DNAse-dependent, NET-independent pathway of thrombus formation in vivo.
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ABSTRACT:
SUBMITTER: Carminita E
PROVIDER: S-EPMC8285961 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The role of the collagen receptor glycoprotein VI (GPVI) in arteriolar thrombus formation was studied in FcRgamma-null mice (FcRgamma(-/-)) lacking platelet surface GPVI. Thrombi were induced with severe or mild FeCl(3) injury. Collagen exposure was significantly delayed and diminished in mild compared with severe FeCl(3) injury. Times to initial thrombus formation and vessel occlusion were delayed in FcRgamma(-/-) compared with wild-type mice after severe injury. Platelet accumulation in wild-type mice was decreased after mild compared with severe injury. However, there was little difference between platelet accumulation after severe or mild injury in FcRgamma(-/-). These data indicate a significant role for GPVI in FeCl(3)-induced thrombus formation. Pretreatment of wild-type mice with lepirudin further impaired mild FeCl(3)-induced thrombus formation, demonstrating a role for thrombin. Laser-induced thrombus formation in wild-type and FcRgamma(-/-) was comparable. Collagen exposure to circulating blood was undetectable after laser injury. Normalized for thrombus size, thrombus-associated tissue factor was 5-fold higher in laser-induced thrombi than in severe FeCl(3)-induced thrombi. Thus, platelet activation by thrombin appears to be more important after laser injury than platelet activation by GPVI-collagen. It may thus be important when considering targets for antithrombotic therapy to use multiple animal models with diverse pathways to thrombus formation.
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Project description:CANTOS reported reduced secondary atherothrombotic events in patients with residual inflammatory risk treated with the inhibitory anti-IL-1β antibody, Canakinumab. Yet, mechanisms that underlie this benefit remain elusive. Recent work has implicated formation of neutrophil extracellular traps (NETosis) in arterial thrombosis. Hence, the present study explored the potential link between IL-1β, NETs, and tissue factor (TF)-the key trigger of the coagulation cascade-in atherothrombosis. To this end, ST-elevation myocardial infarction (STEMI) patients from the Swiss multicenter trial SPUM-ACS were retrospectively and randomly selected based on their CRP levels. In particular, 33 patients with STEMI and high C-reactive protein (CRP) levels (≥ 10 mg/L) and, 33 with STEMI and low CRP levels (≤ 4 mg/L) were investigated. High CRP patients displayed elevated circulating IL-1β, NETosis, and NET-associated TF plasma levels compared with low CRP ones. Additionally, analysis of patients stratified by circulating IL-1β levels yielded similar results. Moreover, NETosis and NET-associated TF plasma levels correlated positively in the whole population. In addition to the above, translational research experiments provided mechanistic confirmation for the clinical data identifying IL-1β as the initial trigger for the release of the pro-coagulant, NET-associated TF. In conclusion, blunted TF presentation by activated neutrophils undergoing NETosis may provide a mechanistic explanation to reduced secondary atherothrombotic events as observed in canakinumab-treated patients in CANTOS.
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Project description:Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a cell surface glycoprotein receptor expressed on a range of blood cells, including platelets, and on vascular endothelial cells. PECAM-1 possesses adhesive and signaling properties, the latter being mediated by immunoreceptor tyrosine-based inhibitory motifs present on the cytoplasmic tail of the protein. Recent studies in vitro have demonstrated that PECAM-1 signaling inhibits the aggregation of platelets. In the present study we have used PECAM-1-deficient mice and radiation chimeras to investigate the function of this receptor in the regulation of thrombus formation. Using intravital microscopy and laser-induced injury to cremaster muscle arterioles, we show that thrombi formed in PECAM-1-deficient mice were larger, formed more rapidly than in control mice, and were more stable. Larger thrombi were also formed in control mice that received transplants of PECAM-1-deficient bone marrow, in comparison to mice that received control transplants. A ferric chloride model of thrombosis was used to investigate thrombus formation in carotid arteries. In PECAM-1-deficient mice the time to 75% vessel occlusion was significantly shorter than in control mice. These data provide evidence for the involvement of platelet PECAM-1 in the negative regulation of thrombus formation.
