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.

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.

Oxidative Stress-Induced Platelet Apoptosis/Activation: Alleviation by Purified Curcumin via ASK1-JNK/p-38 Pathway.

Platelets are known for their indispensable role in hemostasis and thrombosis. However, alteration in platelet function due to oxidative stress is known to mediate various health complications, including cardiovascular diseases and other health complications. To date, several synthetic molecules have displayed antiplatelet activity; however, their uses are associated with bleeding and other adverse effects. The commercially available curcumin is generally a mixture of three curcuminoids: curcumin, demethoxycurcumin, and bisdemethoxycurcumin. Although crude curcumin is known to inhibit platelet aggregation, the effect of purified curcumin on platelet apoptosis, activation, and aggregation remains unclear. Therefore, in this study, curcumin was purified from a crude curcumin mixture and the effects of this preparation on the oxidative stress-induced platelet apoptosis and activation was evaluated. 2,2'-Azobis(2-methylpropionamidine) dihydrochloride (AAPH) compound was used as an inducer of oxidative stress. Purified curcumin restored AAPH-induced platelet apoptotic markers like reactive oxygen species, intracellular calcium level, mitochondrial membrane potential, cardiolipin peroxidation, cytochrome c release from mitochondria to the cytosol, and phosphatidyl serine externalization. Further, it inhibited the agonist-induced platelet activation and aggregation, demonstrating its antiplatelet activity. Western blot analysis confirms protective effect of the purified curcumin against oxidative stress-induced platelet apoptosis and activation via downregulation of MAPKs protein activation, including ASK1, JNK, and p-38. Together, these results suggest that the purified curcumin could be a potential therapeutic bioactive molecule to treat the oxidative stress-induced platelet activation, apoptosis, and associated complications.

Qilong capsule prevents myocardial ischemia/reperfusion injury by inhibiting platelet activation via the platelet CD36 signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Qilong capsule (QC) is developed from the traditional Chinese medicine formula Buyang Huanwu Decoction, which has been clinically used to invigorate Qi and promote blood circulation to eliminate blood stasis. Myocardial ischemia‒reperfusion injury (MIRI) can be attributed to Qi deficiency and blood stasis. However, the effects of QC on MIRI remain unclear. AIM OF THE STUDY: This study aimed to investigate the protective effect and possible mechanism of QC on platelet function in MIRI rats. MATERIALS AND METHODS: The left anterior descending artery of adult Sprague‒Dawley rats was ligated for 30 min and then reperfused for 120 min with or without QC treatment. Then, the whole blood viscosity, plasma viscosity, coagulation, platelet adhesion rate, platelet aggregation, and platelet release factors were evaluated. Platelet CD36 and its downstream signaling pathway-related proteins were detected by western blotting. Furthermore, the active components of QC and the molecular mechanism by which QC regulates platelet function were assessed via molecular docking, platelet aggregation tests in vitro and BLI analysis. RESULTS: We found that QC significantly reduced the whole blood viscosity, plasma viscosity, platelet adhesion rate, and platelet aggregation induced by ADP or AA in rats with MIRI. The inhibition of platelet activation by QC was associated with reduced levels of ß-TG, PF-4, P-selectin and PAF. Mechanistically, QC effectively attenuated the expression of platelet CD36 and thus inhibited the activation of Src, ERK5, and p38. The active components of QC apparently suppressed platelet aggregation in vitro and regulated the CD36 signaling pathway. CONCLUSIONS: QC improves MIRI-induced hemorheological disorders, which might be partly attributed to the inhibition of platelet activation via CD36-mediated platelet signaling pathways.

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.

Apolipoprotein A-IV polymorphisms Q360H and T347S attenuate its endogenous inhibition of thrombosis.

