scholarly journals Neutrophil Extracellular Traps Induced By Activated Platelets Contribute to Hypercoagulable State in Patients with Colorectal Cancer

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1229-1229
Author(s):  
Yan Zhang ◽  
Baorong Li ◽  
Yingmiao Liu ◽  
Cong Zhang ◽  
Yan Kou ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Endothelial Cells ◽  
Neutrophil Extracellular Traps ◽  
Hypercoagulable State ◽  
Enzyme Linked Immunosorbent Assay ◽  
Healthy Controls ◽  
Coagulation Time ◽  
Extracellular Traps ◽  
Activated Platelets

Abstract Background: Patients with colorectal cancer (CRC) are at increased risk of venous thromboembolism (VTE), but the precise mechanisms of hypercoagulability in CRC remain largely unknown. Neutrophil extracellular traps (NETs) are web-like chromatin structures decorated with cytoplasmic, granular and nuclear components of neutrophils, which can participate in both antimicrobial responses and contribute to a number of autoimmune and thrombotic diseases. However, a definitive role of NETs in the hypercoagulable state in CRC patients is still unclear. The aims of this study were to identify the novel role of NET in the induction of procoagulant activity (PCA) in CRC, and to evaluate its interactions with platelets and endothelial cells (ECs). Methods: Ninety-two CRC patients and 30 healthy controls were included. The presence of NETs was assessed using immunofluorescence microscopy. Cell-free DNA (cf-DNA) was quantified using the Quant-iT PicoGreen dsDNA Assay Kit, myeloperoxidase (MPO)-DNA complex was measured using a capture enzyme linked immunosorbent assay (ELISA). Thrombin-antithrombin (TAT) complex of NETs was evaluated by ELISA. Coagulation time of NETs, platelets and ECs was assessed by coagulation time (CT) using one-stage recalcification time assays, purified coagulation complex and fibrin turbidity were measured using ELISA. PS exposure on platelets and ECs, and fibrin formation on ECs were detected with flow cytometry and confocal microscopy. Results: We showed that the levels of cf-DNA and MPO-DNA complexes in the peripheral blood of CRC patients were increased in parallel with cancer progression and reached significance in stage III and IV patients compared to healthy subjects (all P<0.01). In addition, NETs released by CRC patients shortened coagulation time (CT), significantly enhanced the generation of TAT complexes and the formation of fibrin fibrils compared to healthy controls (all P<0.05). Moreover, DNase1-mediated degradation of NETs resulted in decreased PCA in patients with CRC (P<0.001). Furthermore, platelets from CRC patients stimulated healthy neutrophils to extrude NETs, which could be inhibited by the depletion of HMGB1 (P<0.01). Conversely, NETs from CRC patients could also induce the exposure of PS on platelets and the release of platelet MPs (PMPs), leading to markedly enhanced intrinsic/extrinsic FXa and FIIa, as well as shortened CT (all P<0.05). Importantly, endothelial cells (ECs) were converted to a procoagulant phenotype when exposed to NETs from CRC patients. The PCA of NETs-activated platelets or ECs could be inhibited either by the cleavage of NETs with DNase1 or the blockage of histone with activated protein C (APC) (all P<0.05). Our study also showed that the levels of NETs in CRC patients was positively correlated with TAT complexes and D-dimer (all P<0.05). Conclusion: Our results suggest that activated platelets promote NETs formation through the release of HMGB1 and result in an elevated PCA in CRC patients. In turn, NETs induce platelet PS exposure and PMPs release, forming a vicious cycle. In addition, NETs could also induce a procoagulant phenotype of ECs, indicating the complex relationship among these cellular constituents and highlighting the procoagulant role and cytotoxic effects of NETs in CRC. We propose that the rapid developments in the field of NETs may provide new therapeutic targets to combat the thrombotic consequences of CRC. Disclosures No relevant conflicts of interest to declare.

scholarly journals Targeting myeloid-cell specific integrin α9β1 inhibits arterial thrombosis in mice

