 1, Randomized, Placebo-Controlled Study.

BACKGROUND: ADP and ATP are importantly involved in vascular and thrombotic homeostasis, via multiple receptor pathways. Blockade of ADP P2Y12 receptors inhibits platelet aggregation and represents an effective cardiovascular disease prevention strategy. AZD3366 (APT102), a long-acting recombinant form of an optimized CD39L3 human apyrase, has effectively reduced ATP, ADP, and platelet aggregation and provided tissue protection in preclinical models, features that could be very beneficial in treating patients with cardiovascular disease. METHODS AND RESULTS: We conducted this phase 1, first-in-human study of single ascending doses of intravenous AZD3366 or placebo, including doses added to dual antiplatelet therapy with ticagrelor and acetylsalicylic acid. The primary objective was safety and tolerability; secondary and exploratory objectives included pharmacokinetics, pharmacodynamics (measured as inhibition of platelet aggregation), adenosine diphosphatase (ADPase) activity, and ATP/ADP metabolism. In total, 104 participants were randomized. AZD3366 was generally well tolerated, with no major safety concerns observed. ADPase activity increased in a dose-dependent manner with a strong correlation to AZD3366 exposure. Inhibition of ADP-stimulated platelet aggregation was immediate, substantial, and durable. In addition, there was a prompt decrease in systemic ATP concentration and an increase in adenosine monophosphate concentrations, whereas ADP concentration appeared generally unaltered. At higher doses, there was a prolongation of capillary bleeding time without detectable changes in the ex vivo thromboelastometric parameters. CONCLUSIONS: AZD3366 was well tolerated in healthy participants and demonstrated substantial and durable inhibition of platelet aggregation after single dosing. Higher doses prolonged capillary bleeding time without detectable changes in ex vivo thromboelastometric parameters. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique Identifier: NCT04588727.

Anti-atherogenic role of green tea (Camellia sinensis) in South Indian smokers.

ETHNOPHARMACOLOGICAL RELEVANCE: Green tea (Camellia sinensis) is a popular beverage consumed all over the world due to its health benefits. Many of these beneficial effects of green tea are attributed to polyphenols, particularly catechins. AIM OF THE STUDY: The present study focuses on underlying anti-platelet aggregation, anti-thrombotic, and anti-lipidemic molecular mechanisms of green tea in South Indian smokers. MATERIALS AND METHODS: We selected 120 South Indian male volunteers for this study to collect the blood and categorised them into four groups; control group individuals (Controls), smokers, healthy control individuals consuming green tea, and smokers consuming green tea. Smokers group subjects have been smoking an average 16-18 cigarettes per day for the last 7 years or more. The subjects (green tea consumed groups) consumed 100 mL of green tea each time, thrice a day for a one-year period. RESULTS: LC-MS analysis revealed the presence of multiple phytocompounds along with catechins in green tea extract. Increased plasma lipid peroxidation (LPO), protein carbonyls, cholesterol, triglycerides, and LDL-cholesterol with decreased HDL-cholesterol levels were observed in smokers compared to the control group and the consumption of green tea showed beneficial effect. Furthermore, docking studies revealed that natural compounds of green tea had high binding capacity with 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-CoA) when compared to their positive controls, whereas (-) epigallocatechin-3-gallate (EGCG) and (-) epicatechin-gallate (ECG) had high binding capacity with sterol regulatory element-binding transcription factor 1 (SREBP1c). Further, our ex vivo studies showed that green tea extract (GTE) significantly inhibited platelet aggregation and increased thrombolytic activity in a dose dependent manner. CONCLUSION: In conclusion, in smokers, catechins synergistically lowered oxidative stress, platelet aggregation and modified the aberrant lipid profile. Furthermore, molecular docking studies supported green tea catechins' antihyperlipidemic efficacy through strong inhibitory activity on HMG-CoA reductase and SREBP1c. The mitigating effects of green tea on cardiovascular disease risk factors in smokers that have been reported can be attributed majorly to catechins or to their synergistic effects.

Platelet Biorheology and Mechanobiology in Thrombosis and Hemostasis: Perspectives from Multiscale Computation.

