Inhibition Of Platelet Activation Research Articles

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

Ethnopharmacological relevanceQilong 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 studyThis study aimed to investigate the protective effect and possible mechanism of QC on platelet function in MIRI rats. Materials and methodsThe 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. ResultsWe 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 β-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. ConclusionsQC improves MIRI-induced hemorheological disorders, which might be partly attributed to the inhibition of platelet activation via CD36-mediated platelet signaling pathways.

The bile acid receptor TGR5 inhibits platelet activation and thrombus formation

Liver failure and cirrhosis are characterized by abnormal hemostasis with aberrant platelet activation. In particular, the consequences of cholestatic liver disease and molecular mechanisms, including the role of bile acids leading to impaired platelet responses, are not well understood. Here, we demonstrate that bile acids inhibit human and murine platelet activation, adhesion and spreading, leading to reduced thrombus formation under flow conditions. We identified the G-protein coupled receptor TGR5 in platelets and provide support for its role as mediator of bile acid-induced impairment of platelet activation. In the liver, TGR5 couples to Gαs proteins, activates the adenylate cyclase to induce a transient cAMP rise and stimulates the MAPK signaling pathway to regulate cholangiocyte proliferation, hepatocyte survival and inflammation. In this report, we demonstrate that the genetic deficiency of TGR5 in mice led to enhanced platelet activation and thrombus formation, suggesting that TGR5 plays an important role in hemostasis. Mechanistically, platelet inhibition is achieved by TGR5 mediated PKA activation and modulation of AKT and ERK1/2 phosphorylation. Thus, this report provides evidence for the ability of TGR5 ligands to reduce platelet activation and identifies TGR5 agonism as a new target for the prevention of cardiovascular diseases.

418 The antiplatelet effects of EPA, an omega-3 fatty acid, are mediated by its 12-lipoxygenase metabolite, 12-HEPE

OBJECTIVES/GOALS: To determine whether cardioprotective effects observed in individuals taking dietary supplementation with eicosapentaenoic acid (EPA), an ω-3 polyunsaturated fatty acid, are realized by altering platelet function, and if these effects are mediated through the 12-lipoxygenase derived metabolite, 12-hydroxyeicosapentaenoic acid (12-HEPE). METHODS/STUDY POPULATION: Washed platelets or platelet rich plasma from healthy human donors were treated with EPA and 12-HEPE to assess their ability to inhibit platelet activation. Platelets were stimulated with agonists targeting different steps of the hemostatic response to vascular injury. Platelet aggregation, dense granule secretion, surface expression of integrin αIIbβ3 and P-selectin, and clot retraction were analyzed. To assess signaling through Gαs-GPCRs and protein kinase A activity, phosphorylation of vasodilator-stimulated phosphoprotein (VASP) was examined via western blot following treatment with EPA or 12-HEPE. RESULTS/ANTICIPATED RESULTS: EPA and 12-HEPE dose-dependently inhibit both collagen and thrombin-induced platelet aggregation. Furthermore, 12-HEPE more potently attenuates dense granule secretion and surface expression of platelet activation markers, integrin αIIbβ3 and P-selectin, in comparison to EPA. Plasma treated with EPA delayed thrombin-induced clot retraction, while 12-HEPE had no effect. Additionally, treatment with 12-HEPE increases phosphorylation of VASP, suggesting it could signal through the activation of the eicosanoid Gαs-GPCRs. DISCUSSION/SIGNIFICANCE: Here, we show for the first time that EPA directly inhibits platelet activation through its 12-LOX metabolite, 12-HEPE. These findings provide further insight into the mechanisms underlying the cardioprotective effects of EPA. A better understanding of current PUFA supplementations can inform treatment and prevention of cardiovascular diseases.

Nutrition and Cardiovascular Disease: The Potential Role of Marine Bioactive Proteins and Peptides in Thrombosis Prevention.

