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.

Thrombocytopenia Independently Leads to Changes in Monocyte Immune Function.

BACKGROUND: While platelets have well-studied hemostatic functions, platelets are immune cells that circulate at the interface between the vascular wall and white blood cells. The physiological implications of these constant transient interactions are poorly understood. Activated platelets induce and amplify immune responses, but platelets may also maintain immune homeostasis in healthy conditions, including maintaining vascular integrity and T helper cell differentiation, meaning that platelets are central to both immune responses and immune quiescence. Clinical data have shown an association between low platelet counts (thrombocytopenia) and immune dysfunction in patients with sepsis and extracorporeal membrane oxygenation, further implicating platelets as more holistic immune regulators, but studies of platelet immune functions in nondisease contexts have had limited study. METHODS: We used in vivo models of thrombocytopenia and in vitro models of platelet and monocyte interactions, as well as RNA-seq and ATAC-seq (assay for transposase-accessible chromatin with sequencing), to mechanistically determine how resting platelet and monocyte interactions immune program monocytes. RESULTS: Circulating platelets and monocytes interact in a CD47-dependent manner to regulate monocyte metabolism, histone methylation, and gene expression. Resting platelet-monocyte interactions limit TLR (toll-like receptor) signaling responses in healthy conditions in an innate immune training-like manner. In both human patients with sepsis and mouse sepsis models, thrombocytopenia exacerbated monocyte immune dysfunction, including increased cytokine production. CONCLUSIONS: Thrombocytopenia immune programs monocytes in a manner that may lead to immune dysfunction in the context of sepsis. This is the first demonstration that sterile, endogenous cell interactions between resting platelets and monocytes regulate monocyte metabolism and pathogen responses, demonstrating platelets to be immune rheostats in both health and disease.

Perioperative Treatment with Rivaroxaban and Dabigatran on Changes of Coagulation and Platelet Activation Biomarkers following Left Atrial Appendage Closure.

Insufficient data exist regarding the investigation of the impact of novel oral anticoagulants (NOACs) on coagulation activation biomarkers in the context of left atrial appendage closure (LAAC) and device-related thrombosis (DRT). The study was designed to investigate the changes and presence of coagulation activation biomarkers between different antithrombotic strategies following LAAC. A total of 120 nonvalvular atrial fibrillation patients intolerant of long-term anticoagulants, who underwent successful WATCHMAN closure implantation, were enrolled (rivaroxaban, n = 82; dabigatran, n = 38). Blood samples were obtained from left atrium (LA) and left atrial appendage (LAA) during the operation and fasting blood samples on the same day of LAAC and 45 days after discharge. The biochemical indicators, thrombin-antithrombin complex (TAT), soluble P-selectin (sP-selectin), von Willebrand factor (vWF), and CD40 ligand (CD40L), were measured by enzyme-linked immunosorbent assay. The primary endpoints of this study were the efficacy and safety characteristics of different antithrombotic strategies, including DRT incidence, stroke or transient ischemic attack, systemic embolism, and clinical major and nonmajor bleeding complications during the follow-up of 180 days. The results revealed that TAT, vWF, sP-selectin, and CD40L levels in vein were significantly reduced by 2.4% (p = 0.043), 5.0% (p < 0.001), 8.7% (p < 0.001), and 2.5% (p = 0.043) from their baseline levels after rivaroxaban treatment. Conversely, no significant changes were detected in the dabigatran group. Furthermore, the plasma levels of platelet activation biomarkers (CD40L and sP-selectin) in both LA and LAA groups were significantly lower after anticoagulation with rivaroxaban, as compared to dabigatran treatment (CD40L: 554.62 ± 155.54 vs. 445.02 ± 130.04 for LA p = 0.0013, 578.51 ± 156.28 vs. 480.13 ± 164.37 for LAA p = 0.0052; sP-selectin: 2849.07 ± 846.69 vs. 2225.54 ± 799.96 for LA p = 0.0105, 2915.52 ± 1402.40 vs. 2203.41 ± 1061.67 for LAA p = 0.0022). Notably, the present study suggests that rivaroxaban may be more effective in the prevention of DRT for patients undergoing LAAC.

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.

