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Neutrophil Extracellular Traps Promote Hypercoagulability in Patients With Sepsis

Yang, Shuofei; Qi, Haozhe; Kan, Kejia; Chen, Jiaquan; Xie, Hui; Guo, Xiangjiang; Zhang, Lan

doi: 10.1097/SHK.0000000000000741
Clinical Aspects
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Background: Patients with sepsis commonly exhibit a hypercoagulability with high risk of venous thromboembolism (VTE). Neutrophil extracellular traps (NETs) are found to trigger inflammation and coagulation. We aim to determine whether NETs promoted the hypercoagulability and early anticoagulation reduced NETs releasing during sepsis.

Methods: In this prospective study, septic patients between September 2013 and June 2015 were included. Patients of age <18 years, acute organ failure, pregnancy, coagulation disorders, receiving anticoagulation before admission were excluded. Blood was sampled in 52 sepsis and 10 non-sepsis patients and 40 healthy controls, clinical, and hematological parameters were collected. The ability of plasma and platelets to prime neutrophils to release NETs and contribution of NETs to coagulation were assessed. NETs releasing was compared in patients with or without early coagulation, and its correlation with the risk of VTE was also evaluated.

Results: NETs formation in septic patients was significantly higher than controls and non-sepsis patients. Neutrophils from septic patients had significantly enhanced NETs releasing compared with those from controls or non-sepsis patients. Plasma or platelets obtained from patients induced control neutrophils to release NETs. Notably, NETs released by neutrophils from septic patients significantly increased the potency of control plasma to generate thrombin and fibrin, and this effect was attenuated by administration of DNase I. Post-treatment NETs releasing in septic patients receiving early anticoagulation within 6 h was significantly lower than patients without early anticoagulation. The NETs formation correlated positively with the VTE risk, rather than the parameters of inflammation or disease severity.

Conclusions: The systemic inflammation during sepsis primes neutrophils to release NETs with increased risk of VTE. Early anticoagulation (6 h) reduces NETs releasing and may improve the coagulopathy of septic patients.

Department of Vascular Surgery, Renji Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China

Address reprint requests to Lan Zhang, MD, PhD, Department of Vascular Surgery, Renji Hospital, School of Medicine, Shanghai Jiaotong University, Dongfang Road 1630, Shanghai 200127, China. E-mail: zhanglanrjxg@gmail.com

Received 13 June, 2016

Revised 5 July, 2016

Accepted 23 August, 2016

The authors report no conflicts of interest.

Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's Website (www.shockjournal.com).

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INTRODUCTION

Sepsis is a life-threatening condition characterized by systemic activation of inflammation in response to microbe infection, with a daunting mortality rate of 25% to 50% for adults and 15% to 70% for children (1, 2). Patients with sepsis have coagulation abnormalities ranging from modest decrease in platelet count and prolongation of clotting times to disseminated intravascular coagulopathy. Septic patients are at high risk of venous thromboembolism (VTE) manifesting as deep vein thrombosis (DVT) or pulmonary embolism (PE), which is the third leading cause of cardiovascular mortality after myocardial infarction and stroke (3). Based on the guidelines for sepsis management, prophylaxis of VTE is recommended with daily subcutaneous low-molecular weight heparin (LMWH) (4). However, early anticoagulant use in these patients remains a significant challenge owing to unequal distribution of venous thrombosis incidence and risks of bleeding related to anticoagulation.

Many clinical trials exploring the agents designed to attenuate coagulation disorders during sepsis have failed, and the outcome remains poor. Thus, a better understanding of the pathogenesis of hypercoagulable state in septic patients is urgently needed. Neutrophil extracellular traps (NETs) generated from activated neutrophils have recently been shown to offer a novel mechanism between inflammation and thrombosis (5). NETs are part of the innate immune response to microbe infections and consist of cell-free DNA (cfDNA), histones, and neutrophil granule proteins (e.g., elastase, myeloperoxidase, and cathepsin G) (6). Neutrophils are the major source of DNA released from activated whole blood, and circulating levels of cfDNA are significantly elevated in septic patients (7). High levels of cfDNA are associated with impaired fibrinolytic activity in septic patients (8).