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Project description:NADPH oxidase (NOX) enzymes are involved in a various physiological and pathological processes such as platelet activation and inflammation. Interestingly, we found that the pan-NOX inhibitors VAS compounds (VAS2870 and its analog VAS3947) exerted a highly potent antiplatelet effect. Unlike VAS compounds, concurrent inhibition of NOX1, 2, and 4 by treatment with ML171, GSK2795039, and GKT136901/GKT137831 did not affect thrombin and U46619-induced platelet aggregation. These findings suggest that VAS compounds may inhibit platelet aggregation via a NOX-independent manner. Thus, we aimed to investigate the detailed antiplatelet mechanisms of VAS compounds. The data revealed that VAS compounds blocked various agonist-induced platelet aggregation, possibly via blocking PKC downstream signaling, including IKKβ and p38 MAPK, eventually reducing platelet granule release, calcium mobilization, and GPIIbIIIa activation. In addition, VAS compounds inhibited mouse platelet aggregation-induced by collagen and thrombin. The in vivo study also showed that VAS compounds delayed thrombus formation without affecting normal hemostasis. This study is the first to demonstrate that, in addition to inhibiting NOX activity, VAS compounds reduced platelet activation and thrombus formation through a NOX-independent pathway downstream of PKC. These findings also indicate that VAS compounds may be safe and potentially therapeutic agents for treating patients with cardiovascular diseases.
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Project description:Modeling the transport, activation, and adhesion of platelets is crucial in predicting thrombus formation and growth following a thrombotic event in normal or pathological conditions. We propose a shear-dependent platelet adhesive model based on the Morse potential that is calibrated by existing in vivo and in vitro experimental data and can be used over a wide range of flow shear rates ([Formula: see text]). We introduce an Eulerian-Lagrangian model where hemodynamics is solved on a fixed Eulerian grid, while platelets are tracked using a Lagrangian framework. A force coupling method is introduced for bidirectional coupling of platelet motion with blood flow. Further, we couple the calibrated platelet aggregation model with a tissue-factor/contact pathway coagulation cascade, representing the relevant biology of thrombin generation and the subsequent fibrin deposition. The range of shear rates covered by the proposed model encompass venous and arterial thrombosis, ranging from low-shear-rate conditions in abdominal aortic aneurysms and thoracic aortic dissections to thrombosis in stenotic arteries following plaque rupture, where local shear rates are extremely high.
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Project description:BackgroundThe process of thrombus formation is thought to involve interactions between platelets and leukocytes. Leukocyte incorporation into growing thrombi has been well established in vivo, and a number of properties of platelet-leukocyte interactions critical for thrombus formation have been characterized in vitro in thromboinflammatory settings and have clinical relevance. Leukocyte activity can be impaired in distinct hereditary and acquired disorders of immunological nature, among which is Wiskott-Aldrich Syndrome (WAS). However, a more quantitative characterization of leukocyte behavior in thromboinflammatory conditions has been hampered by lack of approaches for its study ex vivo. Here, we aimed to develop an ex vivo model of thromboinflammation, and compared granulocyte behavior of WAS patients and healthy donors.ResultsThrombus formation in anticoagulated whole blood from healthy volunteers and patients was visualized by fluorescent microscopy in parallel-plate flow chambers with fibrillar collagen type I coverslips. Moving granulocytes were observed in hirudinated or sodium citrate-recalcified blood under low wall shear rate conditions (100 s-1). These cells crawled around thrombi in a step-wise manner with an average velocity of 90-120 nm/s. Pre-incubation of blood with granulocyte priming agents lead to a significant decrease in mean-velocity of the cells and increase in the number of adherent cells. The leukocytes from patients with WAS demonstrated a 1.5-fold lower mean velocity, in line with their impaired actin polymerization. It is noteworthy that in an experimental setting where patients' platelets were replaced with healthy donor's platelets the granulocytes' crawling velocity did not change, thus proving that WASP (WAS protein) deficiency causes disruption of granulocytes' behavior. Thereby, the observed features of granulocytes crawling are consistent with the neutrophil chemotaxis phenomenon. As most of the crawling granulocytes carried procoagulant platelets teared from thrombi, we propose that the role of granulocytes in thrombus formation is that of platelet scavengers.ConclusionsWe have developed an ex vivo experimental model applicable for observation of granulocyte activity in thrombus formation. Using the proposed setting, we observed a reduction of motility of granulocytes of patients with WAS. We suggest that our ex vivo approach should be useful both for basic and for clinical research.