Thrombus and cardiovascular diseases pose a significant health threat, and dietary interventions have shown promising potential in reducing the incidence of these diseases. Marine bioactive proteins and peptides have been extensively studied for their antithrombotic properties. They can inhibit platelet activation and aggregation by binding to key receptors on the platelet surface. Additionally, they can competitively anchor to critical enzyme sites, leading to the inhibition of coagulation factors. Marine microorganisms also offer alternative sources for the development of novel fibrinolytic proteins, which can help dissolve blood clots. The advancements in technologies, such as targeted hydrolysis, specific purification, and encapsulation, have provided a solid foundation for the industrialization of bioactive peptides. These techniques enable precise control over the production and delivery of bioactive peptides, enhancing their efficacy and safety. However, it is important to note that further research and clinical studies are needed to fully understand the mechanisms of action and therapeutic potential of marine bioactive proteins and peptides in mitigating thrombotic events. The challenges and future application perspectives of these bioactive peptides also need to be explored.

Thrombin-induced platelet inhibition by 2′,4′-dihydroxychalcone and 2′,4′-dihydroxydihydrochalcone is mediated by inhibition of ROS production and Thromboxane synthase a activity: structure-activity relationship of polyphenol secondary metabolites from Empetrum nigrum

In this study, we expanded our previous work by testing compounds 1–12 for their ability to inhibit platelet activation at low (30 µM) concentration by inhibition of ROS production, thromboxane synthase (TxS) activity, and activation of cyclic nucleotide pathways. We also investigated whether some of these compounds could potentiate the effects of P2Y12 ADP receptor inhibitor action and discussed possible structure-activity relationships of the tested compounds. We showed that at this concentration only compounds 7 and 12 significantly inhibited thrombin-induced platelet activation which was accompanied by inhibition of ROS production and thromboxane synthase activity. Correspondingly, these compounds significantly potentiated the inhibitory effect of cangrelor on thrombin-induced platelet activation. In some other cases, inhibition of ROS production and thromboxane synthase activity did not correlate with platelet inhibition, indicating that these compounds could affect some, still unidentified, activatory pathways in platelets that counteract their inhibitory effects.

Elucidating the distinctive regulatory effects and mechanisms of active compounds in Salvia miltiorrhiza Bunge via network pharmacology: Unveiling their roles in the modulation of platelet activation and thrombus formation

Salvia miltiorrhiza Bunge. (DS), as an important traditional Chinese medicine (TCM), has a long history of usage for promoting blood circulation and removing blood stasis. Modern studies have shown that the chemical components of DS have many biological activities such as cardiovascular protection, anti-arrhythmia, anti-atherosclerosis, improvement of microcirculation, protection of myocardium, inhibition and removal of platelet aggregation. Nevertheless, the action mechanism of DS as well its active compounds on platelet activation has not been fully uncovered. This study aimed to find out the potential targets and mechanisms of DS in the modulation of platelet activation and thrombosis, using network pharmacology and biological experimental. These compounds with anti-thrombotic activity in DS, cryptotanshinone (CPT), isoeugenol (ISO) and tanshinone IIA (TSA), together with the corresponding targets being Src, Akt and RhoA are screened by network pharmacology. We confirmed that ISO, CPT and TSA dose-dependently inhibited platelet activation in vitro, mainly by inhibiting agonist-induced clot retraction, aggregation and P-selectin and ATP release. The western blot findings indicated that ISO, CPT, and TSA led to reduced levels of p-Akt and p-ERK in activated platelets. Additionally, ISO and TSA were observed to decrease p-cSrc expression while increasing RhoA expression. ISO, CPT, and TSA demonstrated a potential to restrict the advancement of carotid arterial thrombosis in vivo. We confirm that ISO, CPT and TSA are the key anti-thrombotic active compounds in DS. These active compounds exhibit unique inhibitory effects on platelet activation and thrombus formation by modulating the Akt/ERK and cSrc/RhoA signaling pathways.

Therapeutic effects and molecular mechanisms of natural products in thrombosis.

Thrombotic disorders, such as myocardial infarction and stroke, are the leading cause of death in the global population and have become a health problem worldwide. Drug therapy is one of the main antithrombotic strategies, but antithrombotic drugs are not completely safe, especially the risk of bleeding at therapeutic doses. Recently, natural products have received widespread interest due to their significant efficacy and high safety, and an increasing number of studies have demonstrated their antithrombotic activity. In this review, articles from databases, such as Web of Science, PubMed, and China National Knowledge Infrastructure, were filtered and the relevant information was extracted according to predefined criteria. As a result, more than 100 natural products with significant antithrombotic activity were identified, including flavonoids, phenylpropanoids, quinones, terpenoids, steroids, and alkaloids. These compounds exert antithrombotic effects by inhibiting platelet activation, suppressing the coagulation cascade, and promoting fibrinolysis. In addition, several natural products also inhibit thrombosis by regulating miRNA expression, anti-inflammatory, and other pathways. This review systematically summarizes the natural products with antithrombotic activity, including their therapeutic effects, mechanisms, and clinical applications, aiming to provide a reference for the development of new antithrombotic drugs.