Thrombosis is the pathological clot formation under abnormal hemodynamic conditions, which can result in vascular obstruction, causing ischemic strokes and myocardial infarction. Thrombus growth under moderate to low shear (<1000 s-1) relies on platelet activation and coagulation. Thrombosis at elevated high shear rates (>10,000 s-1) is predominantly driven by unactivated platelet binding and aggregating mediated by von Willebrand factor (VWF), while platelet activation and coagulation are secondary in supporting and reinforcing the thrombus. Given the molecular and cellular level information it can access, multiscale computational modeling informed by biology can provide new pathophysiological mechanisms that are otherwise not accessible experimentally, holding promise for novel first-principle-based therapeutics. In this review, we summarize the key aspects of platelet biorheology and mechanobiology, focusing on the molecular and cellular scale events and how they build up to thrombosis through platelet adhesion and aggregation in the presence or absence of platelet activation. In particular, we highlight recent advancements in multiscale modeling of platelet biorheology and mechanobiology and how they can lead to the better prediction and quantification of thrombus formation, exemplifying the exciting paradigm of digital medicine.

1.8-cineole prevents platelet activation and aggregation by activating the cAMP pathway via the adenosine A2A receptor.

AIMS: Dysregulated platelet aggregation is a fatal condition in many bacterial- and virus-induced diseases. However, classical antithrombotics cannot completely prevent immunothrombosis, due to the unaddressed mechanisms towards inflammation. Thus, targeting platelet hyperactivation together with inflammation might provide new treatment options in diseases, characterized by immunothrombosis, such as COVID-19 and sepsis. The aim of this study was to investigate the antiaggregatory effect and mode of action of 1.8-cineole, a monoterpene derived from the essential oil of eucalyptus leaves, known for its anti-inflammatory proprieties. MAIN METHODS: Platelet activity was monitored by measuring the expression and release of platelet activation markers, i.e., P-selectin, CD63 and CCL5, as well as platelet aggregation, upon treatment with 1.8-cineole and stimulation with several classical stimuli and bacteria. A kinase activity assay was used to elucidate the mode of action, followed by a detailed analysis of the involvement of the adenylyl-cyclase (AC)-cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) pathway by Western blot and ELISA. KEY FINDINGS: 1.8-cineole prevented the expression and release of platelet activation markers, as well as platelet aggregation, upon induction of aggregation with classical stimuli and immunological agonists. Mechanistically, 1.8- cineole influences the activation of the AC-cAMP-PKA pathway, leading to higher cAMP levels and vasodilator-stimulated phosphoprotein (VASP) phosphorylation. Finally, blocking the adenosine A2A receptor reversed the antithrombotic effect of 1.8-cineole. SIGNIFICANCE: Given the recognized anti-inflammatory attributes of 1.8-cineole, coupled with our findings, 1.8-cineole might emerge as a promising candidate for treating conditions marked by platelet activation and abnormal inflammation.

Evaluation of coagulation and platelet activation state and function in heartworm-infected dogs.

BACKGROUND: Enhanced platelet responses have been demonstrated in heartworm-infected (HWI) dogs; however, the cause and clinical implications of altered platelet function have not been fully elucidated. OBJECTIVE: This study evaluated platelet function in HWI dogs. METHODS: Anticoagulated whole blood collected from eight HWI and eight uninfected dogs was evaluated using turbidometric platelet aggregometry, a platelet function analyzer (PFA-100), a total thrombus analysis system (T-TAS), tissue factor-activated and tissue plasminogen activator modified thromboelastography (TF- and tPA-TEG), CBC, von Willebrand Factor activity, and fibrinogen concentrations. Platelet activation state and the presence of reticulated platelets were assessed via flow cytometric expression of P-selection (CD-62P) and thiazole orange staining. RESULTS: Platelet aggregation responses to adenosine diphosphate (ADP, 10 µM) or collagen (20 µg/mL), PFA-100 closure times, and T-TAS occlusion times did not differ between groups. TEG values TF-R, tPA-R, TF-K, and TF-LY60 were decreased (P = .025, P = .047, P = .038, P = .025) and TF-MA, tPA-MA, TF-G, tPA-G and TF-alpha angle were increased (P < .04) in HWI dogs. HWI dogs had higher fibrinogen concentrations (465.6 ± 161 mg/dL vs 284.5 ± 38 mg/dL, P = .008) and eosinophil counts (0.686 ± 0.27 × 103/µL vs 0.267 ± 0.20 × 103/µL, P = .003). There was no difference in hematocrit, activation state, or percent of reticulated platelets. Non-activated reticulated platelets exhibited higher CD62P expression compared with mature platelets. CONCLUSIONS: Chronic canine heartworm disease was accompanied by hypercoagulability, hyperfibrinogenemia, and decreased fibrinolysis. Enhanced platelet activation was not identified in this group of HWI dogs.