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Project description:Aluminium salts have been used in vaccines for decades. However, the mechanisms underlying their adjuvant effect are still unclear. Neutrophils, the first immune cells at the injection site, can release cellular DNA together with granular material, so-called neutrophil extracellular traps (NETs). In mice, NETs apparently play a role in aluminium hydroxide (alum)-adjuvant immune response to vaccines. Although no experimental data exist, this effect is assumed to be operative also in humans. As a first step to verify this knowledge in humans, we demonstrate that the injection of alum particles into human skin biopsies ex vivo leads to similar tissue infiltration of neutrophils and NET-formation. Moreover, we characterized the mechanism leading to alum-induced NET-release in human neutrophils as rapid, NADPH oxidase-independent process involving charge, phagocytosis, phagolysosomal rupture, Ca2+ -flux, hyperpolarization of the mitochondrial membrane, and mitochondrial ROS. Extracellular flow and inhibition experiments suggested that no additional energy from oxidative phosphorylation or glycolysis is required for NET-release. This study suggests a so far unappreciated role for neutrophils in the initial phase of immune responses to alum-containing vaccines in humans and provides novel insights into bioenergetic requirements of NET-formation.
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Project description:Salidroside is the main bioactive component in Rhodiola rosea and possesses multiple biological and pharmacological properties. However, whether salidroside affects platelet function remains unclear. Our study aims to investigate salidroside's effect on platelet function. Human or mouse platelets were treated with salidroside (0-20 ?M) for 1 hour at 37°C. Platelet aggregation, granule secretion, and receptors expression were measured together with detection of platelet spreading and clot retraction. In addition, salidroside (20 mg/kg) was intraperitoneally injected into mice followed by measuring tail bleeding time, arterial and venous thrombosis. Salidroside inhibited thrombin- or CRP-induced platelet aggregation and ATP release and did not affect the expression of P-selectin, glycoprotein (GP) Ib?, GPVI and ?IIb?3. Salidroside-treated platelets presented decreased spreading on fibrinogen or collagen and reduced clot retraction with decreased phosphorylation of c-Src, Syk and PLC?2. Additionally, salidroside significantly impaired hemostasis, arterial and venous thrombus formation in mice. Moreover, in thrombin-stimulated platelets, salidroside inhibited phosphorylation of AKT (T308/S473) and GSK3? (Ser9). Further, addition of GSK3? inhibitor reversed the inhibitory effect of salidroside on platelet aggregation and clot retraction. In conclusion, salidroside inhibits platelet function and thrombosis via AKT/GSK3? signaling, suggesting that salidroside may be a novel therapeutic drug for treating thrombotic or cardiovascular diseases.
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Project description:Despite the absence of the genome in platelets, transcription profiling provides important insights into platelet function and can help clarify abnormalities in platelet disorders. The Bloodomics Consortium performed whole-genome expression analysis comparing in vitro-differentiated megakaryocytes (MKs) with in vitro-differentiated erythroblasts and different blood cell types. This allowed the identification of genes with upregulated expression in MKs compared with all other cell lineages, among the receptors BAMBI, LRRC32, ESAM, and DCBLD2. In a later correlative analysis of genome-wide platelet RNA expression with interindividual human platelet reactivity, LLRFIP and COMMD7 were additionally identified. A functional genomics approach using morpholino-based silencing in zebrafish identified various roles for all of these selected genes in thrombus formation. In this review, we summarize the role of the six identified genes in zebrafish and discuss how they correlate with subsequently performed mouse experiments.
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Project description:Neutrophil extracellular traps (NETs) are extracellular chromatin fibers adorned with antimicrobial proteins, such as myeloperoxidase (MPO), which are extruded from activated neutrophils. NETosis is the metamorphosis of neutrophils with NET formation that follows decondensation of DNA and rupture of the plasma membrane. Although NETs play important roles in innate immunity, excessive formation of NETs can be harmful to the hosts. Until now, various methods for evaluation of NETs have been reported. Although each has a virtue, the gold standard has not been established. Here we demonstrate a simple, objective, and quantitative method to detect NETs using flow cytometry. This method uses a plasma membrane-impermeable DNA-binding dye, SYTOX Green. SYTOX Green-positive cells were detected in human peripheral polymorphonuclear cells exposed to a NET inducer, phorbol 12-myristate 13-acetate (PMA). The number of SYTOX Green-positive cells was increased depending on the exposure duration and concentrations of PMA. Furthermore, co-localization of MPO and plasma membrane-appendant DNA of SYTOX Green-positive cells was demonstrated. Moreover, a NET inhibitor, diphenylene iodonium, could significantly reduce the number of SYTOX Green-positive cells induced by PMA. The collective evidence suggests that SYTOX Green-positive cells include neutrophils that formed NETs. The established method could detect neutrophils that underwent NETosis but not early apoptosis with equivalence in quantification to another well-used image analysis, which is based on fluorescent staining. Additionally, NETs that were formed in vivo were also detectable by this method. It is conceivable that the established method will bring us better understanding of the relation between NETosis and human diseases. © 2017 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of ISAC.
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