Platelets are small anucleate cells that play a key role in thrombosis and hemostasis. Our group previously identified apolipoprotein A-IV (apoA-IV) as an endogenous inhibitor of thrombosis by competitive blockade of the αIIbß3 integrin on platelets. ApoA-IV inhibition of platelets was dependent on the N-terminal D5/D13 residues, and enhanced with absence of the C-terminus, suggesting it sterically hinders its N-terminal platelet binding site. The C-terminus is also the site of common apoA-IV polymorphisms apoA-IV-1a (T347S) and apoA-IV-2 (Q360H). Interestingly, both are linked with an increased risk of cardiovascular disease, however, the underlying mechanism remains unclear. Here, we generated recombinant apoA-IV and found that the Q360H or T347S polymorphisms dampened its inhibition of platelet aggregation in human platelet-rich plasma and gel-filtered platelets, reduced its inhibition of platelet spreading, and its inhibition of P-selectin on activated platelets. Using an ex vivo thrombosis assay, we found that Q360H and T347S attenuated its inhibition of thrombosis at both high (1800s-1) and low (300s-1) shear rates. We then demonstrate a conserved monomer-dimer distribution among apoA-IV WT, Q360H, and T347S and use protein structure modelling software to show Q360H and T347S enhance C-terminal steric hindrance over the N-terminal platelet-binding site. These data provide critical insight into increased cardiovascular risk for individuals with Q360H or T347S polymorphisms.

Antiplatelet and Anticoagulant Effects of Two New Phenylpropanoid Sucrose Esters and Other Secondary Metabolites from the Aerial Part of Canna edulis.

This study isolated pure compounds from Canna edulis aerial parts and assessed their antiplatelet and anticoagulant potential. Structural elucidation resulted in the identification of two new compounds: caneduloside A (1) and caneduloside B (2), and eleven known compounds: 6'-acetyl-3,6,2'-tri-p-coumaroyl sucrose (3), 6'-acetyl-3,6,2'-triferuloyl sucrose (4), tiliroside (5), afzelin (6), quercitrin (7), 2-hydroxycinnamaldehyde (8), cinnamic acid (9), 3,4-dimethoxycinnamic acid (10), dehydrovomifoliol (11), 4-hydroxy-3,5-dimethoxybenzaldehyde (12), and (S)-(-)-rosmarinic acid (13). Compounds 3, 4, 6-9, 13 were previously reported for antithrombotic properties. Hence, antithrombotic tests were conducted for 1, 2, 5, 10-12. All tested compounds demonstrated a dose-dependent antiaggregatory effect, and 10 and 12 were the most potent for both ADP and collagen activators. Additionally, 10 and 12 showed anticoagulant effects, with prolonged prothrombin time and activated partial thromboplastin time. The new compound 1 displayed antiplatelet and anticoagulant activity, while 2 mildly inhibited platelet aggregation. C. edulis is a potential source for developing antithrombotic agents.

Factors associated with clopidogrel resistance and clinical outcomes in ischemic cerebrovascular disease: A retrospective study.

OBJECTIVE: Clopidogrel resistance may lead to the recurrence of cerebrovascular diseases. We aimed to identify potential factors associated with clopidogrel resistance and evaluate the clinical outcomes of the patients. MATERIALS AND METHODS: In this retrospective study, patients with ischemic cerebrovascular disease treated with clopidogrel were included and classified into 2 groups according to the adenosine diphosphate (ADP)-induced platelet aggregation. Patients with the ADP inhibition rate of <30 % were included in clopidogrel resistance group, otherwise were included in clopidogrel sensitive group. CYP2C19 genotype and other clinical data were analyzed to identify factors and clinical features in the multivariate analysis. The outcomes were vascular events in 6 months. RESULTS: In total, 139 patients were enrolled with 81 (58.27 %) in clopidogrel sensitive group and 58 (41.73 %) in clopidogrel resistance group. Female and CYP2C19 *2*3 carrying were risk factors for clopidogrel resistance, and female was an independent risk factor (OR 2.481, 95 % CI 1.066-5.771, P=0.035). The clopidogrel resistance group showed a higher use rate of argatroban (P=0.030) and a lower arachidonic acid-induced inhibition of platelet aggregation (P=0.036). Clopidogrel resistance was related to the progressing stroke (HR 3.521, 95 % CI 1.352-9.170, P=0.010), but had no influence on the bleeding events (P>0.05). CONCLUSIONS: The risk of clopidogrel resistance increased significantly in female patients. Patients with clopidogrel resistance may have an increased incidence of stroke progression in the acute phase.

Recombinant human soluble domain of CD39L3 and ticagrelor: cardioprotective effects in experimental myocardial infarction.