Effect of a diet rich in potato peel on platelet aggregation / Efecto de una dieta rica en cáscara de papa sobre la agregación plaquetaria

Background: Potato peel extract has demonstrated the ability to reduce platelet aggregation in vitro, suggesting its potential as a dietary intervention for preventing atherothrombotic disorders. Objective: This study aims to evaluate the impact of a potato peel-rich diet on platelet aggregation. Methods: A randomized, crossover-controlled, open two-period study was carried out with the participation of 12 healthy volunteers. Platelet aggregation was assessed before and after a seven-day dietary intervention. Participants consumed either a diet rich in potato peel (2 g/kg/d) or acetylsalicylic acid (ASA) as a reference (100 mg/d). Platelet aggregation percentages were measured following stimulation with arachidonic acid (AA, 150 µg/mL), adenosine diphosphate (ADP, 10 µM), and collagen (COL, 10 µg/mL). Results: The potato peel-rich diet resulted in a slight but significant reduction in platelet aggregation when stimulated with arachidonic acid compared to baseline values (85.0±2.0% vs. 91.3±1.7%, p<0.05). This effect was less pronounced than the reduction achieved with ASA (16±1.9%, p<0.001). Conclusion: The administration of a diet rich in potato peel reduces platelet aggregation induced by arachidonic acid, suggesting its potential role in the prevention of atherothrombotic disorders.

Introducción: El extracto de cáscara de patata ha demostrado su capacidad para reducir la agregación plaquetaria in vitro, lo que sugiere su potencial como intervención dietética para prevenir trastornos aterotrombóticos. Objetivo: Evaluar el impacto de una dieta rica en cáscara de patata en la agregación plaquetaria. Materiales y métodos: Se llevó a cabo un estudio aleatorizado, controlado, cruzado y abierto con la participación de 12 voluntarios sanos. Se evaluó la agregación plaquetaria antes y después de una intervención dietética de siete días. Los participantes consumieron una dieta rica en cáscara de patata (2 g/kg/d) o ácido acetilsalicílico (ASA) como referente (100 mg/d). Se midieron los porcentajes de agregación plaquetaria después de la estimulación con ácido araquidónico (AA, 150 µg/mL), difosfato de adenosina (ADP, 10 µM) y colágeno (COL, 10 µg/mL). Resultados: La dieta rica en cáscara de patata resultó en una ligera pero significativa reducción en la agregación plaquetaria cuando se estimuló con ácido araquidónico en comparación con los valores iniciales (85,0 ± 2,0% vs. 91,3 ± 1,7%, p <0,05). Este efecto fue menos pronunciado que la reducción lograda con ASA (16 ± 1,9%, p <0,001). Conclusión: La administración de una dieta rica en cáscara de patata reduce la agregación plaquetaria inducida por ácido araquidónico, lo que sugiere su papel potencial en la prevención de trastornos aterotrombóticos.

C-type lectin-like receptor-2 in platelets mediates ferric chloride-induced platelet activation and attenuates ferroptosis of endothelial cells.