Immune Thrombocytopenic Purpura and Intracranial Stenting.

Patients with immune thrombocytopenic purpura (ITP) presenting with indications for dual antiplatelet therapy (DAPT) can be difficult to manage due to the precarious balance of managing the need for increased platelet counts as well as inhibition of platelet activity. This case represents a 65year old woman with ITP who presented with a bilateral subarachnoid hemorrhage secondary to a left ophthalmic aneurysm that required placement of a pipeline embolization device (PED) necessitating DAPT. After treatment of her ITP with pulse dexamethasone for fourdays, she was safely discharged on onemonth of DAPT with aspirin and ticagrelor then switched to aspirin monotherapy without any immediate complications. During her period of DAPT, she did not receive additional medical treatment for her ITP. This case successfully presents a high-risk ITP patient requiring DAPT for a neurosurgical procedure and illustrates that these patients can be safely and successfully treated with DAPT once their ITP is stabilized.

Multi-Omics Approaches for Liver Reveal the Thromboprophylaxis Mechanism of Aspirin Eugenol Ester in Rat Thrombosis Model.

Aspirin eugenol ester (AEE) is a novel medicinal compound synthesized by esterifying aspirin with eugenol using the pro-drug principle. Pharmacological and pharmacodynamic experiments showed that AEE had excellent thromboprophylaxis and inhibition of platelet aggregation. This study aimed to investigate the effect of AEE on the liver of thrombosed rats to reveal its mechanism of thromboprophylaxis. Therefore, a multi-omics approach was used to analyze the liver. Transcriptome results showed 132 differentially expressed genes (DEGs) in the AEE group compared to the model group. Proteome results showed that 159 differentially expressed proteins (DEPs) were identified in the AEE group compared to the model group. Six proteins including fibrinogen alpha chain (Fga), fibrinogen gamma chain (Fgg), fibrinogen beta chain (Fgb), orosomucoid 1 (Orm1), hemopexin (Hpx), and kininogen-2 (Kng2) were selected for parallel reaction monitoring (PRM) analysis. The results showed that the expression of all six proteins was upregulated in the model group compared with the control group. In turn, AEE reversed the upregulation trend of these proteins to some degree. Metabolome results showed that 17 metabolites were upregulated and 38 were downregulated in the model group compared to the control group. AEE could reverse the expression of these metabolites to some degree and make them back to normal levels. The metabolites were mainly involved in metabolic pathways, including linoleic acid metabolism, arachidonic acid metabolism, and the tricarboxylic acid (TCA) cycle. Comprehensive analyses showed that AEE could prevent thrombosis by inhibiting platelet activation, decreasing inflammation, and regulating amino acid and energy metabolism. In conclusion, AEE can have a positive effect on thrombosis-related diseases.

Distinct mechanisms underlying the therapeutic effects of low-molecular-weight heparin and chondroitin sulfate on Parkinson's disease

Parkinson's disease (PD) is a neurodegenerative disorder influenced by various factors, including age, genetics, and the environment. Current treatments provide symptomatic relief without impeding disease progression. Previous studies have demonstrated the therapeutic potential of exogenous heparin and chondroitin sulfate in PD. However, their therapeutic mechanisms and structure-activity relationships remain poorly understood. In this study, low-molecular-weight heparin (L-HP) and chondroitin sulfate (L-CS) exhibited favorable therapeutic effects in a mouse model of PD. Proteomics revealed that L-HP attenuated mitochondrial dysfunction through its antioxidant properties, whereas L-CS suppressed neuroinflammation by inhibiting platelet activation. Two glycosaminoglycan (GAG)-binding proteins, manganese superoxide dismutase (MnSOD2) and fibrinogen beta chain (FGB), were identified as potential targets of L-HP and L-CS, and we investigated their structure-activity relationships. The IdoA2S-GlcNS6S/GlcNAc6S unit in HP bound to SOD2, whereas the GlcA-GalNAc4S and GlcA-GalNAc4S6S units in CS preferred FGB. Furthermore, N-S and 2-O-S in L-HP, and 4-O-S, 6-O-S, and -COOH in L-CS contributed significantly to the binding process. These findings provide new insights and evidence for the development and use of glycosaminoglycan-based therapeutics for PD.

