A Novel Assay for Neutrophil Extracellular Trap Formation Independently Predicts Disseminated Intravascular Coagulation and Mortality in Critically Ill Patients

Abstract

Rationale: Neutrophil extracellular traps (NETs) are important in the host defense against infection, but they also promote intravascular coagulation and multiorgan failure in animal models. Their clinical significance remains unclear, and available assays for patient care lack specificity and reliability. Objectives: To establish a novel assay and test its clinical significance. Methods: A prospective cohort of 341 consecutive adult ICU patients was recruited. The NET-forming capacity of ICU admission blood samples was semiquantified by directly incubating patient plasma with isolated neutrophils ex vivo. The association of NETforming capacity with Sequential Organ Failure Assessment scores, disseminated intravascular coagulation, and 28-day mortality was analyzed and compared with available NET assays. Measurements and Main Results: Using the novel assay, we could stratify ICU patients into four groups with absent (22.0%), mild (49.9%), moderate (14.4%), and strong (13.8%) NET formation, respectively. Strong NET formation was predominantly found in sepsis (P , 0.0001). Adjusted by Acute Physiology and Chronic Health Evaluation II score, multivariate regression showed that the degree of NET formation could independently predict disseminated intravascular coagulation and mortality, whereas other NET assays (e.g., cell-free DNA, myeloperoxidase, and myeloperoxidase-DNA complexes) could not. IL-8 concentrations were found to be strongly associated with NET formation, and inhibiting IL-8 significantly attenuated NETosis. Mitogen-activated protein kinase activation by IL-8 has been identified as a major pathway of NET formation in patients. Conclusions: This assay directly measures the NET-forming capacity in patient plasma. This could guide clinical management and enable identification of NET-inducing factors in individual patients for targeted treatment and personalized ICU medicine.

Increased Circulating Levels of Neutrophil Extracellular Traps During Cardiopulmonary Bypass

“…Recent evidence has shown that NETs formation by neutrophils collected from a cohort of critically ill patients independently predicts mortality and disseminated intravascular coagulation. 37 Consequently, inhibiting NETs formation by the use of peptidyl arginine deiminase inhibitors 38 or inducing degradation of circulating NETs by using exogenous DNase I may represent promising therapeutic avenues. 39 Our original hypothesis was that NETs formation would be correlated with production of IL-6, an early inflammation marker contributing to neutrophil activation.…”

The Emerging Role of Vitamin C as a Treatment for Sepsis

“…Furthermore, vitamin C protects against the loss of epithelial and endothelial barriers and enhances neutrophil function in a multidimensional way. Moreover, vitamin C promotes lymphocytic and neutrophilic activity while attenuating neutrophil necrosis and NETosis (neutrophil extracellular trap), which contributes to multiorgan failure [69][70][71][72][73][74][75][76][77][78][79][80][81][82][83][119][120][121][122]. Vitamin C also regulates nuclear cellular responses to stress and hypoxia by regulating HIF-1α [40,49,52,62,123], produces NF-κB epigenetic modifications [124] through its ability to de-methylate histones [75,82,[87][88][89]125,126], regulates pro-inflammatory and coagulation gene expression [28,41,42,44,123,[125][126][127], and orchestrates the immune system and circulating cytokine homeostasis in pleotropic ways ( Table 1).…”

Flow Cytometry-Based Quantification of Neutrophil Extracellular Traps Shows an Association with Hypercoagulation in Septic Shock and Hypocoagulation in Postsurgical Systemic Inflammation—A Proof-of-Concept Study

“…The capture of pathogens decelerates their spread and concentrates neutrophil antimicrobial activity. However, NETs might contribute to negative effects, such as excessive activation of coagulation, which eventually results in disseminated intravascular coagulation [18][19][20]. Several determinants lead to an NET-induced activation of the coagulatory system: Due to their polyanionic surface, NETs activate the intrinsic plasmatic system while the extrinsic pathway is stimulated by tissue factor presentation of NETs and platelets are strongly activated by DNA/histones complexes [21][22][23][24].…”

“…Several determinants lead to an NET-induced activation of the coagulatory system: Due to their polyanionic surface, NETs activate the intrinsic plasmatic system while the extrinsic pathway is stimulated by tissue factor presentation of NETs and platelets are strongly activated by DNA/histones complexes [21][22][23][24]. However, despite an increasing number of studies hinting towards a procoagulatory effect of NETs, the clinical relevance of these findings remains controversial [18,19,23,25].…”