BACKGROUND: An iron overload status induces ferroptosis, an iron-dependent nonapoptotic cell death, in various pathological conditions. We previously reported that hemin (heme), protoporphyrin-IX with ferric iron, activates platelets via C-type lectin-like receptor-2 (CLEC-2) and glycoprotein VI/FcRγ, but protoporphyrin-IX alone blocks CLEC-2-dependent platelet activation. Therefore, we hypothesized that free iron has the ability to activate platelets. OBJECTIVES: This study aimed to elucidate platelet activation mechanisms of iron (ferric chloride), including the identification of signaling pathways and receptors, and to examine whether platelets regulate ferroptosis. METHODS: Platelet aggregometry, platelet activation marker expression, and protein phosphorylation were examined in ferric chloride-stimulated human and murine platelets. Inhibitors of platelet activation signaling pathways and receptor-deleted platelets were utilized to identify the responsible signaling pathway and receptor. The effect of platelets on ferroptosis of endothelial cells was investigated in vitro. RESULTS: Ferric chloride induced platelet activation dependent on Src family kinase pathways in humans and mice. Ferric chloride-induced platelet aggregation was almost lost in CLEC-2-depleted murine platelets and wild-type platelets preincubated with recombinant CLEC-2 proteins. Furthermore, coculture of wild-type platelets, but not CLEC-2-deficient platelets, attenuated ferroptosis of endothelial cells in vitro. CONCLUSION: Ferric chloride activates platelets via CLEC-2 and Src family kinase pathways, and platelets have a protective role in the ferroptosis of endothelial cells dependent on CLEC-2.

Elevated plasma levels of factor VIII enhance arterial thrombus formation on erosive smooth muscle cell-rich neointima but not normal intima in rabbits.

BACKGROUND: Plaque erosion, a type of coronary atherothrombosis, involves superficial injury to smooth muscle cell (SMC)-rich plaques. Elevated levels of coagulation factor VIII (FVIII) correlate with an increased ischemic heart disease risk. FVIII may contribute to thrombus formation on eroded plaques. AIMS: We aimed to elucidate the role of elevated FVIII in arterial thrombus formation within SMC-rich neointima in rabbits. METHODS AND RESULTS: We assessed the effect of recombinant human FVIII (rFVIII) on blood coagulation in vitro and platelet aggregation ex vivo. An SMC-rich neointima was induced through balloon injury to the unilateral femoral artery. Three weeks after the first balloon injury, superficial erosive injury and thrombus formation were initiated with a second balloon injury of the bilateral femoral arteries 45 min after the administration of rFVIII (100 IU/kg) or saline. The thrombus area and contents were histologically measured 15 min after the second balloon injury. rFVIII administration reduced the activated partial thromboplastin time and augmented botrocetin-induced, but not collagen- or adenosine 5'-diphosphate-induced, platelet aggregation. While rFVIII did not influence platelet-thrombus formation in normal intima, it increased thrombus formation on SMC-rich neointima post-superficial erosive injury. Enhanced immunopositivity for glycoprotein IIb/IIIa and fibrin was observed in rFVIII-administered SMC-rich neointima. Neutrophil count in the arterial thrombus on the SMC-rich neointima correlated positively with thrombus size in the control group, unlike the rFVIII group. CONCLUSIONS: Increased FVIII contributes to thrombus propagation within erosive SMC-rich neointima, highlighting FVIII's potential role in plaque erosion-related atherothrombosis.

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.

The prognostic value of platelet aggregation in patients with sepsis.

BACKGROUND: This study aims to investigate the relevance of platelet aggregation markers, specifically arachidonic acid (AA) and adenosine diphosphate (ADP), in relation to the prognosis of sepsis patients. METHODS: A cohort of 40 sepsis patients was included and stratified, based on their 28-day post-treatment prognosis, into two groups: a survival group (n = 31) and a severe sepsis group (n = 9. Then, their various clinical parameters, including patient demographics, platelet counts (PLT), inflammatory markers, and platelet aggregation rates (PAR) induced by AA and ADP between the two groups, were compared. Long-term health implications of sepsis were assessed using the Acute Physiologic Assessment and Chronic Health Evaluation II (APACHE II) score, and logistic regression analysis was conducted to evaluate the prognostic significance of PAR in sepsis patients. RESULTS: Patients with severe sepsis exhibited significantly elevated levels of procalcitonin (PCT), platelet adhesion rates, and PAR induced by ADP (P < 0.05), but having lower PLT (P < 0.05), compared to those in the survival group. Logistic regression analysis demonstrated that PAR induced by ADP was a protective factor in predicting prognosis in sepsis patients (P < 0.01). CONCLUSIONS: Activation of platelets in sepsis intensifies inflammatory response. Patients with sepsis whose ADP-induced PAR was < 60% displayed significant impairment in platelet aggregation function, and had higher mortality rate. Monitoring ADP-induced PAR is crucial for management of sepsis.

