From neutrophil extracellular traps release to thrombosis: an overshooting host-defense mechanism?



    1. Laboratory for Clinical Thrombosis and Hemostasis, Departments of Internal Medicine and Biochemistry, Cardiovascular Research Institute of Maastricht, Maastricht University Medical Center, Maastricht, the Netherlands
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    1. Laboratory for Clinical Thrombosis and Hemostasis, Departments of Internal Medicine and Biochemistry, Cardiovascular Research Institute of Maastricht, Maastricht University Medical Center, Maastricht, the Netherlands
    Search for more papers by this author

Hugo ten Cate, Laboratory for Clinical Thrombosis and Hemostasis, Departments of Internal Medicine and Biochemistry, Cardiovascular Research Institute of Maastricht (CARIM), Maastricht University Medical Center+ (MUMC+), Universiteitsingel 50, PO Box 616, Box 8, 6200 MD Maastricht, the Netherlands.
Tel.: +31 43 38 84262; fax: +31 43 38 84159.

Innate immunity and blood coagulation are evolutionary entangled in an intricate network of molecular and cellular interactions, thus forming an integral part of the host-defense system [1]. Polymorphonuclear cells, in particular neutrophils, are essential for the primary innate immune response against local and systemic infections or tissue injury [2], but are also major cellular mediators supporting inflammation–coagulation interactions [3]. Upon inflammation, multiple chemotactic stimuli (cytokines, chemokines, etc.) are released to promote neutrophil activation, extravazation and migration towards the infectious foci. Neutrophils exert their bactericidal capacity by phagocytizing the disseminating pathogens, releasing numerous cytotoxic mediators and promoting cell death. Scientific evidence suggests that activation of blood coagulation, leading to subsequent fibrin deposition at the sites of inflammation, is an additional protective mechanism serving against microbial dissemination [4,5]. However, a persisting neutrophil hyper-responsiveness may trigger a pronounced oxidative stress and proteolysis through an enhanced synthesis of enzymatic proteins such as myeloperoxidase (MPO), neutrophil elastase and cathepsin G. These molecular mechanisms can result in the inactivation and degradation of important anti-coagulant proteins such as antithrombin, thrombomodulin (TM), protein C and tissue factor pathway inhibitor (TFPI), thus inducing a strong inflammation-driven local or systemic pro-coagulant response [6]. Persisting inflammation may trigger an over-reactive host defense response over time, thus disrupting the immune balance, contributing to tissue injury and thrombosis [5,7]. In fact, neutrophils have been indicated to play a role in the pathophysiology of several pathologic conditions including venous thrombosis, acute coronary syndromes and stroke [8–13].

Besides the abundant amount of reactive oxygen species (ROS) that neutrophils can generate [14], substantial evidence has emerged over the years showing the potential of neutrophils to kill pathogens also via the release of so-called neutrophil extracellular traps (NETs) [15,16]. The latter represent extracellular chromatin threads with potent cytotoxic effects, comprised of both histones and granular proteins. Recent studies have shown that NETs formation is a well-regulated process [17], and not only part of a cell-death program [18]. During activation, neutrophil elastase and MPO are released from the azurophilic granules and translocate to the nucleus, where they act in synergy to promote chromatin decondensation and histone degradation [17]. Furthermore, NETs establish a new interface between inflammation and blood coagulation (Figure 1). NETs are able to entrap and kill bacteria, but also to induce numerous pro-thrombotic effects such as:

Figure 1.

 The continuous cycle between inflammation, innate immunity and blood coagulation. NETs, neutrophil extracellular traps; MPO, neutrophil myeloperoxidase; TF, tissue factor; TM, thrombomodulin; PC, protein C; TFPI, tissue factor pathway inhibitor; factor (F)V/Va.

Platelet adhesion, activation and aggregation

Both platelets and neutrophils have the potential to activate each other through different molecular mechanisms. Once activated, platelets and neutrophils interact mainly via P-selectin, β2- and β3-integrin receptors. Besides the classic pathways of activation involving cytokines [e.g. interleukin (IL)-1β and tumor necrosis factor-alpha (TNF-α)], growth factors (e.g. G-CSF), the complement system (e.g. C5a), etc., neutrophils can be activated by platelets via binding to the TREM-1 receptor located on the neutrophil surface. The latter can evoke a massive oxidative burst and IL-8 release [19], thus contributing to the recruitment of even more neutrophils to the site of tissue injury/inflammation. The secretion of neutrophil-derived proteases (neutrophil elastase and matrix metalloproteinases) can also degrade the proteoglycans; hence, von Willebrand factor (VWF) gets exposed to promote platelet adhesion [20]. During severe inflammatory conditions such as sepsis, lipopolysaccharide (LPS) binds to toll-like receptor 4 (TLR4) on platelets, hence stimulating neutrophil activation and NETs formation [21]. TLR2 and TLR4 receptors are also involved in the histone-induced platelet activation and increased platelet pro-coagulant activity. Histones trigger platelet aggregation, P-selectin, phosphatidylserine and factor V/Va expression [22]. Of note, experimental studies have shown that NETs can serve as a surface for red blood cell and platelet adhesion, platelet activation and aggregation, thus resulting in thrombosis [23].