Qihuang Zhuyu formula alleviates coronary microthrombosis by inhibiting PI3K/Akt/αIIbß3-mediated platelet activation.

BACKGROUND: Coronary 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/PURPOSE: This 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. METHODS: We 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. RESULTS: Combining 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 αIIbß3 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 αIIbß3, 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/αIIbß3 pathways, mainly by inhibiting PI3K and Akt phosphorylation. CONCLUSION: QHZYF 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.

Ayurvedic preparations of Raudra Rasa inhibit agonist-mediated platelet activation and restrict thrombogenicity without affecting cell viability.

Ayurveda is considered to be one of the most ancient forms of medicine still practiced. The Ayurvedic preparation Raudra Rasa and its derivatives have been widely employed against cancer since the 12th century, but the effect of these traditional formulations on platelet function and signaling has not previously been examined. Here we demonstrate that Raudra Rasa and its derivatives significantly reduce thrombin-induced integrin activation and granule secretion in platelets, as observed by reduced PAC-1 binding and P-selectin externalization, respectively. These formulations also inhibited thrombin-stimulated phosphatidylserine exposure, mitochondrial reactive oxygen species generation, and mitochondrial transmembrane potential in platelets. Consistent with the above, Raudra Rasa significantly reduced thrombin-induced tyrosine phosphorylation of the platelet proteins, as well as phosphorylation of the enzymes AKT and GSK-3ß. In summary, Raudra Rasa inhibits agonist-mediated platelet activation without affecting cell viability, suggesting it may have therapeutic potential as an anti-platelet/anti-thrombotic agent.

Alpha-ketoglutarate supplementation reduces inflammation and thrombosis in type 2 diabetes by suppressing leukocyte and platelet activation.

The interplay between platelets and leukocytes contributes to the pathogenesis of inflammation, thrombosis, and cardiovascular diseases (CVDs) in type 2 diabetes (T2D). Our recent studies described alpha-ketoglutarate (αKG), a Krebs cycle intermediate metabolite as an inhibitor to platelets and leukocytes activation by suppressing phosphorylated-Akt (pAkt) through augmentation of prolyl hydroxylase-2 (PHD2). Dietary supplementation with a pharmacological concentration of αKG significantly inhibited lung inflammation in mice with either SARS-CoV-2 infection or exposed to hypoxia treatment. We therefore investigated if αKG supplementation could suppress hyperactivation of these blood cells and reduce thromboinflammatory complications in T2D. Our study describes that dietary supplementation with αKG (8 mg/100 g body wt. daily) for 7 days significantly reduced the activation of platelets and leukocytes (neutrophils and monocytes), and accumulation of IL1ß, TNFα, and IL6 in peripheral blood of T2D mice. αKG also reduced the infiltration of platelets and leukocytes, and accumulation of inflammatory cytokines in lungs by suppressing pAkt and pP65 signaling. In a cross-sectional investigation, our study also described the elevated platelet-leukocyte aggregates and pro-inflammatory cytokines in circulation of T2D patients. T2D platelets and leukocytes showed an increased aggregation and thrombus formation in vitro. Interestingly, a pre-incubation of T2D blood samples with octyl αKG significantly suppressed the activation of these blood cells and ameliorated aggregate/thrombus formation in vitro. Thus, suggesting a potential therapeutic role of αKG against inflammation, thrombosis, and CVDs in T2D.

MAGT1 Deficiency Dysregulates Platelet Cation Homeostasis and Accelerates Arterial Thrombosis and Ischemic Stroke in Mice.