Material strengths of shear-induced platelet aggregation clots and coagulation clots.

Arterial occlusion by thrombosis is the immediate cause of some strokes, heart attacks, and peripheral artery disease. Most prior studies assume that coagulation creates the thrombus. However, a contradiction arises as whole blood (WB) clots from coagulation are too weak to stop arterial blood pressures (> 150 mmHg). We measure the material mechanical properties of elasticity and ultimate strength for Shear-Induced Platelet Aggregation (SIPA) type clots, that form under stenotic arterial hemodynamics in comparison with coagulation clots. The ultimate strength of SIPA clots averaged 4.6 ± 1.3 kPa, while WB coagulation clots had a strength of 0.63 ± 0.3 kPa (p < 0.05). The elastic modulus of SIPA clots was 3.8 ± 1.5 kPa at 1 Hz and 0.5 mm displacement, or 2.8 times higher than WB coagulation clots (1.3 ± 1.2 kPa, p < 0.0001). This study shows that the SIPA thrombi, formed quickly under high shear hemodynamics, is seven-fold stronger and three-fold stiffer compared to WB coagulation clots. A force balance calculation shows a SIPA clot has the strength to resist arterial pressure with a short length of less than 2 mm, consistent with coronary pathology.

Regulation of platelet activation and thrombus formation in acute non-ST segment elevation myocardial infarction: Role of Beclin1.

This study aims to investigate the mechanism of platelet activation-induced thrombosis in patients with acute non-ST segment elevation myocardial infarction (NSTEMI) by detecting the expression of autophagy-associated proteins in platelets of patients with NSTEMI. A prospective study was conducted on 121 patients with NSTEMI who underwent emergency coronary angiography and optical coherence tomography. The participants were divided into two groups: the ST segment un-offset group (n = 64) and the ST segment depression group (n = 57). We selected a control group of 60 patients without AMI during the same period. The levels of autophagy-associated proteins and the expression of autophagy-associated proteins in platelets were measured using immunofluorescence staining and Western blot. In NSTEMI, the prevalence of red thrombus was higher in the ST segment un-offset myocardial infarction (STUMI) group, whereas white thrombus was more common in the ST segment depression myocardial infarction (STDMI) group. Furthermore, the platelet aggregation rate was significantly higher in the white thrombus group compared with the red thrombus group. Compared with the control group, the autophagy-related protein expression decreased, and the expression of αIIbß3 increased in NSTEMI. The overexpression of Beclin1 could activate platelet autophagy and inhibit the expression of αIIbß3. The results suggested that the increase in platelet aggregation rate in patients with NSTEMI may be potentially related to the change in autophagy. And the overexpression of Beclin1 could reduce the platelet aggregation rate by activating platelet autophagy. Our findings demonstrated that Beclin1 could be a potential therapeutic target for inhibiting platelet aggregation in NSTEMI.

Palliative effects of carnosol on lung-deposited pollutant particles-induced thrombogenicity and vascular injury in mice.

