Thromb Haemost 2020; 120(03): 373-383
DOI: 10.1055/s-0039-3402731
Special Focus
Georg Thieme Verlag KG Stuttgart · New York

Traps N' Clots: NET-Mediated Thrombosis and Related Diseases

Dimitrios Stakos*
1  Department of Cardiology, Democritus University of Thrace, Alexandroupolis, Greece
2  Laboratory of Molecular Hematology, University Hospital of Alexandroupolis, Democritus University of Thrace, Alexandroupolis, Greece
,
Panagiotis Skendros*
2  Laboratory of Molecular Hematology, University Hospital of Alexandroupolis, Democritus University of Thrace, Alexandroupolis, Greece
3  First Department of Internal Medicine, University Hospital of Alexandroupolis, Democritus University of Thrace, Alexandroupolis, Greece
,
Stavros Konstantinides
1  Department of Cardiology, Democritus University of Thrace, Alexandroupolis, Greece
4  Center for Thrombosis and Hemostasis, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany
,
Konstantinos Ritis
2  Laboratory of Molecular Hematology, University Hospital of Alexandroupolis, Democritus University of Thrace, Alexandroupolis, Greece
3  First Department of Internal Medicine, University Hospital of Alexandroupolis, Democritus University of Thrace, Alexandroupolis, Greece
› Author Affiliations
Funding This work was supported by Externally Sponsored Scientific Research Grant number ESR-16–12098 from AstraZeneca, and Research Grant number 80895 from the Scientific Committee of Democritus University of Thrace.
Further Information

Publication History

01 July 2019

26 November 2019

Publication Date:
15 January 2020 (online)

Insights into Neutrophils and NETs: A Historical Perspective

Vessel wall injury and subsequent blood extravasation activates a series of local biological processes to prevent excess blood loss via the formation of hemostatic plug strictly restricted at the site of vascular injury with minimal or no extension in the vessel lumen.[1] In the vast majority of cases, a catastrophic systemic activation of these processes is contained by specific mechanisms. As opposed to hemostasis, thrombosis is characterized by the deregulated clot formation, various degrees of vessel occlusion, tissue ischemia, and necrosis.[1]

A large body of accumulating experimental and clinical data over the past 20 years has clearly indicated the reciprocal relationship and dynamic interplay between inflammation and thrombosis.[2] [3] [4] [5] Today, targeting inflammation to prevent thrombotic events represents a realistic and promising therapeutic approach.[6] Among immune cell subsets that are implicated in multiple molecular pathways during inflammatory response, neutrophils have a crucial role, recruited first to the site of injury following instructive signals from the tissue environment.[7]

The very first observation linking neutrophils with thromboinflammation was reported almost 70 years ago describing granulocytes as a main component of clotted blood in patients suffering from active lupus erythematosus.[8] During the following years, although several studies had described the accumulation of neutrophils at the site of thrombus formation, these cells remained neglected and less studied in many thrombotic diseases. The traditional aspect of neutrophils as dispensable, passive bystanders was dramatically revised after the milestone discovery that they represent a primary source of blood-borne tissue factor (TF), the main in vivo initiator of the extrinsic coagulation cascade, resulting in thrombin generation and ensuing thrombus formation.[9] [10] Later on, several studies from our laboratory and others provided evidence for the critical role of neutrophils in thrombosis and inflammation-mediated thrombotic complications.[3] [11] [12] Intravital microscopy studies in mouse models of venous and arterial thrombosis demonstrated neutrophil recruitment and activation at the site of endothelial damage in the early phase of thrombosis.[13] [14] Of note, neutrophils are not only implicated in thrombotic processes, but also seem to be indispensable for thrombosis. Neutropenia induced in vivo by anti-Ly6G or GR-1 antibody abrogated venous[13] and arterial[14] thrombosis, respectively. When purified neutrophils from wild-type mice were injected into transgenic mice that express no mouse TF and only minimal (< 1%) amounts of human TF (low TF mice), the defective fibrin generation in these animals was restored, indicating that TF expressing neutrophils represent the main source of TF during thrombus formation.[14] Similarly, when normal TF expressing mice were transplanted with low-human TF bone marrow cells they did not develop deep vein thrombosis (DVT).[13] However, the mechanisms underlying the activation and delivery of active TF by neutrophils remained unknown.