Blood ◽  
2020 ◽  
Vol 135 (11) ◽  
pp. 857-861 ◽  
Author(s):  
Nirav Dhanesha ◽  
Manasa K. Nayak ◽  
Prakash Doddapattar ◽  
Manish Jain ◽  
Gagan D. Flora ◽  
...  
Keyword(s):  
Arterial Thrombosis ◽  
Potential Role ◽  
Myeloid Cell ◽  
Neutrophil Extracellular Traps ◽  
Cathepsin G ◽  
Wild Type ◽  
Extracellular Traps ◽  
Laser Injury ◽  
Activated Platelets

Abstract Evidence suggests that neutrophils contribute to thrombosis via several mechanisms, including neutrophil extracellular traps (NETs) formation. Integrin α9β1 is highly expressed on neutrophils when compared with monocytes. It undergoes affinity upregulation on neutrophil activation, and stabilizes adhesion to the activated endothelium. The role of integrin α9 in arterial thrombosis remains unexplored. We generated novel myeloid cell-specific integrin α9−/− mice (α9fl/flLysMCre+) to study the role of integrin α9 in arterial thrombosis. α9fl/fl littermates were used as controls. We report that α9fl/flLysMCre+ mice were less susceptible to arterial thrombosis in ferric chloride (FeCl3) and laser injury-induced thrombosis models with unaltered hemostasis. Neutrophil elastase-positive cells were significantly reduced in α9fl/flLysMCre+ mice concomitant with reduction in neutrophil count, myeloperoxidase levels, and red blood cells in the FeCl3 injury-induced carotid thrombus. The percentage of cells releasing NETs was significantly reduced in α9fl/flLysMCre+ mouse neutrophils stimulated with thrombin-activated platelets. Furthermore, we found a significant decrease in neutrophil-mediated platelet aggregation and cathepsin-G secretion in α9fl/flLysMCre+ mice. Transfusion of α9fl/fl neutrophils in α9fl/flLysMCre+ mice restored thrombosis similar to α9fl/fl mice. Treatment of wild-type mice with anti-integrin α9 antibody inhibited arterial thrombosis. This study identifies the potential role of integrin α9 in modulating arterial thrombosis.

Exosomal MALAT1 derived from ox-LDL-treated endothelial cells induce neutrophil extracellular traps to aggravate atherosclerosis

2020 ◽  
Vol 401 (3) ◽  
pp. 367-376 ◽  
Author(s):  
Hailai Gao ◽  
XiaoLi Wang ◽  
Chaolan Lin ◽  
Zhujun An ◽  
Jiangbo Yu ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Umbilical Vein ◽  
Low Density Lipoprotein ◽  
Atherosclerotic Lesion ◽  
Density Lipoprotein ◽  
Neutrophil Extracellular Traps ◽  
Human Neutrophils ◽  
Enzyme Linked Immunosorbent Assay ◽  
Extracellular Traps ◽  
Umbilical Vein Endothelial Cells

AbstractThe objective of this study was to reveal a novel mechanism underlying the progression of atherosclerosis (AS) associated with endothelial cells (ECs) and neutrophils. Transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) were used to observe the morphology and particle size of isolated exosomes. Western blotting was applied to examine exosomal markers, while the expression of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) was measured by quantitative real-time polymerase chain reaction (qRT-PCR). The production of inflammatory cytokines and reactive oxygen species (ROS) was determined by an enzyme-linked immunosorbent assay (ELISA) and a dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay. Circulating neutrophil extracellular traps (NETs) were represented by myeloperoxidase (MPO)-DNA complexes. NETs formation was assessed using immunofluorescence microscopy. Atherosclerotic lesion development was measured by Oil Red O (ORO) staining. In the results, MALAT1 expression was increased in exosomes extracted from oxidized low-density lipoprotein (ox-LDL)-treated human umbilical vein endothelial cells (HUVECs). When co-cultured with human neutrophils, exosomes derived from ox-LDL-treated HUVECs were revealed to promote NETs formation, which was mediated by exosomal MALAT1. Furthermore, ox-LDL-treated HUVECs-derived exosomes were demonstrated to trigger hyperlipidemia, inflammatory response and NETs release in a mouse model of AS. In conclusion, exosomal MALAT1 derived from ox-LDL-treated ECs initiated NETs formation, which in turn deteriorated AS.