BACKGROUND AND AIMS: The ecto-nucleoside triphosphate diphosphohydrolases of the CD39 family degrade ATP and ADP into AMP, which is converted into adenosine by the extracellular CD73/ecto-5-nucleotidase. This pathway has been explored in antithrombotic treatments but little in myocardial protection. We have investigated whether the administration of solCD39L3 (AZD3366) confers additional cardioprotection to that of ticagrelor alone in a pre-clinical model of myocardial infarction (MI). METHODS: Ticagrelor-treated pigs underwent balloon-induced MI (90 min) and, before reperfusion, received intravenously either vehicle, 1 mg/kg AZD3366 or 3 mg/kg AZD3366. All animals received ticagrelor twice daily for 42 days. A non-treated MI group was run as a control. Serial cardiac magnetic resonance (baseline, Day 3 and Day 42 post-MI), light transmittance aggregometry, bleeding time, and histological and molecular analyses were performed. RESULTS: Ticagrelor reduced oedema formation and infarct size at Day 3 post-MI vs. controls. A 3 mg/kg AZD3366 provided an additional 45% reduction in oedema and infarct size compared with ticagrelor and a 70% reduction vs. controls (P < .05). At Day 42, infarct size declined in all ticagrelor-administered pigs, particularly in 3 mg/kg AZD3366-treated pigs (P < .05). Left ventricular ejection fraction was diminished at Day 3 in placebo pigs and worsened at Day 42, whereas it remained unaltered in ticagrelor ± AZD3366-administered animals. Pigs administered with 3 mg/kg AZD3366 displayed higher left ventricular ejection fraction upon dobutamine stress at Day 3 and minimal dysfunctional segmental contraction at Day 42 (χ2P < .05 vs. all). Cardiac and systemic molecular readouts supported these benefits. Interestingly, AZD3366 abolished ADP-induced light transmittance aggregometry without affecting bleeding time. CONCLUSIONS: Infusion of AZD3366 on top of ticagrelor leads to enhanced cardioprotection compared with ticagrelor alone.

Ethyl-acetate extract of Spatholobi Caulis blocked the pro-metastatic support from the hemato-microenvironment of colon cancer by specific disruption of tumor-platelet adhesion.

BACKGROUND: Within the pro-metastatic hemato-microenvironment, interaction between platelets and tumor cells provides essential support for tumor cells by inducing Epithelial-Mesenchymal Transition (EMT), which greatly increases the stemness of colon cancer cells. Pharmacologically, although platelet deactivation has proved to be benefit against metastasis, its wide application is severely restricted due to the bleeding risk. Spatholobi Caulis, a traditional Chinese herb with circulatory promotion and blood stasis removal activity, has been proved to be clinically effective in malignant medication, leaving its mechanistic relevance to tumor-platelet interaction largely unknown. METHODS: Firstly, MC38-Luc cells were injected into tail-vein in C57BL/6 mice to establish hematogenous metastasis model and the anti-metastasis effects of SEA were evaluated by using a small-animal imaging system. Then, we evaluated the anti-tumor-platelet interaction efficacy of SEA using a tumor-specific induced platelet aggregation model. Platelet aggregation was specifically induced by tumor cells in vitro. Furthermore, to clarify the anti-metastatic effects of SEA is mainly attributed to its blockage on tumor-platelet interaction, after co-culture with tumor cells and platelets (with or without SEA), MC38-Luc cells were injected into the tail-vein and finally count the total of photons quantitatively. Besides, to clarify the blocking pattern of SEA within the tumor-platelet complex, the dependence of SEA on different fractions from activated platelets was tested. Lastly, molecular docking screening were performed to screen potential effective compounds and we used ß-catenin blockers to verify the pathways involved in SEA blocking tumor-platelet interaction. RESULTS: Our study showed that SEA was effective in blocking tumor-platelet specific interaction: (1) Through CCK-8 and LDH assays, SEA showed no cytotoxic effects on tumor cells and platelets. On this basis, by the tail vein injection model, the photon counts in the SEA group was significantly lower than model group, indicating that SEA effectively reduced metastasis. (2) In the "tumor-platelet" co-culture model, SEA effectively inhibited the progression of EMT and cancer stemness signatures of MC38 cells in the model group. (3) In mechanism study, by using the specific inhibitors for galectin-3 (GB1107) andWNT (IWR) respectively, we proved that SEA inhibits the activation of the galectin-3-mediated ß-catenin activation. CONCLUSION: By highlighting the pro-metastatic effects of galectin-3-mediated tumor-platelet adhesion, our study provided indicative evidence for Spatholobi Caulis as the representative candidate for anti-metastatic therapy.