BACKGROUND: MAGT1 (magnesium transporter 1) is a subunit of the oligosaccharide protein complex with thiol-disulfide oxidoreductase activity, supporting the process of N-glycosylation. MAGT1 deficiency was detected in human patients with X-linked immunodeficiency with magnesium defect syndrome and congenital disorders of glycosylation, resulting in decreased cation responses in lymphocytes, thereby inhibiting the immune response against viral infections. Curative hematopoietic stem cell transplantation of patients with X-linked immunodeficiency with magnesium defect causes fatal bleeding and thrombotic complications. METHODS: We studied the role of MAGT1 deficiency in platelet function in relation to arterial thrombosis and hemostasis using several in vitro experimental settings and in vivo models of arterial thrombosis and transient middle cerebral artery occlusion model of ischemic stroke. RESULTS: MAGT1-deficient mice (Magt1-/y) displayed accelerated occlusive arterial thrombus formation in vivo, a shortened bleeding time, and profound brain damage upon focal cerebral ischemia. These defects resulted in increased calcium influx and enhanced second wave mediator release, which further reinforced platelet reactivity and aggregation responses. Supplementation of MgCl2 or pharmacological blockade of TRPC6 (transient receptor potential cation channel, subfamily C, member 6) channel, but not inhibition of store-operated calcium entry, normalized the aggregation responses of Magt1-/y platelets to the control level. GP (glycoprotein) VI activation of Magt1-/y platelets resulted in hyperphosphorylation of Syk (spleen tyrosine kinase), LAT (linker for activation of T cells), and PLC (phospholipase C) γ2, whereas the inhibitory loop regulated by PKC (protein kinase C) was impaired. A hyperaggregation response to the GPVI agonist was confirmed in human platelets isolated from a MAGT1-deficient (X-linked immunodeficiency with magnesium defect) patient. Haploinsufficiency of TRPC6 in Magt1-/y mice could normalize GPVI signaling, platelet aggregation, and thrombus formation in vivo. CONCLUSIONS: These results suggest that MAGT1 and TRPC6 are functionally linked. Therefore, deficiency or impaired functionality of MAGT1 could be a potential risk factor for arterial thrombosis and stroke.

The changes of blood platelet reactivity in the presence of Elaeagnus rhamnoides (L.) A. Nelson leaves and twig extract in whole blood.

Uncontrolled blood platelet activation is an important risk factor of cardiovascular disease (CVDs). Various studies on phenolic compounds indicate that they have a protective effect on the cardiovascular system through different mechanisms, including the reduction of blood platelet activation. One of the plants that is particularly rich in phenolic compounds is sea buckthorn (Elaeagnus rhamnoides (L.) A. Nelson). The aim of the present study in vitro was to determine the anti-platelet properties of crude extracts isolated from leaves and twigs of E. rhamnoides (L.) A. Nelson in whole blood using flow cytometric and total thrombus-formation analysis system (T-TAS). In addition, the aim of our study was also analyze of blood platelet proteomes in the presence of different sea buckthorn extracts. A significant new finding is a decrease surface exposition of P-selectin on blood platelets stimulated by 10 µM ADP and 10 µg/mL collagen, and a decrease surface exposition of GPIIb/IIIa active complex on non-activated platelets and platelets stimulated by 10 µM ADP and 10 µg/mL collagen in the presence of sea buckthorn leaf extract (especially at the concentration 50 µg/mL). The twig extract also displayed antiplatelet potential. However, this activity was higher in the leaf extract than in the twig extract in whole blood. In addition, our present findings clearly demonstrate that investigated plant extracts have anticoagulant properties (measured by T-TAS). Therefore, the two tested extracts may be promising candidates for the natural anti-platelet and anticoagulant supplements.

Platelet activation and ferroptosis mediated NETosis drives heme induced pulmonary thrombosis.

Cell-free heme (CFH) is a product of hemoglobin, myoglobin and hemoprotein degradation, which is a hallmark of pathologies associated with extensive hemolysis and tissue damage. CHF and iron collectively induce cytokine storm, lung injury, respiratory distress and infection susceptibility in the lungs suggesting their key role in the progression of lung disease pathology. We have previously demonstrated that heme-mediated reactive oxygen species (ROS) induces platelet activation and ferroptosis. However, interaction of ferroptotic platelets and neutrophils, the mechanism of action and associated complications remain unclear. In this study, we demonstrate that heme-induced P-selectin expression and Phosphatidylserine (PS) externalization in platelets via ASK-1-inflammasome axis increases platelet-neutrophil aggregates in circulation, resulting in Neutrophil extracellular traps (NET) formation in vitro and in vivo. Further, heme-induced platelet activation in mice increased platelet-neutrophil aggregates and accumulation of NETs in the lungs causing pulmonary damage. Thus, connecting CFH-mediated platelet activation to NETosis and pulmonary thrombosis. As lung infections induce acute respiratory stress, thrombosis and NETosis, we propose that heme -mediated platelet activation and ferroptosis might be crucial in such clinical manifestations. Further, considering the ability of redox modulators and ferroptosis inhibitors like FS-1, Lpx-1 and DFO to inhibit heme-induced ferroptotic platelets-mediated NETosis and pulmonary thrombosis. They could be potential adjuvant therapy to regulate respiratory distress-associated clinical complications.