The cellular events during sepsis that trigger VTE in response to systemic inflammation remain poorly defined. Neutrophils and platelets are found to be indispensable for the initiation and propagation of thrombosis through both platelet-dependent and platelet-independent mechanisms (9). A positive correlation between endogenous cfDNA and thrombin generation which is attenuated with DNase infusion is found in platelet poor plasma samples from patients with sepsis (10). NETs stimulate platelets via histones H3 and H4 to promote the thrombotic reaction (11). Elevated levels of circulating nucleosomes and neutrophil elastase (NE)/α1-antitrypsin complexes contribute to a 3-fold risk of DVT (12). Neutrophils are the main sources of cfDNA in DVT patients, and cfDNA correlated positively with D-dimer, von Willebrand factor (vWF), and myeloperoxidase (MPO) (13).

In this study, we hypothesize that NETs formation promotes the hypercoagulable state during sepsis. The ability of plasma and platelets from septic patients to prime neutrophils to release NETs and the contribution of NETs to coagulation in septic patients are assessed in vitro. The markers of NETs releasing are compared in septic patients with or without early coagulation, and their correlation with the risk of VTE is also evaluated.

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PATIENTS AND METHODS

Patients and study design

In this prospective study, consecutive patients with sepsis admitted to surgical intensive care unit (ICU), Renji Hospital, China, between September 2013 and June 2015, were enrolled. The exclusion criteria were: age <18 years; irreversible acute heart, hepatic or renal failure at admission; pregnancy or lactation; thromboembolic complications at admission; platelets and/or blood coagulation disorders; receiving anticoagulant and/or antiplatelet treatment before admission. Diagnosis of sepsis and organ dysfunction was according to the international guidelines for management of severe sepsis and septic shock (4). The initial time of anticoagulation is determined based on the balance of anticoagulation benefit and risk of bleeding in specific patients. The prophylactic anticoagulation is usually started with 80 U/kg to 100 U/kg LMWH every 24 h. Informed consent was obtained from each patient and healthy control. This study protocol was reviewed and approved by the Institutional Review Board of Renji Hospital and conducted in accordance with the principles in the Declaration of Helsinki.

Finally, 52 sepsis patients were enrolled and 10 non-sepsis patients in ICU and 40 healthy controls from the hospital staff were recruited. Pretreatment (on ICU admission) and post-treatment (36 h after ICU admission) blood samples were collected from patients. Count of neutrophils and plasma levels of D-dimers and C-reactive protein (CRP) were determined in hospital's routine laboratories. Demographic information (e.g., age, gender, disease, or family history) was assessed at admission. Adapted Caprini score for VTE risk assessment and acute physiology and chronic health evaluation (APACHE) II score were calculated at ICU admission. Patients with Caprini score >5 were associated with high risk of VTE. The septic patients were stratified by Caprini score ≤ or >5, and their plasma levels of cf-DNA, MPO-DNA, and NE were compared. In addition, the correlation between NETs releasing markers (cf-DNA, MPO-DNA, NE) and APACHE II score or CRP level was analyzed.

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Sample collection and isolation of platelets and neutrophils

All procedures for the collection and centrifugation of plasma were performed at 0°C. Fresh whole venous blood samples were collected into 3.2% sodium citrate and centrifuged at 150 × g for 10 min to obtain platelet-rich plasma (PRP). Platelets were isolated immediately from PRP by centrifugation at 600 × g for 10 min, and then resuspended in HEPES buffer and used immediately for analysis or in vitro stimulations. Platelet-free plasma was prepared with two serial centrifugations at 2,500 × g for 15 min and stored in aliquots at −80°C until used (14). Neutrophils were isolated by a discontinuous Percoll gradient as described previously (15). Purity of neutrophil was assessed by Diff–Quik stain and viability by Trypan blue stain (both >98%).

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In vitro stimulation studies

Purified neutrophils (1 × 106) isolated from patients with sepsis or healthy controls were incubated for 3 h at 37°C in 5% CO2. For in vitro studies, only neutrophils from control individuals (n = 5) were treated with 6% plasma isolated from individual patient (n = 52) or from healthy control individual (n = 40) or phosphate-buffered saline (PBS). They were also treated with platelets derived from patients (n = 52) or healthy controls (n = 40) individually in a ratio of 1:50 for 3 h.

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NETs production and isolation

NETs were prepared and isolated as previously described (16). In brief, 1.5 × 106in vitro-stimulated neutrophils were left in culture for 4 h. Then the medium was removed and cells were washed with RPMI. Two milliliters RPMI was added to each well and NETs were collected on supernatant medium after vigorous agitation. The medium was centrifuged at 20 × g for 5 min and supernatant phase containing NETs was collected and stored at −20°C. Very low concentration of NETs collected from untreated control neutrophils that undergo spontaneous NETs generation (about 3%) was used as a vehicle control. NETs were produced with neutrophils from both patients and healthy controls.