Phosphotidylserine exposure and neutrophil extracellular traps enhance procoagulant activity in patients with inflammatory bowel disease

2016 ◽  
Vol 115 (04) ◽  
pp. 738-751 ◽  
Author(s):  
Ruishuang Ma ◽  
Yan Zhang ◽  
Muhua Cao ◽  
Tao Li ◽  
Zhipeng Yao ◽  
...  
Keyword(s):  
Inflammatory Bowel Disease ◽  
Bowel Disease ◽  
Thromboembolic Event ◽  
Neutrophil Extracellular Traps ◽  
Hypercoagulable State ◽  
Coagulation Time ◽  
Extracellular Traps ◽  
Fibrin Formation ◽  
Inflammatory Bowel ◽  
Epidemiological Association

SummaryInflammatory bowel disease (IBD)-associated thromboembolic event often lacks precise aetiology. The aim of this study was to investigate the contribution of phosphatidylserine (PS) exposure and neutrophil extracellular traps (NETs) towards the hypercoagulable state in IBD. We demonstrated that the levels of PS exposed MPs and the sources of MP-origin, platelets, erythrocytes, leukocytes and cultured endothelial cells (ECs) were higher in IBD groups than in healthy controls using flow cytometry and confocal microscopy. Wright-Giemsa and immunofluorescence staining demonstrated that the elevated NETs were released by activated IBD neutrophils or by control neutrophils treated with IBD sera obtained from patients with the active disease. MPs and MP-origin cells in IBD groups, especially in active stage, markedly shortened coagulation time and had increased levels of fibrin, thrombin and FXa production as assessed by coagulation function assays. Importantly, we found that on stimulated ECs, PS rich membranes provided binding sites for FXa and FVa, promoting fibrin formation while TNF blockage or IgG depletion attenuated this effect. Treatment of control neutrophils with TNF and isolated IgG from PR3-ANCA-positive active IBD patients also resulted in the release of NETs. Blockade of PS with lactadherin prolonged coagulation time, decreased fibrin formation to control levels, and inhibited the procoagulant enzymes production in the MPs and MP-origin cells. NET cleavage by DNase I partly decreased PCA in IBD or stimulated neutrophils. Our study reveals a previously unrecognised link between hypercoagulable state and PS exposure or NETs, and may further explain the epidemiological association of thrombosis within IBD patients.

Increased Circulating Nucleosomes and Neutrophil Activation As a Measure of the Formation of Neutrophil Extracellular Traps (NETs) During Sickle Cell Painful Crisis

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 821-821
Author(s):  
Marein Schimmel ◽  
Erfan Nur ◽  
Sacha Zeerleder ◽  
Gerard J van Mierlo ◽  
Shabnam Solati ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Steady State ◽  
Sickle Cell ◽  
Plasma Levels ◽  
Neutrophil Extracellular Traps ◽  
Neutrophil Activation ◽  
Acute Chest Syndrome ◽  
Healthy Controls ◽  
Extracellular Traps ◽  
Painful Crisis