Platelet Functional Testing Via High-Throughput Microtiter Plate-Based Assays.

Platelets play a critical role in hemostasis and thrombosis; therefore, in vitro assays that measure platelet reactivity are fundamental tools to gain insight into these physiologic processes, to diagnose platelet disorders, and to develop antithrombotic therapies. However, conventional platelet assays such as aggregometry, the clinical gold standard for assessing platelet function, are low throughput and require specialized equipment. Since platelets have a finite life span ex vivo, processes to miniaturize and multiplex assays allow a much broader overview of platelet function in significantly less time than conventional assays. Several groups have developed simplified, high-throughput approaches to quantify platelet activation with standard laboratory equipment to lower the barrier of entry to study platelet biology. This article describes a panel of optimized and validated high-throughput microplate assays to comprehensively assess platelet functionality, independently or in combination, to increase throughput and reduce costs. Specifically, following stimulation of platelets, a plate reader can be used to measure light transmission aggregation via absorbance; dense-granule secretion based on ATP-dependent luminescence generation; and cytosolic calcium levels with a cell-permeant, fluorescent Ca2+ -sensitive dye. Additionally, platelets are an easily accessible component of the blood that share signaling pathways with other cells, making them ideal for high-throughput drug screens. The highly adaptable and complementary assays presented in this article can be used to decipher the molecular mechanism underlying platelet activation or to identify novel inhibitors. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Microtiter plate-based light transmission aggregometry Basic Protocol 2: Measuring dense-granule secretion in high-throughput microplate assays Basic Protocol 3: Microtiter plate-based calcium mobilization Support Protocol: Platelet isolation and enumeration.

Mechanism of platelet activation and potential therapeutic effects of natural drugs.

BACKGROUND: Cardiovascular disease is one of the most concerning chronic diseases in the world. Many studies have shown that platelet overactivation is a very important factor in the occurrence and development of cardiovascular diseases. At present, the widely used antiplatelet drugs have some defects, such as drug resistance and adverse reactions. PURPOSE: The purpose of this article is to summarize the main mechanisms and pathways of platelet activation, the main targets of antiplatelet aggregation, and the antiplatelet aggregation components of natural drugs and their mechanisms of action to provide new research ideas for the development and application of antiplatelet drugs. STUDY DESIGN AND METHODS: In this review, we systematically searched the PubMed, Google Scholar, Web of Science, and CNKI databases and selected studies based on predefined eligibility criteria. We then assessed their quality and extracted data. RESULTS: ADP, AA, THR, AF, collagen, SDF-1α, and Ca2+ can induce platelet aggregation and trigger thrombosis. Natural drugs have a good inhibitory effect on platelet activation. More than 50 kinds of natural drugs and over 120 kinds of chemical compounds, including flavonoids, alkaloids, saponins, terpenoids, coumarins, and organic acids, have significantly inhibited platelet activation activity. The MAPK pathway, cGMP-PKG pathway, cAMP-PKA pathway, PI3K-AKT pathway, PTK pathway, PLC pathway, and AA pathway are the main mechanisms and pathways of platelet activation. CONCLUSION: Natural drugs and their active ingredients have shown good activity and application prospects in anti-platelet aggregation. We hope that this review provides new research ideas for the development and application of antiplatelet drugs.

Identification of linoleic acid as an antithrombotic component of Wenxin Keli via selective inhibition of p-selectin-mediated platelet activation.