Silencing of FUN14 Domain Containing 1 Inhibits Platelet Activation in Diabetes Mellitus through Blocking Mitophagy.

Platelet hyperactivity represents a deleterious physiological phenomenon in diabetes mellitus (DM). This study aimed to explore the role of FUN14 domain containing 1 (FUNDC1) in platelet activation within the context of DM and to uncover relevant mechanisms, with a focus on mitophagy. A mouse model of DM was established by high-fat feeding and streptozotocin injection. Platelets isolated from whole blood were exposed to carbonyl cyanide-4-(trifluo-romethoxy)phenylhydrazone (FCCP) to induce mitophagy. The relative mRNA expression of FUNDC1 was detected by quantitative real-time PCR (qRT-PCR). Western blotting was employed to measure the protein levels of FUNDC1, the ratio of LC3-II toLC3-I, and cleaved caspase-3. Immunofluorescence and flow cytometry were performed to assess LC3-positive mitochondria and platelet activation factor CD62P, respectively. Additionally, serum levels of β-thrombo-globulin (β-TG) and platelet factor 4 (PF4)were measured by enzyme-linked immunosorbent assay. FUNDC1 expression was elevated in DM mice, and its silencing decreased the body weight and fasting blood glucose. Inhibition of FUNDC1 also significantly attenuated FCCP-induced platelet mitophagy, as evidenced by the down-regulation of the LC3-II/LC3-I ratio, up-regulation of Tomm20, and diminished presence of LC3-positive mitochondria. Moreover, platelet activation was noted in DM mice; this activation was mitigated upon FUNDC1 silencing, which was confirmed by the down-regulation of cleaved caspase-3 and CD62P as well as reductions in β-TG and PF4 serum levels. Silencing of FUNDC1 inhibited platelet hyperactivity in DM by impeding mitophagy. As such, FUNDC1-midiated mitophagy may be a promising target for the treatment of DM and its associated cardiovascular complications related cardiovascular events.

Relieving thrombo-inflammation with acid-triggered polymersomes toward ischemic stroke therapy

In acute ischemic stroke, neutrophils contribute to ischemia-reperfusion injury through producing neutrophil extracellular traps (NETs), which promote the interaction of platelets and neutrophils to elicit thrombo-inflammation and facilitate further infarct development. As the primary degrader of NETs, DNase 1 is restricted by easy deactivation and low efficiency to enrich in the ischemic brain. Herein, a pH-triggered polymersome nanoplatform is fabricated for enhanced brain delivery of DNase 1 to relieve ischemic stroke-related thrombo-inflammation. The polymersomes can efficiently disintegrate upon acidic microenvironment, releasing DNase 1 to clear NETs, which disrupt the thrombus skeleton structure to inhibit microthrombosis. Furthermore, DNase 1 can decrease the stability of histones in NETs to prevent platelet activation through TLR4 pathway, which in turn downregulate HMGB-1 to avoid NETs formation. As a result, the platelets and neutrophils aggregates can be effectively reduced simultaneously through NETs decomposition and inhibition of platelet activation. The acid-triggered polymersomes may present a promising strategy for ischemic stroke therapy.

Toll-like receptor expression and functional behavior in platelets from patients with systemic lupus erythematosus