The toxicity of inhaled particulate air pollution perseveres even at lower concentrations than those of the existing air quality limit. Therefore, the identification of safe and effective measures against pollutant particles-induced vascular toxicity is warranted. Carnosol is a bioactive phenolic diterpene found in rosemary herb, with anti-inflammatory and antioxidant actions. However, its possible protective effect on the thrombotic and vascular injury induced by diesel exhaust particles (DEP) has not been studied before. We assessed here the potential alleviating effect of carnosol (20 mg/kg) administered intraperitoneally 1 h before intratracheal (i.t.) instillation of DEP (20 µg/mouse). Twenty-four hours after the administration of DEP, various parameters were assessed. Carnosol administration prevented the increase in the plasma concentrations of C-reactive protein, fibrinogen, and tissue factor induced by DEP exposure. Carnosol inhibited DEP-induced prothrombotic effects in pial microvessels in vivo and platelet aggregation in vitro. The shortening of activated partial thromboplastin time and prothrombin time induced by DEP was abated by carnosol administration. Carnosol inhibited the increase in pro-inflammatory cytokines (interleukin-6 and tumor necrosis factor α) and adhesion molecules (intercellular adhesion molecule-1, vascular cell adhesion molecule-1, E-selectin, and P-selectin) in aortic tissue. Moreover, it averted the effects of DEP-induced increase of thiobarbituric acid reactive substances, depletion of antioxidants and DNA damage in the aortic tissue. Likewise, carnosol prevented the decrease in the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) caused by DEP. We conclude that carnosol alleviates DEP-induced thrombogenicity and vascular inflammation, oxidative damage, and DNA injury through Nrf2 and HO-1 activation.

1,25-Dihydroxyvitamin D3 attenuates platelet aggregation potentiated by SARS-CoV-2 spike protein via inhibiting integrin αIIbß3 outside-in signaling.

Platelet hyperreactivity contributes to the pathogenesis of COVID-19, which is associated with a hypercoagulability state and thrombosis disorder. It has been demonstrated that Vitamin D deficiency is associated with the severity of COVID-19 infection. Vitamin D supplement is widely used as a dietary supplement due to its safety and health benefits. In this study, we investigated the direct effects and underlying mechanisms of 1,25(OH)2D3 on platelet hyperreactivity induced by SRAS-CoV-2 spike protein via Western blot and platelet functional studies in vitro. Firstly, we found that 1,25(OH)2D3 attenuated platelet aggregation and Src-mediated signaling. We further observed that 1,25(OH)2D3 attenuated spike protein-potentiated platelet aggregation in vitro. Mechanistically, 1,25(OH)2D3 attenuated spike protein upregulated-integrin αIIbß3 outside-in signaling such as platelet spreading and the phosphorylation of ß3, c-Src and Syk. Moreover, using PP2, the Src family kinase inhibitor to abolish spike protein-stimulated platelet aggregation and integrin αIIbß3 outside-in signaling, the combination of PP2 and 1,25(OH)2D3 did not show additive inhibitory effects on spike protein-potentiated platelet aggregation and the phosphorylation of ß3, c-Src and Syk. Thus, our data suggest that 1,25(OH)2D3 attenuates platelet aggregation potentiated by spike protein via downregulating integrin αIIbß3 outside-in signaling.

A Case of Pseudothrombocytopenia due to Pre-Analytical Issues Revealed by the Presence of Fibrin in a Blood Film.

BACKGROUND: Pseudothrombocytopenia (PTCP) can be caused by anticoagulants or pre-analytical issues. The authors present a case of PTCP attributed to pre-analytical issues in a 68-year-old male patient. METHODS: The platelet count results were obtained using both the impedance and fluorescence channels of Sysmex XN-10. The blood film was scanned using both Cellavision DM96 and a microscope. RESULTS: The flag for PLT-Clumps and the scattergram from the PLT-F channel indicated the presence of platelet aggregation. Fibrin could be observed at the feathered end of the blood film. A diagnosis of PTCP resulting from pre-analytical issues was made. CONCLUSIONS: The presence of fibrin in a blood film is a critical indicator for diagnosing PTCP due to pre-analytical issues.

Differential Impact In Vivo of Pf4-ΔCre-Mediated and Gp1ba-ΔCre-Mediated Depletion of Cyclooxygenase-1 in Platelets in Mice.