Atrial fibrillation significantly increases the risk of thromboembolism and stroke. Wenxin Keli (WXKL) is a widely used Chinese patent medicine against arrhythmia but if it has antithrombotic activity is unknown. Since platelet activation is a critical factor in thrombosis and the key target for many antithrombotic drugs, this study aims to demonstrate the antithrombotic efficacy of WXKL. In vitro platelet activation experiments showed that WXKL significantly inhibited platelet adhesion and aggregation. The potential active monomers in WXKL were screened by in silico prediction and in vitro platelet aggregation/adhesion assays. From WXKL chemical fractions and more than 40 monomers, linoleic acid (LA) was identified as the strongest antiplatelet compound. Oral administration of WXKL (1.2 g/kg/day) and LA (50 mg/kg/day) for 7 days significantly improved FeCl3-induced carotid thrombus formation in ICR mice without prolonging bleeding time. Flow cytometry showed that both WXKL and LA inhibited the release of p-selectin after platelet activation. ELISA showed that WXKL and LA also inhibited the expression of 6-Keto-PGF1α in plasma of mice with thrombus, but had no obvious effect on the expression of TXB2. WXKL inhibited platelet activation by broadly inhibiting the phosphorylation of protein kinase B (Akt), mitogen-activated protein kinases (MAPKs) and phospholipase C (PLC) ß3. In contrast, LA only inhibited the phosphorylation of PLCß3. In conclusion, WXKL and its active component LA showed good antiplatelet and antithrombotic efficacy in vivo and in vitro. Mechanistically, the multicomponent Chinese medicine WXKL acts on multiple targets in the platelet activation pathway whereas its active monomer linoleic acid acts specifically on phospholipase C ß3.

Danshensu prevents thrombosis by inhibiting platelet activation via SIRT1/ROS/mtDNA pathways without increasing bleeding risk.

BACKGROUND: Coronary thrombosis and its correlated disorders are main healthcare problems globally. The therapeutic effects of current treatments involving antiplatelet drugs are not fully satisfactory. Danshensu (DSS) is an important monomer obtained from Salvia miltiorrhiza roots that have been widely employed for vascular diseases in medicinal practices. Nonetheless, the underlying mechanisms of DSS are not fully unraveled. PURPOSE: The objective of this study was to penetrate the antithrombotic and antiplatelet mechanisms of DSS. METHODS: Network pharmacology assay was used to forecast the cellular mechanisms of DSS for treating thrombosis. The work focused the impacts of DSS on platelet activation by analyzing aggregation and adhesion in vitro. Flow cytometry, western blotting, CM-H2DCFDA staining and mitochondrial function assays were performed to reveal the molecular mechanisms. The model of common carotid artery thrombus induced by ferric chloride was established. The wet weight of thrombus was measured, and the thrombosis was observed by hematoxylin and eosin (H&E) staining, in order to support the inhibitory effect of DSS on thrombosis. RESULTS: Data mining found the antithrombotic effect of DSS is related to platelet activation and the core target is silent information regulator 1 (SIRT1). We confirmed that DSS dose-dependently inhibited platelet activation in vitro. DSS was further demonstrated to induce the expression of SIRT1 and decreased reactive oxygen species (ROS) burden and thereby prevented mitochondrial dysfunction. Mitochondrial function tests further indicated that DSS prevented mitochondrial DNA (mtDNA) release, which induced activation of platelet in a dendritic cell specific intercellular-adhesion-molecule-3 grabbing non-integrin (DC-SIGN)-dependent manner. In carotid artery injury model induced by ferric chloride, DSS inhibited the development of carotid arterial thrombosis. More encouragingly, in tail bleeding time assay, DSS did not augment bleeding risk. CONCLUSION: These findings indicated that DSS effectively inhibited platelet activation by depressing the collection of ROS and the release of platelet mtDNA without arousing hemorrhage risk. DSS might represent a promising candidate drug for thrombosis and cardiovascular disease therapeutics.

Petasites japonicus extract exerts anti-malarial effects by inhibiting platelet activation.

