scholarly journals Neutrophil Extracellular Traps (NETs) Contribute to the Formation of Microvascular Thrombosis in Immune Thrombotic Thrombocytopenic Purpura

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1020-1020
Author(s):  
Noritaka Yada ◽  
Jingrui Sui ◽  
Liang Zheng ◽  
X. Long Zheng
Keyword(s):  
Neutrophil Extracellular Traps ◽  
Dnase I ◽  
Confocal Imaging ◽  
Adamts13 Activity ◽  
Imaging Analysis ◽  
Thrombocytopenic Purpura ◽  
Pathogenetic Role ◽  
Extracellular Traps ◽  
Endothelial Surface

Abstract Introduction. Immune thrombotic thrombocytopenic purpura (iTTP), a potentially fatal blood disorder, is primarily caused by severe deficiency of plasma ADAMTS13 activity resulting from immunoglobulin (Ig) G-mediated inhibition of plasma ADAMTS13 activity. However, severe ADAMTS13 deficiency is necessary but not sufficient to cause acute iTTP. An environmental factor such as infection or acute inflammation may be necessary to trigger the acute onset of the disease. We and others have previously reported that plasma markers of neutrophil activation and neutrophil extracellular traps (NETs) formation are significantly elevated in patients with acute iTTP, which returns to normal during remission. However, the pathogenetic role of NETs in acute iTTP is not fully understood. Methods and results. Using flow cytometry, microfluidic shear-based assay, and confocal imaging analysis, we determined the in vivo NETosis in blood samples obtained from patients with acute episode of iTTP and ex vivo NETs formation, as well as the therapeutic efficacy of DNase I on thrombus formation under flow. We showed that by flow cytometry that only very few CitH3+/MPO+ positive neutrophils were present in the healthy donor blood. This population of cells dramatically increased after being stimulated with a bacterial toxin (i.e., Shigatoxin-2) at ~100 ng/mL for 15 min. Importantly, the number of CitH3+/MPO+ positive neutrophils in the sample obtained from a patient with acute iTTP was ~1,000 times higher than that in the healthy controls (Fig. 1), suggesting a massive NETosis in patients with acute iTTP. Microfluidic shear-based assay and confocal imaging analysis further confirmed a dramatic increase in adhesion and aggregation of murine platelets (stained with Alexa647 anti-CD41) and neutrophil (stained with Hoechst), as well as formation of NETs (stained with Syto green) following a perfusion of an Adamts13 -/- murine whole blood (anti-coagulated with thrombin inhibitor, PPACK) under arterial shear (15 dyne/cm 2) over a stimulated murine endothelial surface. Interestingly, an addition of DNase I (100 U/mL) significantly reduced the overall surface coverage of platelets and neutrophils on the murine endothelial surface under the same conditions (Fig. 2). Conclusions. These results demonstrate for the first time NETosis and NETs formation are common in patients with acute iTTP and in Adamts13 -/- mice after being stimulated with shigatoxin; DNase I appears to be highly efficacious eliminating the NETs and platelet/neutrophil-dominant thrombosis under arterial flow. Our findings support the pathogenetic role of NETs in the onset and progression of iTTP, and the therapeutic potential of DNase I in such a fatal disease. Figure 1 Figure 1. Disclosures Zheng: Alexion: Speakers Bureau; Sanofi-Genzyme: Honoraria, Speakers Bureau; Takeda: Consultancy, Honoraria; Clotsolution: Other: Co-founder; AJMC: Honoraria.

scholarly journals Neutrophil Extracellular Traps Promote Hypercoagulability in ST-Elevated Myocardial Infarction Following Fibrinolytic Administration

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3797-3797
Author(s):  
Lili Zou ◽  
Hui Liang ◽  
LI Hou ◽  
Tao Li ◽  
Yan Zhang ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Neutrophil Extracellular Traps ◽  
Dnase I ◽  
Myocardial Reperfusion ◽  
Confocal Imaging ◽  
Growth Media ◽  
Extracellular Traps ◽  
Fibrin Formation ◽  
Recurrent Ischemia