BackgroundMultiple blood cell abnormalities participate in the development of inflammation in systemic lupus erythematosus (SLE). Although platelets have been suggested as one of these contributors through the release of their content during activation, there are limited specific data about their role as immune players in SLE. Materials and MethodsThirteen SLE patients were included. Flow cytometry was used to measure Toll-like receptors (TLR) 2, 4, and 9 in resting platelets, platelet-activation markers (PAC-1 binding, P-selectin, CD63, and CD40 ligand -L) and platelet-leukocyte aggregates before and after specific TLR stimulation. Soluble CD40L and von Willebrand factor (vWf) release from stimulated platelets was measured using ELISA. ResultsIn resting conditions, SLE platelets showed normal expression levels of TLR 2, 4 and 9. Platelet surface activation markers, soluble CD40L, and vWf release were normal at baseline and after TLR stimulation. Platelet-monocyte aggregates were elevated in resting conditions in SLE samples and showed only a marginal increase after TLR stimulation, while baseline and stimulated platelet-neutrophil and platelet-lymphocyte aggregates were normal. C-reactive protein levels positively correlated with platelet-monocyte aggregates both at baseline and after stimulation with the TLR-2 agonist PAM3CSK4, suggesting these complexes could reflect the inflammatory activity in SLE. In our cohort, 12 of 13 patients received treatment with hydroxychloroquine (HCQ), a known inhibitor of endosomal activity and a potential inhibitor of platelet activation. The fact that SLE platelets showed an adequate response to TLR agonists suggests that, despite this treatment, they retain the ability to respond to the increased levels of damage-associated molecular patterns (DAMPs), which represent known TLR ligands, present in the circulation of SLE patients. Interestingly, elevated plasma levels of high mobility group box 1 (HMGB1), a classical DAMP, correlated with vWf release from TLR-stimulated platelets, suggesting that HMGB1 may also be released by platelets, thereby creating a positive feedback loop for platelet activation that contributes to inflammation. ConclusionOur study demonstrates normal platelet TLR expression and function together with increased circulating platelet-monocyte aggregates. In addition, a direct correlation was observed between plasma HMGB1 levels and platelet vWf release following TLR2 stimulation. This platelet behavior in a group of patients undergoing HCQ treatment suggests that platelets could play a role in the inflammatory state of SLE.

Vibro-Acoustic Platelet Activation: An Additive Mechanism of Prothrombosis with Applicability to Snoring and Obstructive Sleep Apnea.

Introduction: Obstructive sleep apnea (OSA) and loud snoring are conditions with increased cardiovascular risk and notably an association with stroke. Central in stroke are thrombosis and thromboembolism, all related to and initiaing with platelet activation. Platelet activation in OSA has been felt to be driven by biochemical and inflammatory means, including intermittent catecholamine exposure and transient hypoxia. We hypothesized that snore-associated acoustic vibration (SAAV) is an activator of platelets that synergizes with catecholamines and hypoxia to further amplify platelet activation. Methods: Gel-filtered human platelets were exposed to snoring utilizing a designed vibro-acoustic exposure device, varying the time and intensity of exposure and frequency content. Platelet activation was assessed via thrombin generation using the Platelet Activity State assay and scanning electron microscopy. Comparative activation induced by epinephrine and hypoxia were assessed individually as well as additively with SAAV, as well as the inhibitory effect of aspirin. Results: We demonstrate that snore-associated acoustic vibration is an independent activator of platelets, which is dependent upon the dose of exposure, i.e., intensity x time. In snoring, acoustic vibrations associated with low-frequency sound content (200 Hz) are more activating than those associated with high frequencies (900 Hz) (53.05% vs. 22.08%, p = 0.001). Furthermore, SAAV is additive to both catecholamines and hypoxia-mediated activation, inducing synergistic activation. Finally, aspirin, a known inhibitor of platelet activation, has no significant effect in limiting SAAV platelet activation. Conclusion: Snore-associated acoustic vibration is a mechanical means of platelet activation, which may drive prothrombosis and thrombotic risk clinically observed in loud snoring and OSA.

Meso‐Aminomethyl‐BODIPY as a Scaffold for Nitric Oxide Photo‐Releasers

AbstractNitric oxide (NO) is a unique biochemical mediator involved in the regulation of vital processes. Its short lifetime in physiological conditions and local action impede direct application in medicine because highly targeted NO‐delivery systems are required. Light‐controllable NO releasers are promising for the development of smart therapies. Here we present simply prepared meso‐aminomethyl BODIPY (4,4‐difluoro‐4‐bora‐3a,4a‐diaza‐s‐indacene) derivatives containing N‐nitroso moiety, which show the photoinduced generation of NO in a solution. These compounds can additionally generate singlet oxygen, and NO/1O2 generation efficiency can be tuned by substituents. As an example of NO‐mediated effect, we demonstrate light‐dependent inhibition of platelet activation in vitro. The presented compounds could serve as the basis for the development of novel hybrid therapeutic methods.