Abstract Abstract 821 Introduction: Sickle cell disease (SCD) is characterized by recurrent acute vaso-occlusive painful crisis frequently leading to SCD related complications such as acute chest syndrome, stroke, multi-organ failure and even sudden death. The complex pathophysiology of the vaso-occlusive painful crisis is mediated by activation of endothelial cells, adhesion of sickled erythrocytes and neutrophils, oxidative stress, coagulation activation and increased release of inflammatory mediators, resulting in ischemic organ damage. Recently, neutrophils have been demonstrated to form neutrophil extracellular traps (NETs) upon activation. Nucleosomes and histones exposed together with neutrophil proteases, such as elastase on these NETs have been shown to kill efficiently bacteria. NET formation has been shown to propagate coagulation in sepsis and in deep venous thrombosis. In addition, nucleosomes and histones exposed on NETs have been shown to be strongly cytotoxic to endothelial cells. Beside the exposure on NETs, nucleosomes can be actively released into the circulation from dead cells. Circulating nucleosomes detected in sepsis have been reported to correlate with severity of inflammation, organ dysfunction and mortality. However, no studies are available yet on the dynamics of nucleosomes and NETs in sickle cell patients suffering from painful crisis. The aim of this case-control study was to assess plasma levels of circulating nucleosomes and human neutrophil elastase–α1-antitrypsin (EA) complexes as measure of systemic neutrophil activation, in sickle cell patients during steady state and painful crisis. Methods: Plasma levels of nucleosomes and EA as a measure of neutrophil activation were measured in 74 patients in asymptomatic state (49 HbSS/HbSβ0-thalassemia, and 25 HbSC/HbSβ+-thalassemia), 70 painful crises (53 HbSS/HbSβ°-thalassemia and 17 HbSC/HbSβ+-thalassemia) in 49 patients and in 24 HbAA healthy controls using Enzyme-Linked Immunosorbent Assay (ELISA). Results: Plasma levels of nucleosomes in both HbSS/HbSβ°-thalassemia and HbSC/HbSβ+-thalassemia patients were significantly higher during painful crisis (median; interquartile range, 20.2; 8.9 – 129.0 U/ml, P < 0.0001 and 11.7; 5.1 – 67.7 U/ml, P = 0.045 respectively) as compared to patients in steady state (6.0; 3.0 – 9.8 U/ml and 7.1; 4.6 – 9.6 U/ml respectively). Nucleosomes levels in healthy controls were just above the detection limit of the assay (5.0; 5.0 – 6.5) U/ml). Plasma levels of EA in HbSS/HbSβ°-thalassemia patients were significantly increased during painful crisis as compared to steady state (75.1; 56.5 – 102.4 vs. 45.7; 34.7 – 59.7 ng/ml, P < 0.0001). Also in HbSC/HbSβ+-thalassemia patients, EA levels were higher during painful crisis than in steady state, though the difference did not reach statistical significance (62.0; 48.0 – 96.7 vs. 50.2; 33.3 – 67.7, P = 0.051). Plasma levels of EA in healthy controls (39.9; 31.5 – 62.2 ng/ml) were comparable with those in steady state patients. In a paired analysis of 36 patients, included both during steady state and painful crisis, significant increments were observed during painful crisis in levels of both nucleosomes (from 5.0; 3.0 – 10.8 to 20.2; 6.8 – 94.3 U/ml, P < 0.0001) and EA (from 47.9; 36.0 – 67.6 to 70.6; 55.9 – 101.4 ng/ml, P < 0.0001), as compared to steady state. During painful crisis, EA levels were strongly correlated with levels of nucleosomes in both HbSS/HbSβ°-thalassemia (Spearman's rank (Sr)=0.55, P<0.0001) and HbSC/HbSβ+-thalassemia patients (Sr=0.90, P=<0.0001). In steady state the correlation was significant only in HbSC/HbSβ+-thalassemia patients (Sr=0.63, P=0.001) Four patients who developed an acute chest syndrome during painful crisis were among the patients with the highest nucleosome (359, 130, 128 and 100 U/ml) and EA levels (121, 87, 92 and 64 ng/ml respectively). Conclusion: Sickle cell painful crisis is associated with increased levels of nucleosome and stronger neutrophil activation. This might point to a crucial role of NET formation in the pathogenesis of painful crisis. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Tissue Factor-Enriched Neutrophil Extracellular Traps Promote Immunothrombosis and Disease Progression in Sepsis-Induced Lung Injury

2021 ◽  
Vol 11 ◽  
Author(s):  
Hao Zhang ◽  
Yilu Zhou ◽  
Mengdi Qu ◽  
Ying Yu ◽  
Zhaoyuan Chen ◽  
...  
Keyword(s):  
Lung Injury ◽  
Disease Progression ◽  
Tissue Factor ◽  
Neutrophil Extracellular Traps ◽  
Thrombin Inhibitors ◽  
Extracellular Traps ◽  
Activated Platelets ◽  
Human Blood Samples

