Neutrophil Cathepsin G Enhances Thrombogenicity of Mildly Injured Arteries via ADP-Mediated Platelet Sensitization

Inhalation of particulate matter in polluted air causes direct, size-restricted passage in the circulation and pronounced lung inflammation, provoking platelet activation and (non)-fatal cardiovascular complications. To determine potency and mechanism of platelet sensitization via neutrophil enzymes, we performed in vitro aggregation studies in washed human platelets and in murine and human blood, in the presence of elastase, cathepsin G and regular platelet agonists, present in damaged arteries. The impact of both enzymes on in vivo thrombogenicity was studied in the same thrombosis mouse model, previously having demonstrated that neutrophil activation enhances peripheral thrombogenicity. At 0.05 U/mL, cathepsin G activated washed human platelets via PAR1, whereas at 0.35 U/mL, aggregation occurred via PAR4. In Swiss mouse platelet-rich plasma no aggregation occurred by cathepsin G at 0.4 U/mL. In human and murine blood, aggregations by 0.05–0.1 U/mL cathepsin G were similar and not PAR-mediated, but platelet aggregation was inhibited by ADP antagonists, advocating cathepsin G-released ADP in blood as the true agonist of sustained platelet activation. In the mouse thrombosis model, cathepsin G and elastase amplified mild thrombogenicity at blood concentrations that activated platelets in vitro. This study shows that cathepsin G and elastase secreted in the circulation during mild air pollution-induced lung inflammation lyse red blood cell membrane proteins, leading to ADP-leakage into plasma, sensitizing platelets and amplifying their contribution to cardiovascular complications of ambient particle inhalation.

Exploring the Mechanism of Edaravone for Oxidative Stress in Rats with Cerebral Infarction Based on Quantitative Proteomics Technology

Objective. To explore the mechanism of edaravone in the treatment of oxidative stress in rats with cerebral infarction based on quantitative proteomics technology. Method. The modified Zea Longa intracavitary suture blocking method was utilized to make rat CI model. After modeling, the rat was intragastrically given edaravone for 7 days, once a day. After the 7-day intervention, the total proteins of serum were extracted. After proteomics analysis, the differentially expressed proteins are analyzed by bioinformatics. Then chemoinformatics methods were used to explore the biomolecular network of edaravone intervention in CI. Result. The neurological scores and pathological changes of rats were improved after the intervention of edaravone. Proteomics analysis showed that in the model/sham operation group, 90 proteins in comparison group were upregulated, and 26 proteins were downregulated. In the edaravone/model group, 21 proteins were upregulated, and 41 proteins were downregulated. Bioinformatics analysis and chemoinformatics analysis also show that edaravone is related to platelet activation and aggregation, oxidative stress, intercellular adhesion, glycolysis and gluconeogenesis, iron metabolism, hypoxia, inflammatory chemokines, their mediated signal transduction, and so on. Conclusion. The therapeutic mechanism of edaravone in the treatment of CI may involve platelet activation and aggregation, oxidative stress, intercellular adhesion, glycolysis and gluconeogenesis, iron metabolism, hypoxia, and so on. This study revealed the serum protein profile of edaravone in the treatment of cerebral infarction rats through serum TMT proteomics and discovered the relevant mechanism of edaravone regulating iron metabolism in cerebral infarction, which provides new ideas for the study of edaravone intervention in cerebral infarction and also provides reference information for future research on the mechanism of edaravone intervention in iron metabolism-related diseases.

The Changes of Blood Platelet Reactivity in the Presence of Crude Extracts Isolated from Leaves and Twigs of Elaeagnus Rhamnoides (L.) A. Nelson Measuring 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). The aim of our study was also analyze of blood platelet proteoms in the presence of different sea buckthorn extracts. A significant new finding is a decrease surface expression of P-selectin on blood platelets stimulated by 10 µM ADP and 10 µg/mL collagen, and a decrease surface expression 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.

Temporal Roles of Platelet and Coagulation Pathways in Collagen- and Tissue Factor-Induced Thrombus Formation

In hemostasis and thrombosis, the complex process of thrombus formation involves different molecular pathways of platelet and coagulation activation. These pathways are considered as operating together at the same time, but this has not been investigated. The objective of our study was to elucidate the time-dependency of key pathways of thrombus and clot formation, initiated by collagen and tissue factor surfaces, where coagulation is triggered via the extrinsic route. Therefore, we adapted a microfluidics whole-blood assay with the Maastricht flow chamber to acutely block molecular pathways by pharmacological intervention at desired time points. Application of the technique revealed crucial roles of glycoprotein VI (GPVI)-induced platelet signaling via Syk kinase as well as factor VIIa-induced thrombin generation, which were confined to the first minutes of thrombus buildup. A novel anti-GPVI Fab EMF-1 was used for this purpose. In addition, platelet activation with the protease-activating receptors 1/4 (PAR1/4) and integrin αIIbβ3 appeared to be prolongedly active and extended to later stages of thrombus and clot formation. This work thereby revealed a more persistent contribution of thrombin receptor-induced platelet activation than of collagen receptor-induced platelet activation to the thrombotic process.