Qihuang Zhuyu formula alleviates coronary microthrombosis by inhibiting PI3K/Akt/αIIbβ3-mediated platelet activation

BackgroundCoronary microembolism (CME) is commonly seen in the peri-procedural period of Percutaneous Coronary Intervention (PCI), where local platelet activation and endothelial cell inflammation crosstalk may lead to micro thrombus erosion and rupture, with serious consequences. Qihuang Zhuyu Formula (QHZYF) is a Chinese herbal compound with high efficacy against coronary artery disease, but its antiplatelet mechanism is unclear. Hypothesis/PurposeThis study aimed to elucidate the effects and mechanisms of QHZYF on sodium laurate-induced CME using network pharmacology and in vitro and in vivo experiments. MethodsWe employed high-performance liquid chromatography mass spectrometry to identify the main components of QHZYF. Network pharmacology analysis, molecular docking and surface plasmon resonance (SPR) were utilized to predict the primary active components, potential therapeutic targets, and intervention pathways mediating the effects of QHZYF on platelet activation. Next, we pretreated a sodium laurate-induced minimally invasive CME rat model with QHZYF. In vivo experiments were performed to examine cardiac function in rats, to locate coronary arteries on heart sections to observe internal microthrombi, to extract rat Platelet-rich plasma (PRP) for adhesion assays and CD62p and PAC-1 (ITGB3/ITGA2B) flow assays, and to measure platelet-associated protein expression in PRP. In vitro clot retraction and Co-culture of HUVECs with PRP were performed and the gene pathway was validated through flow cytometry and immunofluorescence. ResultsCombining UPLC-Q-TOF/MS technology and database mining, 78 compounds were finally screened as the putative and representative compounds of QHZYF, with 75 crossover genes associated with CME. QHZYF prevents CME mainly by regulating key pathways of the inflammation and platelets, including Lipid and atherosclerosis, Fluid shear stress, platelet activation, and PI3K-Akt signaling pathways. Five molecules including Calyson, Oroxin A, Protosappanin A,Kaempferol and Geniposide were screened and subjected to molecular docking and SPR validation in combination with Lipinski rules (Rule of 5, Ro5). In vivo experiments showed that QHZYF not only improved myocardial injury but also inhibited formation of coronary microthrombi. QHZYF inhibited platelet activation by downregulating expression of CD62p receptor and platelet membrane protein αIIbβ3 and reduced the release of von Willebrand Factor (vWF), Ca2+ particles and inflammatory factor IL-6. Further analysis revealed that QHZYF inhibited the activation of integrin αIIbβ3, via modulating the PI3K/Akt pathways. In in vitro experiments, QHZYF independently inhibited platelet clot retraction. Upon LPS induction, the activation of platelet membrane protein ITGB3 was inhibited via the PI3K/Akt pathway, revealing an important mechanism for attenuating coronary microthrombosis. We performed mechanistic validation using PI3K inhibitor LY294002 and Akt inhibitor MK-2206 to show that QHZYF inhibited platelet membrane protein activation and inflammation to improved coronary microvessel embolism by regulating PI3K/Akt/αIIbβ3 pathways, mainly by inhibiting PI3K and Akt phosphorylation. ConclusionQHZYF interferes with coronary microthrombosis through inhibition of platelet adhesion, activation and inflammatory crosstalk, thus has potential in clinical anti-platelet applications. Calyson, Oroxin A, Protosappanin A, Kaempferol and Geniposide may be the major active ingredient groups of QHZYF that alleviate coronary microthrombosis.

The improvement of endothelial function by inhibition of platelet activity using acetylsalicylic acid in patients with arterial hypertension