During the last years, advances in molecular biology provided the most exciting update of neutrophil physiology, in particular their capacity to release neutrophil extracellular traps (NETs). NETs are extracellular web-like structures of chromatin fibers lined with various highly active proteases and proteins of nuclear, granular, and cytosolic origin.[15] The release of NETs from activated neutrophils was initially described in 2004 as a novel defense mechanism able to “trap and kill” a wide range of pathogens.[16] However, increasing evidence during the past few years highlighted their fundamental role in the pathogenesis of numerous noninfectious inflammatory disorders, such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), autoimmune vasculitis, gout, ulcerative colitis, interleukin (IL)-1β-mediated autoinflammatory syndromes, and thrombosis.[15] [17] [18] [19]

Activated platelets are able to induce robust NET release within vasculature providing a scaffold for fibrin deposition and stabilization of thrombus.[20] [21] [22] [23] Notably, these NETs are decorated with functionally active TF, which explains its extracellular delivery at the site of tissue injury.[13] [19] [24] Besides TF, NETs were found to deliver several proteins and clot factors involved in thrombosis such as von Willebrand factor (vWF), XII, fibrinogen, and fibronectin.[2] [13] [25] The thrombogenic potential of NETs was further supported by experimental studies indicating that extracellular histones induce endothelial activation, platelet activation/aggregation, and thrombin generation[26] [27] ([Fig. 1]). Phylogenetically, the capacity of NETs to activate coagulation serves to trap and eliminate pathogens, resembling primitive defense systems operating several millions years ago, and it is conserved today in insects.[28] In these organisms, coagulation and immunity use common mechanisms to prevent fluid loss and pathogen invasion. These systems are tightly interrelated, since NET-associated antimicrobial proteases are able to trigger several coagulation pathways,[2] while activation of the coagulation system supports several immune responses such as bacterial compartmentalization, immobilization, and elimination especially in microvasculature (immunothrombosis). Probably, much of the NET-mediated antimicrobial effect is due to entrapment, rather than direct killing[29] [30] ([Fig. 2]).

Zoom Image
Fig. 1 Mechanisms of neutrophil extracellular trap (NET) thrombogenicity. Left: Platelets-PolyP-neutrophils-NETs interactivation leads to NET generation. (–) denotes NET autoregulation. Right: NETs can deliver thrombogenic signals through many different mechanisms presented here. APC, activated protein C; FX, FXII, FV, coagulation factors; PolyP: polyphosphate; TF, tissue factor; TFPI, TF pathway inhibitor; tPA, tissue plasminogen activator.
Zoom Image
Fig. 2 Skewing of immunothrombosis (tissue protection) to pathological thrombosis (tissue damage). In primitive organisms (bottom, A) immunity and coagulation use a common, hemocyte-based system to prevent fluid loss and pathogen invasion. In mammals including humans (B) hemostasis and immunity are operated by distinct systems (platelets and coagulation, left; and white blood cells such as neutrophils, right, respectively). Interaction between platelets/coagulation and neutrophils leads to neutrophil extracellular trap (NET) formation (C). In cases of a pathogen invasion, NETs are involved in pathogen entrapment and elimination in microcirculation (green path, host defense, D). On the contrary, inappropriate NET formation in cases of sterile inflammation (autoimmune or inflammatory environment) leads to thrombotic complications (red path, tissue damage) in microcirculation (E) or large vessels (F). DIC, disseminated intravascular coagulation.

Apart from neutrophils, extracellular traps (ETs) formation has also been described in other types of granulocytes, such as eosinophils and mast cells.[31] [32] Very recently data implicate macrophages, mast cells, and eosinophils through ETs generation in atherosclerotic plaque formation and thrombosis.[33] [34] However, ETs formation in macrophages is controversial and remains unclear whether it is distinct from pyroptosis.[35]

In view of the above, NETs could be perceived as a double-edged sword during disease processes. They may be beneficial by enhancing the antimicrobial potential in numerous infectious diseases or contributing to normal hemostasis and pathogen curbing in neutrophil clots lattice, but also harmful by amplifying systemic or local inflammation leading to tissue damage and thrombosis[15] [36] ([Fig. 2]). Therefore, NETs and their components emerge today as novel candidate for diagnostic and therapeutic targets of thrombosis in many clinical settings.[12] [15] [37] [38]

* These authors contributed equally to this work.