BACKGROUND: New antimalarial agents are needed to combat emerging resistance to the currently available drugs. In the pathology of cerebral malaria, platelets play a central role by binding infected and uninfected red cells and the endothelium. Since Petasites japonicus extract was reported as an effective inhibitor of platelet activation, we examined the antimalarial activities of the P. japonicus extract. PURPOSE: This study aimed to evaluate the impact of P. japonicus extract prepared from whole plants on malarial infection. METHODS: The P. japonicus extract were characterized by high-performance liquid chromatography (HPLC) profiling. Antimalarial activity of the P. japonicus ethanolic extract was evaluated in vitro using chloroquine-sensitive (3D7) and chloroquine-resistant (Dd2) P. berghei strains. Also, the in vivo activity of the extract was evaluated in P. berghei-infected mice via oral administration followed by a four-day suppressive test to measure the hematological parameters. In addition, platelet activation signaling induced by the P. japonicus extract in P. berghei infection was evaluated. RESULTS: In HPLC study, catechin, rutin, liquiritin, 3,4-di-O-caffeoylquinic acid, 3,5-di-O-caffeoylquinic acid, and 4,5-di-O-caffeoylquinic acid were identified in P. japonicus extract. Exposure to the P. japonicus extract significantly inhibited both CQ-sensitive (3D7) and resistant (Dd2) strains of P. falciparum with IC50 values of 8.48 ± 1.70 and 7.83 ± 6.44 µg/ml, respectively. Administration of the P. japonicus extract also resulted in potent antimalarial activities in P. berghei-infected mice with no associated toxicity. The treatment also improved the hematologic parameters. In addition, the survived mice from P. berghei infection exhibited the inhibition of collagen-induced platelet aggregation by attenuated glycoprotein VI (GPVI) downstream signaling. CONCLUSION: P. japonicus extracts promote antimalarial effects both in vitro and in vivo. In addition, the effects appear to be induced by the inhibition of collagen-induced platelet activation related to attenuated GPVI downstream signaling. Further studies to identify and characterize the antimalarial compounds in P. japonicus will promote the development of new drugs.

Potential Role of Mitochondria as Modulators of Blood Platelet Activation and Reactivity in Diabetes and Effect of Metformin on Blood Platelet Bioenergetics and Platelet Activation.

Blood platelet dysfunctions are strongly involved in the development of the micro- and macrovascular complications in diabetes mellitus (DM). However, the molecular causes of abnormal platelet activation in DM remain unclear. Experimental data suggests that platelet mitochondria can regulate the prothrombotic phenotype of platelets, and changes in these organelles may influence platelet activation and modify platelet responses to stimulation. The present study evaluates the impact of DM on mitochondrial respiratory parameters and blood platelet activation/reactivity in a rat model of experimental diabetes following 1, 2.5 and 5 months of streptozotocin (STZ)-induced diabetes. Moreover, a mild inhibition of the mitochondrial respiratory chain with the use of metformin under in vitro and in vivo conditions was tested as a method to reduce platelet activation and reactivity. The platelets were studied with a combination of flow cytometry and advanced respirometry. Our results indicate that prolonged exposure of blood platelets to high concentrations of glucose, as in diabetes, can result in elevated blood platelet mitochondrial respiration; this may be an effect of cell adaptation to the high availability of energy substrates. However, as these alterations occur later than the changes in platelet activation/reactivity, they may not constitute the major reason for abnormal platelet functioning in DM. Moreover, metformin was not able to inhibit platelet activation and reactivity under in vitro conditions despite causing a decrease in mitochondrial respiration. This indicates that the beneficial effect of metformin on the coagulation system observed in vivo can be related to other mechanisms than via the inhibition of platelet activation.

Glaucocalyxin a protect liver function via inhibiting platelet over-activation during sepsis.

BACKGROUND: Rabdosia japonica (Burm. f.) var. glaucocalyx (Maxim.) is a perennial herb, and is traditionally used as folk medicine for treating inflammatory diseases and cancer. Gaucocalyxin A (GLA) is an ent­kaurane diterpenoid that is isolated from the aerial parts of R. japonica (Burm. f.) var. glaucocalyx (Maxim.). In a recent study, we found that GLA protects against acute liver dysfunction induced by Escherichia coli, which is likely related to its anti-inflammatory effects. However, the mechanism by which GLA protects liver injury during sepsis is unknown. AIM: To evaluate the anti-inflammatory function of GLA and its regulatory effect on platelet function. METHOD: An in vivo model of sepsis was established by inoculating mice with E. coli. Live function and platelet activation were evaluated through standard assays. The levels of pro-inflammatory factors were measured through ELISA and qRT-PCR. RESULTS: GLA alleviated liver dysfunction in the mouse model of sepsis. GLA-treated mice displayed lower complement activation and liver dysfunction after E. coli infection. GLA alleviated the decrease in peripheral platelet counts by inhibiting their clearance by Kupffer cells in liver. Furthermore, GLA inhibited platelet activation through the RIP1/RIP3/AKT pathway and downregulated C3aR expression on the platelets, thereby inhibiting liver injury and dysfunction due to excessive complement activation. CONCLUSION: GLA can inhibit platelet activation by reducing surface expression of C3aR, which protect the liver from injury induced by excessive complement activation. GLA is a novel therapeutic agent for controlling sepsis-related liver dysfunction.