In accordance with modern ideas about the pathogenesis of thrombotic complications of cardiovascular diseases (myocardial infarction, stroke), it should be noted that platelets and platelet humoral factors play a key role in the development of thrombosis. Activated platelets are able to activate both endotheliocytes and pro-inflammatory cells - monocytes/macrophages, which take a direct part in the formation and progression of atherosclerotic plaque. The purpose of the study is to investigate the potential improvement of endothelial function through the inhibition of platelet activity using acetylsalicylic acid in patients with arterial hypertension and established atherosclerotic cardiovascular diseases. Materials and methods. We enrolled 41 patients with arterial hypertension and established atherosclerotic cardiovascular diseases in our study. The participants were divided into two groups. Group 1 comprised 20 patients who were already taking acetylsalicylic acid (ASA) before the study, while Group 2 consisted of 21 patients who had not received ASA before participating. During the 6-month study period, patients from both groups received ASA (75 mg once a day) as part of their basic therapy, which included antihypertensive and statin therapy. Platelet activity was assessed in all patients before the study and at the final stage by determining the expression of glycoproteins GPIIb-IIIa and P-selectin on their surface. Additionally, the content of endothelial progenitor cells (phenotype CD45-CD31+CD133+) and desquamated endothelial cells (phenotype CD45-CD31+CD133-) in the blood was analyzed using flow cytometry. ELISA was employed to measure the content of C-reactive protein, cytokines TNF-α and IL-10, as well as asymmetric dimethylarginine (ADMA) in the blood. Finally, all patients underwent a test with flow-dependent vasodilation of the brachial artery. Results. In patients who did not receive ASA before the study, there was a higher level of platelet activity in peripheral blood flow, along with signs of more pronounced endothelial dysfunction compared to those who received it. After 6 months of taking ASA alongside standard antihypertensive therapy, the activation level of circulating blood platelets decreased in both groups. Specifically, in patients of group 1, the expression level of CD41 (GPIIb) decreased by 31.8 % (p < 0.01), and CD61 (GPIIIa) decreased by 15.2 % (p < 0.01). In group 2 patients, the suppression of platelet activity was even more pronounced, with the expression level of CD41 (GPIIb) decreasing by 55.2 % (p < 0.001), and CD61 (GPIIIa) decreasing by 27.5 % (p < 0.05). Furthermore, in patients of group 1, the percentage of platelets carrying P-selectin on the surface decreased by 78.1 % (p < 0.01). In group 2, the number of such platelets also significantly decreased by 42.5 % (p < 0.05). The number of progenitor cells of endothelial cells in the circulating blood increased significantly in both groups, showing a 3-fold increase in patients of group 1 (p < 0.001) and a 2.3-fold increase in patients of group 2 ( p< 0.001). In patients of both groups, a significant 2-fold increase in the endothelium-dependent vasodilatation index was observed (p < 0.01). At the end of the study, there was a decrease in the blood level of CRP by 12.2 % and 18.8 %, and pro-inflammatory cytokine TNF-α decreased by 50% and 57 %, respectively, in patients of groups 1 and 2 (p < 0.001). Conclusion. The reduction in blood platelet activity triggered by ASA in patients with arterial hypertension and atherosclerotic cardiovascular diseases was associated with notable alterations in the intensity of systemic inflammation and the restoration of endothelial functions. These findings suggest a potential therapeutic role for ASA in modulating both platelet function and endothelial health in individuals with these conditions.

CS585 Demonstrates Favorable Selectivity and Sustained In Vivo Action in Preventing Platelet Activation and Thrombosis Compared to Existing IP Receptor Agonists