BackgroundPatients with sepsis may progress to acute respiratory distress syndrome (ARDS). Evidence of neutrophil extracellular traps (NETs) in sepsis-induced lung injury has been reported. However, the role of circulating NETs in the progression and thrombotic tendency of sepsis-induced lung injury remains elusive. The aim of this study was to investigate the role of tissue factor-enriched NETs in the progression and immunothrombosis of sepsis-induced lung injury.MethodsHuman blood samples and an animal model of sepsis-induced lung injury were used to detect and evaluate NET formation in ARDS patients. Immunofluorescence imaging, ELISA, Western blotting, and qPCR were performed to evaluate in vitro NET formation and tissue factor (TF) delivery ability. DNase, an anti-TF antibody, and thrombin inhibitors were applied to evaluate the contribution of thrombin to TF-enriched NET formation and the contribution of TF-enriched NETs to immunothrombosis in ARDS patients.ResultsSignificantly increased levels of TF-enriched NETs were observed in ARDS patients and mice. Blockade of NETs in ARDS mice alleviated disease progression, indicating a reduced lung wet/dry ratio and PaO2 level. In vitro data demonstrated that thrombin-activated platelets were responsible for increased NET formation and related TF exposure and subsequent immunothrombosis in ARDS patients.ConclusionThe interaction of thrombin-activated platelets with PMNs in ARDS patients results in local NET formation and delivery of active TF. The notion that NETs represent a mechanism by which PMNs release thrombogenic signals during thrombosis may offer novel therapeutic targets.

scholarly journals Neutrophil extracellular traps induce MCP-1 release from endothelial cells at the plaque rupture site in acute myocardial infarction

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
A Ondracek ◽  
T.M Hofbauer ◽  
A Mangold ◽  
T Scherz ◽  
V Seidl ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Acute Myocardial Infarction ◽  
Endothelial Cells ◽  
Plaque Rupture ◽  
Ex Vivo ◽  
Coronary Intervention ◽  
Neutrophil Extracellular Traps ◽  
Funding Source ◽  
Extracellular Traps

Abstract Introduction Leukocyte-mediated inflammation is crucial in acute myocardial infarction (AMI). We recently observed that neutrophil extracellular traps (NETs) are increased at the culprit site, promoting activation and differentiation of fibrocytes, cells with mesenchymal and leukocytic properties. Fibrocyte migration is mediated by monocyte chemoattractant protein (MCP)-1 and C-C chemokine receptor type 2 (CCR2). We investigated the interplay between NETs, fibrocyte function, and MCP-1 in AMI. Methods Culprit site and femoral blood of AMI patients was drawn during percutaneous coronary intervention. We characterized CCR2 expression of fibrocytes by flow cytometry. MCP-1 and the NET marker citrullinated histone H3 (citH3) were measured by ELISA. Fibrocytes were treated in vitro with MCP-1. Human coronary arterial endothelial cells (hCAECs) were stimulated with isolated NETs, and MCP-1 was measured by ELISA and qPCR. The influence of MCP-1 on NET formation in vitro was assessed using isolated neutrophils. Results We have included 50 consecutive AMI patients into the study. NETs and concentrations of MCP-1 were increased at the CLS. NET stimulation of hCAECs induced MCP-1 on mRNA and protein level. Increasing MCP-1 gradient was associated with fibrocyte accumulation at the site of occlusion. In the presence of higher MCP-1 these fibrocytes expressed proportionally less CCR2 than peripheral fibrocytes. In vitro, MCP-1 dose-dependently decreased fibrocyte CCR2 and reduced ex vivo NET release of healthy donor neutrophils. Conclusions NETs induce endothelial MCP-1 release, presumably promoting a chemotactic gradient for leukocyte and fibrocyte migration. MCP-1 mediated inhibition of NET formation could point to a negative feedback loop. These data will shed light on vascular healing. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Austrian Science Fund

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