BACKGROUND: Low-dose aspirin is widely used for the secondary prevention of cardiovascular disease. The beneficial effects of low-dose aspirin are attributable to its inhibition of platelet Cox (cyclooxygenase)-1-derived thromboxane A2. Until recently, the use of the Pf4 (platelet factor 4) Cre has been the only genetic approach to generating megakaryocyte/platelet ablation of Cox-1 in mice. However, Pf4-ΔCre displays ectopic expression outside the megakaryocyte/platelet lineage, especially during inflammation. The use of the Gp1ba (glycoprotein 1bα) Cre promises a more specific, targeted approach. METHODS: To evaluate the role of Cox-1 in platelets, we crossed Pf4-ΔCre or Gp1ba-ΔCre mice with Cox-1flox/flox mice to generate platelet Cox-1-/- mice on normolipidemic and hyperlipidemic (Ldlr-/-; low-density lipoprotein receptor) backgrounds. RESULTS: Ex vivo platelet aggregation induced by arachidonic acid or adenosine diphosphate in platelet-rich plasma was inhibited to a similar extent in Pf4-ΔCre Cox-1-/-/Ldlr-/- and Gp1ba-ΔCre Cox-1-/-/Ldlr-/- mice. In a mouse model of tail injury, Pf4-ΔCre-mediated and Gp1ba-ΔCre-mediated deletions of Cox-1 were similarly efficient in suppressing platelet prostanoid biosynthesis. Experimental thrombogenesis and attendant blood loss were similar in both models. However, the impact on atherogenesis was divergent, being accelerated in the Pf4-ΔCre mice while restrained in the Gp1ba-ΔCres. In the former, accelerated atherogenesis was associated with greater suppression of PGI2 biosynthesis, a reduction in the lipopolysaccharide-evoked capacity to produce PGE2 (prostaglandin E) and PGD2 (prostanglandin D), activation of the inflammasome, elevated plasma levels of IL-1ß (interleukin), reduced plasma levels of HDL-C (high-density lipoprotein receptor-cholesterol), and a reduction in the capacity for reverse cholesterol transport. By contrast, in the latter, plasma HDL-C and α-tocopherol were elevated, and MIP-1α (macrophage inflammatory protein-1α) and MCP-1 (monocyte chemoattractant protein 1) were reduced. CONCLUSIONS: Both approaches to Cox-1 deletion similarly restrain thrombogenesis, but a differential impact on Cox-1-dependent prostanoid formation by the vasculature may contribute to an inflammatory phenotype and accelerated atherogenesis in Pf4-ΔCre mice.

Platelet activity, coagulation, and fibrinolysis in long-term users of anabolic-androgenic steroids compared to strength-trained athletes.

INTRODUCTION: Use of anabolic-androgenic steroids (AAS) is associated with adverse cardiovascular (CV) effects, including potential prothrombotic effects. This study aimed to assess platelet activation and aggregation, coagulation, and fibrinolysis, in long-term AAS users compared to non-using strength-trained athletes. MATERIALS AND METHODS: Thirty-seven strength-trained men using AAS were compared to seventeen non-using professional strength-trained athletes at similar age (median 33 years). AAS use was verified by blood and urine analyses. Platelet Function Analyzer 100 (PFA-100) and whole blood impedance aggregometry with thrombin, arachidonic acid, and ADP as agonists, were performed to evaluate platelet aggregation. ELISA methods were used for markers of platelet activation. Fibrinogen, D-dimer, the coagulation inhibitors protein S and C activity, and antithrombin were measured by routine. Fibrinolysis was evaluated by Plasminogen Activator Inhibitor-1 (PAI-1) activity. RESULTS: There were no significant differences in platelet aggregation between the two groups. Von Willebrand factor was lower among the AAS users (p < 0.01), and P-Selectin was slightly higher (p = 0.05), whereas CD40 Ligand, ß-thromboglobulin, and thrombospondin did not differ significantly. No differences were found in the assessed coagulation inhibitors. Higher D-dimer levels (p < 0.01) and lower PAI-1 activity (p < 0.01) were found among the AAS users. CONCLUSIONS: The investigated long-term users of AAS did not exhibit elevated platelet activity compared to strength-trained non-using athletes. However, AAS use was associated with higher D-dimer levels and lower PAI-1 activity. These findings suggest that any prothrombotic effect of long-term AAS use may predominantly involve other aspects of the hemostatic system than blood platelets.