Discovery of Novel Hybrids of Edaravone and 6-Phenyl-4,5-dihydropyridazin-3(2H)-one with Antiplatelet Aggregation and Neuroprotection for Ischemic Stroke Treatment.

Drugs with anti-platelet aggregation and neuroprotection are of great significance for the treatment of ischemic stroke. A series of edaravone and 6-phenyl-4,5-dihydropyridazin-3(2H)-one hybrids were designed and synthesized. Among them, 6g showed the most effective cytoprotective effect against oxygen-glucose deprivation/reoxygenation-induced damage in BV2 cells and an excellent inhibitory effect on platelet aggregation induced by adenosine diphosphate and arachidonic acid. Additionally, 6g could prevent thrombosis caused by ferric chloride in rats and pose a lower risk of causing bleeding compared with aspirin. It provides better protection against ischemia/reperfusion injury in rats compared with edaravone and alleviates the oxidative stress related to cerebral ischemia/reperfusion by increasing the GSH and SOD levels and decreasing the MDA concentration. Finally, molecular docking results showed that 6g probably acts on PDE3 A and plays an anti-platelet aggregation effect. Overall, 6g could be a potential candidate compound for the treatment of ischemic stroke.

The effect of oscillation depolymerization on ethylenediaminetetraacetic acid-dependent platelet aggregation samples: A cross-over study.

INTRODUCTION: Ethylenediaminetetraacetic acid (EDTA)-dependent platelet aggregation (PA) can cause medical errors. Currently, there is no reliable method for completely solving this problem. This study aims to solve this problem that has plagued clinical practice for many years by using oscillation method. METHODS: Sixty-one EDTA-PA samples were collected, divided, and disaggregated using the oscillation method at various times and speeds. The samples were analyzed using routine blood tests and blood smears. RESULTS: Platelet counts (PLT) were increased significantly after oscillation. PLT in the 3000 rpm for 0.5 min group was significantly higher than that in the 500 rpm for 0.5 min group (p < 0. 01). After 3000 rpm oscillation, the PLT gradually increased with time, while compared with the 10-min group, the PLT in the 13-min group showed no significant differences. The effective disaggregation rates in the EDTA-PA samples using the oscillation method and sodium citrate anticoagulant were 96.72% and 65.57%, respectively. There were no significant changes in white blood cell (WBC) or red blood cell (RBC) counts or morphology after the use of the oscillation method. CONCLUSION: The oscillation method effectively depolymerized EDTA-PA without adverse effects on WBC and RBC. The implementation of this technique promises to resolve the issue of EDTA-PA.

Use of clinical biological tests of haemostasis to evaluate topical haemostatics.

INTRODUCTION: In addition to traditional means, topical haemostatics are currently used to avoid haemorrhage during surgery. Although they have been reported to be effective, there is a low level of proof of their clinical efficacy, which is at odds with their levels of use. This study used two methods to better understand their in vitro mechanism of action. METHODS: Two clinical biology assays were used to measure the action of topical haemostatics on primary and secondary haemostasis. Calibrated samples of collagen sponges and polypropylene non-woven gauze were tested. Platelet aggregation was assessed using a multichannel aggregometer. A thrombin generation assay (TGA) was used with a fluorogenic readout. Tissue factor solutions were used to activate coagulation. RESULTS: In terms of primary haemostasis, collagen sponges stimulated platelet aggregation, in particular between 2 and 5 min after incubation with platelet-rich plasma and with no dose effect. In regard to coagulation, the kinetics of thrombin generation was enhanced. Polypropylene non-woven gauze did not exhibit any effect on platelet aggregation, although it did have a weak effect on the kinetics of thrombin generation. CONCLUSION: Collagen is well known to exert a haemostatic effect due to its action on platelet aggregation. By contrast, polypropylene non-woven gauze has not been shown to have any effect on platelet aggregation other than a minor impact on thrombin generation. The results obtained with the devices tested are in agreement with the literature. Platelet aggregation biological assays and TGA measurements appear to be suitable for evaluation of these medical products.