Abstract Introduction:Fibrinolysis plays an important role in the treatment of ST-elevated myocardial infarction (STEMI) when percutaneous coronary intervention is not readily available. Early and successful myocardial reperfusion with thrombolytic therapy effectively reduces the infarct size and improves the clinical outcome. However, the process of restoring blood flow to the ischemic myocardium can induce injury and reduce the beneficial effects of myocardial reperfusion. Previous studies had shown that platelets, leukocytes and TF play important role in thrombotic complications after fibrinolysis in AMI. However, there are still 10-15% patients who have risk for re-occlusion after antiplatelet and anticoagulant therapies. Thus, we speculate that there may be other mechanisms involved in the hypercoagulability after STEMI fibrinolysis. Neutrophil extracellular traps (NETs) are double-edge swords that could ensnare and kill microbial pathogens but also contribute to thrombosis. However, the role of NETs during STEMI fibrinolysis-induced re-occlusion is largely unknown. Our aims were to determine the procoagulant role of NETs after successful thrombolysis, and to elucidate its interaction with endothelial cells (ECs). Methods:31 STEMI patients with successfully fibrinolysis and 12 healthy controls were enrolled. Patient blood samples were collected at 0 h, 2 h, 6 h, 12 h and 24 h after fibrinolysis. Cell-free DNA (cf-DNA) was quantified using the Quant-iT PicoGreen dsDNA Assay Kit. ELISA was used to detect MPO-DNA complexes and TAT (thrombin-antithrombin) complexes. Wright-Giemsa and immunofluorescence confocal microscope were used to analyze and quantify NETs formation in neutrophil cells. ECs were incubated in growth media containing 20% pooled serum obtained from healthy donors in the presence or absence of 20-fold concentrated neutrophil extracellular chromatin. The procoagulant activity (PCA) of neutrophils and ECs was measured by clotting time and purified coagulation complex assays. DNase I or anti-TF were included in the inhibition assays. Results: We found that cf-DNA, MPO-DNA and TAT are significantly reduced at 2 hours in STEMI patients with successful fibrinolysis. Their levels then increased and peaked at 6 hours (Figure 1A, B, E). Interestingly, the level of cf-DNA at 6 hours in STEMI thrombotic patients was positively correlated with TAT (r=0.959; p<0.01; Figure 1G). Wright-Giemsa and immunofluorescence staining showed that NETs were released by STEMI reperfusion neutrophils or by control neutrophils treated with plasma obtained from STEMI patients with fibrinolysis (Figure 1D,F), and the percentage of NETs-releasing PMNs was about 30% (Figure 1C). Isolated neutrophils from fibrinolytic patients in vitro demonstrated significantly shortened coagulation time and increased fibrin formation after 2 hours fibrinolysis, and peaked at 6 hours. DNase I but not anti-tissue factor antibody could inhibit these effects. Co-incubation assays revealed that NETs triggered PS exposure on ECs, converting them to a procoagulant phenotype. Confocal imaging of NETs-treated ECs illustrated that bound FVa and FXa colocalized within PS-enriched areas of ECs to form prothrombinase, and further supported fibrin formation. Moreover, patients with recurrent ischemia showed significantly higher NETs release and thrombin generation than non-recurrent ischemia. Conclusions: Our study reveals that the PCA of STEMI following fibrinolytic administration decrease after 2 hours, then increase and peak at 6 hours, which is at least partly due to the release of NETs induced by activated PMNs. Additionally, NETs partly contribute to ECs injury after myocardial reperfusion. DNase I can disconnect NETs and may therefore serve as a promising therapeutic target in STEMI reinfarction and recurrent ischemia. Disclosures No relevant conflicts of interest to declare.

scholarly journals Neutrophil Extracellular Traps (NETs): Opportunities for Targeted Therapy

Acta Naturae ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 15-23
Author(s):  
Dmitry V. Volkov ◽  
George V. Tetz ◽  
Yury P. Rubtsov ◽  
Alexey V. Stepanov ◽  
Alexander G. Gabibov
Keyword(s):  
Solid Tumors ◽  
Nuclear Dna ◽  
Neutrophil Extracellular Traps ◽  
Dnase I ◽  
Extracellular Traps ◽  
Leading Role ◽  
Deoxyribonuclease I ◽  
Car T ◽  
Main Component