Isolation of formononetin-7-O-β-D-glucopyranoside from the grass of Ononis arvensis L. and the assessment of its effect on induced platelet activation

Introduction. Analysis of the clinical and laboratory picture of the SARS-CoV-2 infection suggests the presence of microcirculation and oxygen transport disorders, hemolysis of erythrocytes, intra-alveolar fibrin formation and microthrombus formation in the patient’s pathogenesis. Accordingly, the search for potential anticoagulants, erythrocyte antiplatelet agents, membrane stabilizing drugs and mild thrombolytic drugs can prevent the development of life-threatening complications and reduce the mortality of COVID-19 patients.Aim. Isolation of formononetin-7-O-β-D-glucopyranoside from the grass of Ononis arvensis L. and identification of the molecular mechanisms of its effect on platelet activation in vitro, induced by TRAP-6 (Thrombin receptor activated peptide) and ADP (adenosine diphosphate).Materials and methods. Terrestrial parts of Ononis arvensis L. were collected in the SPCPU nursery of medicinal plants (Leningrad region, Vsevolozhsky district, Priozerskoe highway, 38 km). Isolation of formononetin-7-O-β-D-glucopyranoside was carried out by preparative high performance liquid chromatography on a Smartline device (Knauer, Germany) equipped with a spectrophotometric detector. The structure of formononetin-7-O-β-D-glucopyranoside was confirmed by one-dimensional and two-dimensional NMR spectroscopy (Bruker Avance III, 400 MHz, Germany), as well as high-resolution mass spectrometry (HR-ESI-MS) (Bruker Micromass Q-TOF, Germany). The study of the effect of formononetin- 7-O-β-D-glucopyranoside on induced platelet activation was carried out on human platelets isolated from the blood of healthy volunteers. To research the effect of formononetin-7-О-β-D-glucopyranoside on platelet aggregation flow cytofluorometry with Cyto-FLEX (Beckman-Coulter, USA) was used.Results and discussion. According to the method of fractionation and purification of the total extract of O. arvensis developed in previous studies, formononetin-7-O-β-D-glucopyranoside was isolated in an individual form for subsequent biological studies with a total yield of 30 % in comparison with its content in the original extract. In samples with formononetin-7-O-β-D-glucopyranoside and ADP, there is a pronounced inhibition of platelet activation – the percentage of active platelets ranges from 6.3–6.6 % at doses of formononetin-7-O-β-D-glucopyranoside 1 μM, 3 μM and 30 μM. The inhibitory effect of formononetin-7-O-β-D-glucopyranoside is not dose-dependent (p ≤ 0.05). In samples with formononetin-7-O-β-D-glucopyranoside and TRAP, there is also a pronounced inhibition of platelet activation. The percentage of active platelets is 8 % at 1 μM formononetin-7-O-β-D-glucopyranoside doses, 15 % at 3 μM doses, and 16 % at 30 μM doses.Conclusion. Administration of formononetin-7-O-β-D-glucopyranoside at doses of 1 μM, 3 μM, 30 μM strongly inhibits platelet activation induced by ADP and TRAP-6. For ADP, there is no dose-dependent effect, while for TRAP there is a weak dose-dependent effect, the greatest inhibition efficiency is achieved with the minimum investigated dose of 1 μM. In all cases, the results obtained are statistically significant.

Antiplatelet and Antithrombotic Effects of Isaridin E Isolated from the Marine-Derived Fungus via Downregulating the PI3K/Akt Signaling Pathway

Isaridin E, a cyclodepsipeptide isolated from the marine-derived fungus Amphichorda felina (syn. Beauveria felina) SYSU-MS7908, has been demonstrated to possess anti-inflammatory and insecticidal activities. Here, we first found that isaridin E concentration-dependently inhibited ADP-induced platelet aggregation, activation, and secretion in vitro, but did not affect collagen- or thrombin-induced platelet aggregation. Furthermore, isaridin E dose-dependently reduced thrombosis formation in an FeCl3-induced mouse carotid model without increasing the bleeding time. Mechanistically, isaridin E significantly decreased the ADP-mediated phosphorylation of PI3K and Akt. In conclusion, these results suggest that isaridin E exerts potent antithrombotic effects in vivo without increasing the risk of bleeding, which may be due to its important role in inhibiting ADP-induced platelet activation, secretion and aggregation via the PI3K/Akt pathways.

Neutrophil-Derived Extracellular Vesicles Activate Platelets after Pneumolysin Exposure

Pneumolysin (PLY) is a pore-forming toxin of Streptococcus pneumoniae that contributes substantially to the inflammatory processes underlying pneumococcal pneumonia and lung injury. Host responses against S. pneumoniae are regulated in part by neutrophils and platelets, both individually and in cooperative interaction. Previous studies have shown that PLY can target both neutrophils and platelets, however, the mechanisms by which PLY directly affects these cells and alters their interactions are not completely understood. In this study, we characterize the effects of PLY on neutrophils and platelets and explore the mechanisms by which PLY may induce neutrophil–platelet interactions. In vitro studies demonstrated that PLY causes the formation of neutrophil extracellular traps (NETs) and the release of extracellular vesicles (EVs) from both human and murine neutrophils. In vivo, neutrophil EV (nEV) levels were increased in mice infected with S. pneumoniae. In platelets, treatment with PLY induced the cell surface expression of P-selectin (CD62P) and binding to annexin V and caused a significant release of platelet EVs (pl-EVs). Moreover, PLY-induced nEVs but not NETs promoted platelet activation. The pretreatment of nEVs with proteinase K inhibited platelet activation, indicating that the surface proteins of nEVs play a role in this process. Our findings demonstrate that PLY activates neutrophils and platelets to release EVs and support an important role for neutrophil EVs in modulating platelet functions in pneumococcal infections.