Conformational activation and inhibition of von Willebrand factor by targeting its autoinhibitory module.

ABSTRACT: Activation of von Willebrand factor (VWF) is a tightly controlled process governed primarily by local elements around its A1 domain. Recent studies suggest that the O-glycosylated sequences flanking the A1 domain constitute a discontinuous and force-sensitive autoinhibitory module (AIM), although its extent and conformation remains controversial. Here, we used a targeted screening strategy to identify 2 groups of nanobodies. One group, represented by clone 6D12, is conformation insensitive and binds the N-terminal AIM (NAIM) sequence that is distal from A1; 6D12 activates human VWF and induces aggregation of platelet-rich plasma at submicromolar concentrations. The other group, represented by clones Nd4 and Nd6, is conformation sensitive and targets the C-terminal AIM (CAIM). Nd4 and Nd6 inhibit ristocetin-induced platelet aggregation and reduce VWF-mediated platelet adhesion under flow. A crystal structure of Nd6 in complex with AIM-A1 shows a novel conformation of both CAIM and NAIM that are primed to interact, providing a model of steric hindrance stabilized by the AIM as the mechanism for regulating GPIbα binding to VWF. Hydrogen-deuterium exchange mass spectrometry analysis shows that binding of 6D12 induces the exposure of the GPIbα-binding site in the A1 domain, but binding of inhibitory nanobodies reduces it. Overall, these results suggest that the distal portion of NAIM is involved in specific interactions with CAIM, and binding of nanobodies to the AIM could either disrupt its conformation to activate VWF or stabilize its conformation to upkeep VWF autoinhibition. These reported nanobodies could facilitate future studies of VWF functions and related pathologies.

Exacerbation of Thrombotic Responses to Silver Nanoparticles in Hypertensive Mouse Model.

With advent of nanotechnology, silver nanoparticles, AgNPs owing majorly to their antibacterial properties, are used widely in food industry and biomedical applications implying human exposure by various routes including inhalation. Several reports have suggested AgNPs induced pathophysiological effects in a cardiovascular system. However, cardiovascular diseases such as hypertension may interfere with AgNPs-induced response, yet majority of them are understudied. The aim of this work was to evaluate the thrombotic complications in response to polyethylene glycol- (PEG-) coated AgNPs using an experimental hypertensive (HT) mouse model. Saline (control) or PEG-AgNPs (0.5 mg/kg) were intratracheally (i.t.) instilled four times, i.e., on days 7, 14, 21, and 28 post-angiotensin II-induced HT, or vehicle (saline) infusion. On day 29, various parameters were assessed including thrombosis in pial arterioles and venules, platelet aggregation in whole blood in vitro, plasma markers of coagulation, and fibrinolysis and systemic oxidative stress. Pulmonary exposure to PEG-AgNPs in HT mice induced an aggravation of in vivo thrombosis in pial arterioles and venules compared to normotensive (NT) mice exposed to PEG-AgNPs or HT mice given saline. The prothrombin time, activated partial thromboplastin time, and platelet aggregation in vitro were exacerbated after exposure to PEG-AgNPs in HT mice compared with either NT mice exposed to nanoparticles or HT mice exposed to saline. Elevated concentrations of fibrinogen, plasminogen activator inhibitor-1, and von Willebrand factor were seen after the exposure to PEG-AgNPs in HT mice compared with either PEG-AgNPs exposed NT mice or HT mice given with saline. Likewise, the plasma levels of superoxide dismutase and nitric oxide were augmented by PEG-AgNPs in HT mice compared with either NT mice exposed to nanoparticles or HT mice exposed to saline. Collectively, these results demonstrate that PEG-AgNPs can potentially exacerbate the in vivo and in vitro procoagulatory and oxidative stress effect in HT mice and suggest that population with hypertension are at higher risk of the toxicity of PEG-AgNPs.

Diindolylmethane ameliorates platelet aggregation and thrombosis: In silico, in vitro, and in vivo studies.