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Quantification of cfDNA, MPO-DNA complex, and NE

Circulating cfDNA, MPO-DNA complex, and NE have been studied as surrogate markers for NETs releasing. The supernatants of media were collected by centrifugation at 1,500 × g for 10 min, and cfDNA was quantified with fluorescence quantification. Briefly, 50 μL samples were mixed with 50 μL SytoxGreen (Invitrogen) to label the DNA. Fluorescence was recorded with a Spectramax microplate fluorometer (Fluoroskan; Thermo Fisher Scientific, Waltham, Mass). Autofluorescence was considered as background and determined in samples mixed with PBS without SytoxGreen. DNA concentrations were calculated based on a standard curve of known concentrations of DNA. To quantify NETs release in supernatant, the amount of circulating MPO-DNA complex was measured using a modified capture ELISA technique as previously described (17). Values for soluble NET release are expressed as percentage increase in absorbance above control. NE (BlueGene Biotech) was measured using ELISA kits according to the manufacturer's instructions.

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Immunofluorescence assay

NETs were visualized by immunofluorescence confocal microscopy as previously described (18). Samples were stained using antihuman neutrophil elastase (Abcam) and antihuman myeloperoxidase (BD Bioscience) antibodies. The primary antibodies were detected with the following secondary antibodies: Alexa Fluor 488–conjugated donkey antimouse and Alexa Fluor 568-conjugated donkey anti-rabbit (both from Invitrogen). Visualization was performed in Nikon ECLIPSE Ti microscope (Tokyo, Japan). The percentage of NET-releasing cells was determined by examining 200 cells in a double-blind experimental procedure.

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Thrombin and fibrin generation test

Thrombin generation was assessed using the thrombin–antithrombin complex (TAT) as previously described (19). Briefly, 80 mL plasma obtained from patients or controls or control plasma stimulated in vitro with NET structures (20%) was incubated with PBS or DNase I (400 μg/mL, sigma) for 30 min at 37°C in a 96-well plate. Clotting was initiated by the addition of CaCl2 (0.1 M). The reaction was performed for 5 min at 37°C. Samples were immediately transferred into ice to stop the reaction. TAT level was measured according to manufacturer's instructions (Assaypro). To further test the procoagulant role of NETs, fibrin formation was monitored by measuring the optical density (405 nm) of the plasma on a Spectramax microplate reader at 37°C for 1 h as previously described (20).

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Statistical analysis

Statistical analysis was performed with at least three observations in each experiment. Continuous variables were defined as means ± standard deviation if they were normally distributed and t tests were used; otherwise, median values and interquartile ranges (IQR; 25th percentile to 75th percentile) were represented and Mann–Whitney tests were used. Groups for categorical variables were analyzed by Chi-square or Chi-square correction methods. To explore the relationship between variables, Spearman tests were used. The SPSS software (version 12.0; SPSS Inc, Chicago, Ill) was used for statistical analysis. Statistical significance was accepted for a P value of 0.05.

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RESULTS

Demographic information and hematological parameters

The demographic information and hematological parameters of septic patients and healthy subjects are summarized in Table 1 and supplementary table, http://links.lww.com/SHK/A468. There were no significant differences of age and gender between patients and healthy controls. In addition to cell counts of neutrophils and platelets, plasma levels of CRP, D-dimer, and TAT were dramatically higher in septic patients than healthy controls (P <0.01). The Caprini and APACHE II score of septic patients were 6.96 ± 4.66 and 16.6 ± 5.03, respectively. These data suggest a hypercoagulable state and high risk of VTE in patients with sepsis.

Table 1

Table 1

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Neutrophils are primed to form NETs during sepsis

The percentage of ex vivo NET releasing neutrophils and plasma cfDNA levels from patients with sepsis were significantly higher than that from healthy controls (P <0.01, Table 1). Significant elevation of cfDNA and MPO-DNA complex levels, and percentage of NETs releasing cells was observed in control neutrophils following stimulation of plasma or platelets from septic patients in vitro compared with control neutrophils stimulated with plasma or platelets from healthy individuals and non-sepsis ICU patients, which further illustrate the circulation environment of sepsis induces neutrophils to release NETs (P <0.01, Fig. 1).