Both G protein-coupled and immunoreceptor tyrosine-based activation motif receptors mediate venous thrombosis in mice.

Platelets are critical in hemostasis and a major contributor to arterial thrombosis (AT). (Pre)clinical studies suggest platelets also contribute to venous thrombosis (VT), but the mechanisms are largely unknown. We hypothesized that in VT, platelets use signaling machinery distinct from AT. Here we aimed to characterize the contributions of platelet G protein-coupled (GPCR) and immunoreceptor tyrosine-based activation motif (ITAM) receptor signaling to VT. Wild-type (WT) and transgenic mice were treated with inhibitors to selectively inhibit platelet-signaling pathways: ITAM-CLEC2 (Clec2mKO), glycoprotein VI (JAQ1 antibody), and Bruton's tyrosine kinase (ibrutinib); GPCR-cyclooxygenase 1 (aspirin); and P2Y12 (clopidogrel). VT was induced by inferior vena cava stenosis. Thrombin generation in platelet-rich plasma and whole-blood clot formation were studied ex vivo. Intravital microscopy was used to study platelet-leukocyte interactions after flow restriction. Thrombus weights were reduced in WT mice treated with high-dose aspirin + clopidogrel (dual antiplatelet therapy [DAPT]) but not in mice treated with either inhibitor alone or low-dose DAPT. Similarly, thrombus weights were reduced in mice with impaired ITAM signaling (Clec2mKO + JAQ1; WT + ibrutinib) but not in Clec2mKO or WT + JAQ1 mice. Both aspirin and clopidogrel, but not ibrutinib, protected mice from FeCl3-induced AT. Thrombin generation and clot formation were normal in blood from high-dose DAPT- or ibrutinib-treated mice; however, platelet adhesion and platelet-neutrophil aggregate formation at the vein wall were reduced in mice treated with high-dose DAPT or ibrutinib. In summary, VT initiation requires platelet activation via GPCRs and ITAM receptors. Strong inhibition of either signaling pathway reduces VT in mice.

Differential Expression of Platelet Activation Markers, CD62P and CD63, after Exposure to Breast Cancer Cells Treated with Kigelia Africana, Ximenia Caffra and Mimusops Zeyheri Seed Oils In Vitro.

Cancer patients, including breast cancer patients, live in a hypercoagulable state. Chemo- and hormone- therapy used in the treatment of breast cancer increases the risk of thrombosis. Due to differences in health care services between developed and developing countries, the survival rate of women with breast cancer in developing countries is low. Consequently, ethnomedicines are used and their efficacy as potential alternatives are being scientifically explored. The seed oils of Kigelia africana, Ximenia caffra and Mimusops zeyheri have anti-proliferative effects on hormone-dependent (MCF-7) and cytotoxic effects on hormone-independent (MDA-MB-231) breast cancer cells. In this study, we determined if these seed oils reduce the thrombogenic ability of breast cancer cells by measuring the platelet surface expression of the activation-specific antigens CD62P and CD63. MDA-MB-231 and MCF-7 cells were pretreated with the seed oils before being exposed to whole blood of human female volunteers. An increase in CD62P and CD63 expression following whole blood exposure to untreated breast cancer cells was observed. Treated MDA-MB-231 cells reduced CD62P and CD63 expression while treated MCF-7 cells increased CD62P and decreased CD63 expression. Kigelia africana, Ximenia caffra and Mimusops zeyheri seed oils are able to reduce the thrombogenic ability of MDA-MB-231 breast cancer cells.