The inter-relationship between inflammation, coagulation, and thrombosis, mediated through the action of NETs has been the focus of much recent attention. Recently, the term “immunothrombosis” designating an innate immune response that induces thrombosis and scaffold generation within microvessels by immune cells and specific thrombosis-related molecules has been introduced (21). Persisting inflammation triggers an over-reactive host defense response disrupting the immune balance and contributing to thrombosis (22). It has been confirmed in animal models and human thrombus that NETs are crucial for pathological thrombosis and NETs components are generally present within the thrombi (23). Extensive microvascular thrombosis contributes to diminished oxygen delivery and subsequent organ dysfunction during sepsis. Levels of cfDNA have discriminative power to predict mortality in patients with sepsis (24). In a model of polymicrobial sepsis, cfDNA level rises within a few hours, accompanied by elevations in interleukin-6 and TAT complex (25). Moreover, high levels of cfDNA in plasma correlate with impaired fibrinolytic activity in septic patients (8). In this study, the procoagulant activity of NETs releasing in patients with sepsis is disclosed for the first time. This phenomenon is infection/sepsis related and not general in all critically ill patients.
There is complicated interaction between neutrophil, platelets, and vascular endothelial cells for the mechanism of thrombosis. Using a mouse model of flow restriction-induced DVT, monocytes and neutrophils are identified as the first blood cells recruited to the vessel wall within the initial hours, contributing to DVT through the delivery of tissue factor and the release of NETs (9). It has been recently reported that the interaction of thrombin-activated platelets with neutrophils at the site of plaque rupture during acute ST-segment elevation myocardial infarction results in local NETs formation and delivery of active tissue factor (19). Furthermore, histone/DNA complexes are potent activators of human platelets via Toll-like receptor-2 and -4, and histones can directly induce the endothelial cell cytotoxicity and maximize the platelet–endothelial interaction (16). The vWF binds and immobilizes extracellular DNA released from neutrophils and acts as a linker for neutrophils adhesion to endothelial cells (30).
NETs can promote coagulation and approaches to destabilize NETs have been explored to reduce thrombosis and treat sepsis. Recent studies highlight heparinoids with low intrinsic anticoagulant activity as antihistone and NETs disruption therapies on the treatment of sepsis and disseminated intravascular coagulation (31). In this study, we found that patients with sepsis receiving early anticoagulation within 6 h were associated with significantly lower level of NETs releasing than patients without early anticoagulation. It has been shown that heparin can block the direct binding of vWF and cfDNA from NETs (30). Anticoagulation by unfractionated heparin and LMWH will be compromised by high affinity binding to circulating histones even in the presence of DNA, relevant to circulating histone concentrations during disease states (32). This may provide a rationale for understanding the sources of heparin resistance in situations where circulating histones are present, and anticoagulation should be instituted early before accumulation of histones from NETs.
To degrade the thrombi to restore the blood flow, fibrin and vWF as the main scaffolds need to be fragmented by the plasmin, disintegrin, or metalloproteinase. NETs provide a newly recognized scaffold for blood clots that is resistant to tissue plasminogen activator (tPA)-induced thrombolysis (29). In the presence of tPA, blood clots lacking fibrin are held together by a scaffold of cfDNA. In a murine model of flow restriction-induced DVT, the venous thrombi contained extracellular citrullinated histone H3 (CitH3), and infusion of DNase I can protect mice from DVT (33). In this study, we found that NETs contributed to the hypercoagulable state in patients with sepsis, and the effect could be inhibited with DNase treatment in vitro. DNase I is the predominant nuclease in plasma with only limited activity to degrade chromatin because it preferentially degrades protein-free DNA. Activated plasminogen degrades histones and therefore allows for degradation of DNA by DNase I (34). Dissolution of NETs might thus facilitate thrombolysis and provides new perspectives for therapeutic advances. Delayed administration of recombinant DNase can reduce plasma cfDNA level, decrease bacterial load, and attenuate organ damage (25). Thus, the timing of administration may be a crucial element in future investigation of the therapeutic potential of DNase for sepsis.
In conclusion, systemic inflammation primes neutrophils to release NETs that contribute to the hypercoagulable state in patients with sepsis. The effect can be inhibited with DNase I treatment in vitro. Moreover, NETs releasing is associated with increased risk of VTE in septic patients. Early anticoagulation within 6 h may reduce the NETs releasing during sepsis. Accordingly, targeting NETs could attenuate the hypercoagulability and result in a declined incidence of VTE in septic patients.
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