Diindolylmethane (DIM), a major metabolite of indole-3-carbinol (I3C), plays a vital role in the pharmacological actions of I3C. The role of DIM in the inhibition of platelet aggregation and thrombus generation is yet to be revealed. However, how DIM and I3C modulate the interaction of platelets with the glycoproteinVI (GPVI) and purinergic receptor Y12 (P2Y12) receptors is unknown. In silico studies revealed that the indole group of DIM and indole and the hydroxyl group of I3C are responsible for modulating platelet interaction with GPVI and P2Y12 receptors. In silico studies further predicted that DIM more superiorly modulates platelet interaction with GPVI and P2Y12 receptors than I3C. In vitro studies identified that DIM significantly inhibited platelet aggregation induced by adenosine diphosphate (ADP), collagen, thrombin, and arachidonic acid, increasing the thrombin-induced clot retraction size and clot retraction weight. Moreover, in vivo results of ferric chloride (FeCl3) induced carotid artery thrombus generation indicate that DIM significantly reduced the reactive oxygen species (ROS), hydrogen peroxide (H2O2), thromboxane 2 (TXB2), cyclooxygenase 1 (COX-1), prostaglandin E2 (PGE2), thrombus weight, increased the cyclic adenosine monophosphate (cAMP), and extended the time to occlusion (TTO). Furthermore, DIM did not show thrombolytic activity. Therefore, DIM acts as an antiplatelet aggregation and antithrombotic agent. Moreover, DIM is responsible for the antiplatelet aggregation and antithrombotic activity of I3C. Therefore, DIM could be used to treat thrombotic diseases.

Flavonoids: Antiplatelet Effect as Inhibitors of COX-1.

Flavonoids are compounds with a benzopyranic structure that exhibits multiple pharmacological activities. They are known for their venotonic activity, but their mechanism of action remains unclear. It is thought that, as this mechanism is mediated by prostaglandins, these compounds may interfere with the arachidonic acid (AA) cascade. These assays are designed to measure the antiplatelet aggregation capacity of quercetin, rutin, diosmetin, diosmin, and hidrosmin, as well as to evaluate a potential structure-activity ratio. In this paper, several studies on platelet aggregation at different concentrations (from 0.33 mM to 1.5 mM) of different flavone compounds are conducted, measuring platelet aggregation by impedance aggregometry, and the cyclooxygenase (COX) activity by metabolites generated, including the activity of the pure recombinant enzyme in the presence of these polyphenols. The results obtained showed that quercetin and diosmetin aglycones have a greater antiplatelet effect and inhibit the COX enzyme activity to a greater extent than their heterosides; however, the fact that greater inhibition of the pure recombinant enzyme was achieved by heterosides suggests that these compounds may have difficulty in crossing biological membranes. In any case, in view of the results obtained, it can be concluded that flavonoids could be useful as coadjuvants in the treatment of cardiovascular pathologies.

Anti-Thrombotic Effects of Artesunate through Regulation of cAMP and PI3K/MAPK Pathway on Human Platelets.

Normal activation of platelets and their aggregation are crucial for proper hemostasis. It appears that excessive or abnormal aggregation of platelets may bring about cardiovascular diseases such as stroke, atherosclerosis, and thrombosis. For this reason, finding a substance that can regulate platelet aggregation or suppress aggregation will aid in the prevention and treatment of cardiovascular diseases. Artesunate is a compound extracted from the plant roots of Artemisia or Scopolia, and its effects have shown to be promising in areas of anticancer and Alzheimer's disease. However, the role and mechanisms by which artesunate affects the aggregation of platelets and the formation of a thrombus are currently not understood. This study examines the ways artesunate affects the aggregation of platelets and the formation of a thrombus on platelets induced by U46619. As a result, cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) production were increased significantly by artesunate relative to the doses, as well as phosphorylated vasodilator-stimulated phosphoprotein (VASP) and inositol 1,4,5-trisphosphate receptor (IP3R), substrates to cAMP-dependent kinase and cGMP-dependent kinase, in a significant manner. The Ca2+, normally mobilized from the dense tubular system, was inhibited due to IP3R phosphorylation from artesunate, and phosphorylated VASP aided in inhibiting platelet activity via αIIb/ß3 platelet membrane inactivation and inhibiting fibrinogen binding. In addition, MAPK and PI3K/Akt phosphorylation was inhibited via artesunate in a significant manner, causing the production of TXA2 and intracellular granular secretion (serotonin and ATP release) to be reduced. Therefore, we suggest that artesunate has value as a substance that inhibits platelet aggregation and thrombus formation through an antiplatelet mechanism.