Anti-platelet therapeutics are common tools in the treatment of thrombotic diseases, decreasing morbidity and mortality resulting from cardiovascular complications. However, many patients remain at risk for a cardiovascular event while others experience bleeding due to hemostatic dysregulation. The need for target specificity and lasting stability presents a major challenge in the development of novel antithrombotic therapeutics. While increasing platelet cAMP levels through activation of the prostacyclin (IP) receptor is a viable therapeutic approach, the IP receptor is currently considered a challenging target due to the non-selectivity and instability of existing IP agonists. Our lab has developed a novel IV and orally available IP receptor agonist, CS585, with selectivity towards the IP receptor and sustained in vivo protection from injury-induced thrombosis without observed bleeding. CS585 demonstrates IP receptor-dependent activity, leading to activation of protein kinase A (PKA), a key player in inhibitory signaling. While indications of current IP receptor agonists are limited to pulmonary arterial hypertension and temporary relief of peripheral artery disease, CS585 presents a new approach to selectively regulate platelet activation and thrombosis. CS585 and other IP receptor agonists, iloprost and selexipag, were assessed by direct comparison to elucidate potential differences in selectivity and sustained action. Selectivity was determined using aggregation and VASP phosphorylation. The effects of CS585 are inhibited by pharmacological inhibition of the IP receptor, but not by inhibition of the DP1, EP2, or EP4 prostaglandin receptors. Meanwhile, the activities of iloprost and selexipag were affected by other prostaglandin inhibitors, supporting the improved selectivity toward activation of the IP receptor with CS585. Arterial thrombosis was measured and quantified in a laser-induced cremaster thrombosis assay in IP agonist-treated WT mice to assess platelet activation and thrombus formation in vivo in mouse models of thrombosis. In WT mice treated with CS585, we observed sustained effects of CS585 following both IV and oral administration. Platelets and fibrin were labeled and accumulation at the site of injury was measured using intravital microscopy. In contrast to the FDA-approved IP receptor agonists (iloprost and selexipag) which showed short-term protection from thrombosis, treatment with CS585 resulted in sustained inhibition of clot and fibrin formation at the site of injury in the same model between 24 and 48 hours post-administration. To determine if the anti-thrombotic effects of CS585 impact coagulation, coagulation parameters were assessed in whole blood using thromboelastography (TEG). Treatment with CS585 did not impact coagulation parameters in whole blood, while a significant increase in lysis was observed with iloprost. Here, we show for the first time a head-to-head comparison of CS585, a novel IP receptor agonist, with selexipag and iloprost. Our preclinical results with CS585 indicate a favorable profile for inhibiting platelet activation and clot formation and demonstrate a sustained duration of action in mice in the ability of CS585 to inhibit platelet activation through multiple routes of administration. Importantly, CS585 does not affect coagulation parameters and previous studies demonstrated no potential for increased bleeding in mouse models. Overall, CS585 provides a new option of activating the IP receptor to decrease platelet reactivity and could represent the first viable option for targeting the IP receptor on platelets for primary inhibition of thrombosis with a reduced risk of bleeding.

Statins Effects on Blood Clotting: A Review.

Statins are powerful lipid-lowering drugs that inhibit cholesterol biosynthesis via downregulation of hydroxymethylglutaryl coenzyme-A reductase, which are largely used in patients with or at risk of cardiovascular disease. Available data on thromboembolic disease include primary and secondary prevention as well as bleeding and mortality rates in statin users during anticoagulation for VTE. Experimental studies indicate that statins alter blood clotting at various levels. Statins produce anticoagulant effects via downregulation of tissue factor expression and enhanced endothelial thrombomodulin expression resulting in reduced thrombin generation. Statins impair fibrinogen cleavage and reduce thrombin generation. A reduction of factor V and factor XIII activation has been observed in patients treated with statins. It is postulated that the mechanisms involved are downregulation of factor V and activated factor V, modulation of the protein C pathway and alteration of the tissue factor pathway inhibitor. Clinical and experimental studies have shown that statins exert antiplatelet effects through early and delayed inhibition of platelet activation, adhesion and aggregation. It has been postulated that statin-induced anticoagulant effects can explain, at least partially, a reduction in primary and secondary VTE and death. Evidence supporting the use of statins for prevention of arterial thrombosis-related cardiovascular events is robust, but their role in VTE remains to be further elucidated. In this review, we present biological evidence and experimental data supporting the ability of statins to directly interfere with the clotting system.

Unraveling the Mechanism of Platelet Aggregation Suppression by Thioterpenoids: Molecular Docking and In Vivo Antiaggregant Activity.

Natural monoterpenes and their derivatives are widely considered the effective ingredients for the design and production of novel biologically active compounds. In this study, by using the molecular docking technique, we examined the effects of two series of "sulfide-sulfoxide-sulfone" thioterpenoids containing different (e.g., bornane and pinane) monoterpene skeletons on the platelet's aggregation. Our data revealed that all the synthesized compounds exhibit inhibitory activities on platelet aggregation. For example, compound 1 effectively inhibited platelet activation and demonstrated direct binding with CD61 integrin, a well-known platelet GPIIb-IIIa receptor on platelets. We further examined the antiaggregant activity of the most active compound, 1, in vivo and compared its activity with that of acetylsalicylic acid and an oral GPIIb-IIIa blocker, orbofiban. We found that compound 1 demonstrates antiaggregant activity in rats when administered per os and its activity was comparable with that of acetylsalicylic acid and orbofiban. Moreover, similarly, tirofiban, a well-known GPIIb-IIIa blocker, compound 1, effectively decreased the expression of P-selectin to the values similar to those of the intact platelets. Collectively, here, we show, for the first time, the potent antiaggregant activity of compound 1 both in vitro and in vivo due to its ability to bind with the GPIIb-IIIa receptor on platelets.