Antitumor therapy, including adoptive immunotherapy, inevitably faces powerful counteraction from advanced cancer. If hematological malignancies are currently amenable to therapy with CAR-T lymphocytes (T-cells modified by the chimeric antigen receptor), solid tumors, unfortunately, show a significantly higher degree of resistance to this type of therapy. As recent studies show, the leading role in the escape of solid tumors from the cytotoxic activity of immune cells belongs to the tumor microenvironment (TME). TME consists of several types of cells, including neutrophils, the most numerous cells of the immune system. Recent studies show that the development of the tumor and its ability to metastasize directly affect the extracellular traps of neutrophils (neutrophil extracellular traps, NETs) formed as a result of the response to tumor stimuli. In addition, the nuclear DNA of neutrophils the main component of NETs erects a spatial barrier to the interaction of CAR-T with tumor cells. Previous studies have demonstrated the promising potential of deoxyribonuclease I (DNase I) in the destruction of NETs. In this regard, the use of eukaryotic deoxyribonuclease I (DNase I) is promising in the effort to increase the efficiency of CAR-T by reducing the NETs influence in TME. We will examine the role of NETs in TME and the various approaches in the effort to reduce the effect of NETs on a tumor.

scholarly journals The state of art of neutrophil extracellular traps in protozoan and helminthic infections

2019 ◽  
Vol 39 (1) ◽  
Author(s):  
César Díaz-Godínez ◽  
Julio C. Carrero
Keyword(s):  
Neutrophil Extracellular Traps ◽  
Parasitic Infections ◽  
Parasitic Diseases ◽  
Helminth Parasites ◽  
Extracellular Traps ◽  
Helminthic Infections ◽  
Bacteria And Fungi ◽  
Net Release

AbstractNeutrophil extracellular traps (NETs) are DNA fibers associated with histones, enzymes from neutrophil granules and anti-microbial peptides. NETs are released in a process denominated NETosis, which involves sequential steps that culminate with the DNA extrusion. NETosis has been described as a new mechanism of innate immunity related to defense against different pathogens. The initial studies of NETs were carried out with bacteria and fungi, but currently a large variety of microorganisms capable of inducing NETs have been described including protozoan and helminth parasites. Nevertheless, we have little knowledge about how NETosis process is carried out in response to the parasites, and about its implication in the resolution of this kind of disease. In the best case, the NETs entrap and kill parasites in vitro, but in others, immobilize the parasites without affecting their viability. Moreover, insufficient studies on the NETs in animal models of infections that would help to define their role, and the association of NETs with chronic inflammatory pathologies such as those occurring in several parasitic infections have left open the possibility of NETs contributing to pathology instead of protection. In this review, we focus on the reported mechanisms that lead to NET release by protozoan and helminth parasites and the evidence that support the role of NETosis in the resolution or pathogenesis of parasitic diseases.

scholarly journals The Emerging Role of Neutrophil Extracellular Traps in Arterial, Venous and Cancer-Associated Thrombosis

2021 ◽  
Vol 8 ◽  
Author(s):  
Yilu Zhou ◽  
Weimin Tao ◽  
Fuyi Shen ◽  
Weijia Du ◽  
Zhendong Xu ◽  
...  
Keyword(s):  
Current Knowledge ◽  
Neutrophil Extracellular Traps ◽  
Vital Role ◽  
Extracellular Traps ◽  
Protein Components ◽  
Cancer Associated Thrombosis ◽  
Coagulation Pathway

Neutrophils play a vital role in the formation of arterial, venous and cancer-related thrombosis. Recent studies have shown that in a process known as NETosis, neutrophils release proteins and enzymes complexed to DNA fibers, collectively called neutrophil extracellular traps (NETs). Although NETs were originally described as a way for the host to capture and kill bacteria, current knowledge indicates that NETs also play an important role in thrombosis. According to recent studies, the destruction of vascular microenvironmental homeostasis and excessive NET formation lead to pathological thrombosis. In vitro experiments have found that NETs provide skeletal support for platelets, red blood cells and procoagulant molecules to promote thrombosis. The protein components contained in NETs activate the endogenous coagulation pathway to promote thrombosis. Therefore, NETs play an important role in the formation of arterial thrombosis, venous thrombosis and cancer-related thrombosis. This review will systematically summarize and explain the study of NETs in thrombosis in animal models and in vivo experiments to provide new targets for thrombosis prevention and treatment.

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