Tissue factor and factor XII-dependent coagulation

Neutrophils can actively participate in clotting through expression of tissue factor (TF) [24]. Neutrophil-derived externalized nucleosomes can trigger both TF- and contact activation pathways of coagulation [5]. These pro-coagulant actions can be exerted either through the formation of NETs or through direct effects of the neutrophil serine proteases (neutrophil elastase, cathepsin G) on blood coagulation, e.g. proteolytic cleavage of C1 inhibitor, TFPI, etc. [5,25]. Besides the pro-coagulant nature of cathepsin G, the latter can also function as a potent platelet activator [26–28]. Furthermore, externalized nucleosomes have the potential to activate factor (F)XII directly most probably via the negatively charged surfaces of the nucleic acids rather than the histone components [5,29]. Histones accelerate thrombin formation in platelet-rich plasma possibly via platelet-derived polyphosphates [22], but this process seems independent of FXII as it has been demonstrated previously [30].

Impairment of antithrombin and TFPI anticoagulant pathways

In vitro evidence suggests that neutrophil elastase can inactivate both antithrombin and TFPI [31,32]. Neutrophil-derived externalized nucleosomes promote recruitment and proteolytic deactivation of TFPI in vivo, mediated by both the action of the extracellular DNA fragments but also the serine proteases [5].

Impairment of TM/Protein C Axis

TM is a cellular transmembrane protein, predominantly expressed on vascular endothelium, but also found in other compartments of the vasculature [33]. TM protects against neutrophil-induced tissue damage by attenuating several molecular pathways, which mediate pro-inflammatory effects [34], whereas activated protein C (APC) can cleave histones reducing histone-induced cytotoxicity [35]. In this issue of the Journal of Thrombosis and Hemostasis, Ammollo et al. [36] present novel mechanistic data on the role of histones in modulating the anti-coagulant protein C pathway. Using the calibrated automated thrombography (CAT) method, the authors show that histones can induce a concentration-dependent increase in thrombin generation, which appears to be a result of a direct interaction with TM and/or the Gla-domain of protein C, thus resulting in an impairment of the TM-dependent protein C activation. This interaction may have profound consequences in conditions such as sepsis [37], where a deficit of protein C, hence the APC generating potential, occurs in the course of disseminated intravascular coagulation. Experimental studies have clearly shown that diminished PC increases mortality in primates, whereas the administration of (A) PC could prevent mortality from sepsis in the same model [38]. While the protective effects of recombinant APC in humans with severe sepsis are still equivocal [39], the experimental data are quite convincing in establishing a strong link between APC generation and tissue damage in systemic inflammatory conditions such as sepsis.

Besides systemic inflammation, histones also appear to be involved in localized tissue damage such as related to ischemia–reperfusion (I/R) injury [40,41]. In fact, one may speculate that some of the described protective effects of APC in I/R injury of the brain and the heart may in part be related to compensatory mechanisms for histone-mediated tissue damage. It raises the question whether antagonizing the effects of histones could be beneficial in limiting organ damage under inflammatory conditions? Several serum proteins and glycosaminoglycans may be able to neutralize histone activity [42]. Whether any of these effects can be exploited to improve the management of patients with sepsis or localized organ damage such as stroke remains to be investigated. This will certainly require additional insight into the molecular mechanisms and clinical relevance of NETs formation.

These data implicate an important role for NETs in the bidirectional inflammation-coagulation interplay. Overall, the evidence suggests that NETs may represent a promising therapeutic target against inflammation, tissue injury, multi-organ failure and thrombosis.


J.I. Borissoff is supported by a Marie Curie fellowship (MEST-CT-2005-020706), granted by the European Commission and is the recipient of a Kootstra Talent Fellowship (2011) from Maastricht University.

Disclosure of Conflict of Interest

The authors state that they have no conflict of interest.