Fig. 1

Fig. 1

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Procoagulant activity of NETs derived from patients with sepsis

TAT levels and potency of fibrin generation were tested to indicate the procoagulant activity of NETs. TAT levels increased and time to peak of fibrin generation shortened significantly in control plasma treated with NETs prepared with neutrophils from septic patients than those from healthy controls and non-sepsis ICU patients (Fig. 2). This effect of NETs produced by neutrophils derived from patients with sepsis was significantly inhibited by treatment with DNase I, further certifying the procoagulant role of NETs (Fig. 2).

Fig. 2

Fig. 2

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Early anticoagulation (<6 h) is associated with reduced NETs releasing during sepsis

To further study the effect of early anticoagulation on NETs releasing, the levels of circulating NETs markers were compared between septic patients with (n = 27) or without (n = 25) early use of LMWH within 6 h. No significant differences of age, hematological parameters, markers of NETs releasing, Caprini and APACHE II score at baseline between two groups were detected. The VTE incidence in patients with late anticoagulation was significantly higher than patients without early anticoagulation (P = 0.04, Table 2, Fig. 3). Post-treatment circulating levels of cfNDA, NE, and MPO-NDA complex from patients receiving early anticoagulation were all significantly lower than those of patients without early anticoagulation within 6 h (Fig. 3).

Table 2

Table 2

Fig. 3

Fig. 3

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Correlation of NETs releasing and risk of VTE in patients with sepsis

The circulating levels of cfNDA, NE, and MPO-NDA complex of septic patients with Caprini score ≥5 were significantly higher than patients with Caprini score <5 (Fig. 4). Furthermore, the circulating levels of cfNDA, NE, and MPO-NDA complex correlate positively with the plasma levels of TAT, D-dimer, and Caprini score, respectively, rather than the parameters of disease severity and inflammatory reaction, the APACHE II score, and CRP level (Fig. 5). These data strongly suggest that NETs contribute to coagulation activating and high risk of VTE in septic patients.

Fig. 4

Fig. 4

Fig. 5

Fig. 5

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DISCUSSION

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.

Plasma or platelets obtained from patients with sepsis were found to be able to induce neutrophils from healthy controls to release NETs in vitro in this study. In addition, neutrophils from septic patients could induce enhanced procoagulant NETs formation than healthy controls. It is still unknown how neutrophil activation and NETs formation contribute to thrombus initiation and propagation during sepsis. NETs form predominantly during the organizing stage of VTE development (26). It has been found that nucleosomes exposed on NETs form a template for neutrophil proteases to inactivate tissue factor pathway inhibitor, thereby propagating coagulation (27). A similar inactivation mechanism could be postulated for antithrombin or protein C, whereas coagulation via NETs-induced factor XII activation represents another pathway (28). Of note, experimental studies have shown that NETs can serve as a surface for red blood cell and platelet adhesion, activation, and aggregation, thus resulting in thrombosis (29).

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.

Nonetheless, NETs may also promote thrombolysis. In vitro, the proteases within NETs can degrade fibrin and enhance fibrinolysis. NETs recruit plasminogen from the plasma, and histone H2B serves as a receptor for plasminogen on the surface of human monocytes/macrophages (35). NETs and fibrin degradation by plasmin and DNase result in simultaneous release of DNA and fibrin fragments. In baboon model of DVT, plasma DNA increases with similar kinetics to the fibrin degradation product D-dimers (29). Here, we found that levels of NETs releasing in plasma correlated positively with levels of TAT, D-dimer, and Caprini score, rather than the APACHE II score and CRP level, which suggests that plasma markers of NETs may be appropriate predictors of VTE in septic patients. Further studies are necessary to elucidate the magnitude of contribution of neutrophils to coagulation activation. The issues that how NETs and their components intensify activation of the coagulation cascade and prime other cell populations (e.g., platelets and endothelial cells), and how neutrophil-derived proteases (e.g., neutrophil elastase and cathepsin G) accelerate these sequential reactions should be further addressed. Given the increasing evidence for the prothrombotic activity, host inflammatory, and immune responses of NETs, our finding can provide the foundation for its further functional study to better understand disease pathogenesis and develop new therapies.

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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Keywords:

Deep vein thrombosis; low-molecular heparin; neutrophil extracellular traps; procoagulant activity; sepsis

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