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The interpretations below are provided by Donna Castellone, MS, MT (ASCP) SH for Aniara Diagnostica, Inc.

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Introduction:

Neutrophils and neutrophil extracellular traps (NETs) play an important role in abnormal thrombus formation. NETs are extracellular structures that are released by neutrophils when stimulated.¹ When neutrophils become activated, they are flattened within minutes, and their nuclear lobes disappear within an hour. The chromatin decondenses and the inner and outer lobes of the nuclear membrane separates into vesicles. The nucleoplasm and cytoplasm merge into homogeneous clumps. Eventually the cells condense and become round, the cytoplasmic membrane ruptures and releases the intracellular components, forming fibrous bundles known as NETs.²

NETs include the presence of pathogens, histones and bacteriostatic proteins resulting in the ability for NETs to confine and prevent the spread of pathogens and assist phagocytes in killing pathogens. The structure of NETs can provide a basis for the aggregation of RBC and platelets and can also activate the coagulation pathway and be involved in thrombosis formation.¹ NETs are fibrous network structure which contain DNA, histone, myeloperoxidase, neutrophil elastase, and cathepsin G which not only kill bacteria but also promote a prothrombotic state by further activating platelets. P-selectin which is found on the platelets attach to the neutrophil surface receptor PSGL-1 to release more NETs.³

NETs and Immunothrombosis

A link between inflammation and thrombosis is found in neutrophils. An inflammatory response occurs when the body encounters exogenous or endogenous stimuli through the action of phagocytes and cytokines which initiate the immune response. Excessive cytokine production results in vascular permeability as well as interaction with platelets and results in a hypercoagulable state predisposing the body to thrombosis. Platelets, which are involved in hemostasis also are involved in host defense producing a wide range of immune mediators including growth factors, cytokines and chemokines.³

Disorders that have reactive or clonal expansion of neutrophils are associated with an increased risk for thrombosis. It is known that monocytes can exhibit a procoagulant phenotype that directly upregulate TF, however it appears that neutrophils acquire TF from activated monocytes. Coagulation occurs on negatively charged surfaces which can be activated by neutrophil derived DNA which could be released after cell death or activation which have been found in disease states associated with an increased risk for thrombosis. The process of NETosis was discovered in 2004 which is when neutrophils expel their nuclear material in a meshwork called neutrophil extracellular traps or NETs.⁴

There are two types of NET formation: suicidal NETosis which activate Fc receptors, toll-like receptors, and complement receptors as a result of the stimulation of interleukin-8 and bacterial surface antigens.⁵ Survival NETs release neutrophils activate TLR-4 and TLR-2 receptors under the stimulus of bacterial lipopolysaccharide and gram-negative bacteria, resulting in PAD4 activation resulting in DNA unwinding.⁶

Immunothrombosis is defined as the process of NETs role in host defense by trapping and killing pathogens at the expense of promoting thrombosis. The exact mechanism by which NETs promote thrombosis is not clear and appear to involve blood cells, soluble components of the plasma, vessel wall and blood flow disturbances. Evidence indicates that intravascular release of NETs promotes vascular occlusion with or without fibrin generation.⁴

Role In Coagulation

Studies have shown that NETs are involved in human arteriovenous thrombosis samples, mouse DVT and other disease models. They have been found to attract platelet activation as well as activating internal and external coagulation pathways.¹

NETs may activate coagulation vial contact activation of FXII, however it was also discovered that NETs can occlude vessels independent of activation of coagulation. When gDNA is introduced into plasma the clotting time is shortened which is similar to bacterial and mitochondrial DNA which are also procoagulant. This occurs due to the direct activation of the contact system leading to FXIIa formation which then activated PK and FXI resulting in thrombin generation. Fibrinolysis is also impaired by gDNA by forming a complex with plasmin and fibrin prevention plasmin-mediated fibrin degradation.

NETs serve as a cell receptor for FVII/VIIa along with TF activates FX and FIX, which FXa with cofactor Va cleaves prothrombin to thrombin, resulting in cleaving fibrinogen to fibrin. FXIII then stabilizes the thrombin clot. NETs network structure provides a framework for platelets, RBC, fibrinogen, vWF and extracellular bodies conducive to thrombosis. NETs contain histones which can attract platelet aggregation and activation through the interaction of fibrinogen, TLR2 and TLR4 and promote an increase in thrombin generation. Histones can also promote the expression of tissue factor on vascular endothelium and macrophages and promote coagulation as well as inhibit activated protein C.⁷

NETs have also been shown to promote erythrocyte rich thrombi in vitro and are directly bound to NETs. They also interact with fibronectin and vWF to attract and promote platelet adhesion and activation. Due to thrombus stability, NETs are more sensitive to tPA due to fibrin deposition as opposed to thrombi with fibrin as the main component.¹

Mechanisms of Neutrophil-Driven Coagulation

NETs have a significant effect on coagulation and promote thrombosis by interacting with endothelial cells, platelets, RBC and coagulation factors. Histones activate platelets, inhibit aPC and activate thrombin. DNA can activate factor XII and initiate coagulation.³

Neutrophils directly activate coagulation pathways via:

1. Tissue Factor (TF) Expression: Activated neutrophils surface-expose TF, initiating thrombin generation and fibrin deposition. A seminal study by Darbousset et al. demonstrated that neutrophils arriving at sites of laser-induced vascular injury precede platelets, releasing TF and thrombin to nucleate thrombus formation.⁸
2. NETosis: NETs—chromatin fibers decorated with histones, NE and MPO—serve as scaffolds for platelet adhesion and amplify thromboinflammation. H3/H4 histones bind von Willebrand factor vWF and platelet receptors, triggering aggregation, while NE degrades anticoagulants like tissue factor pathway inhibitor (TFPI).⁹
3. Biomechanical Signaling: Mechanical stress (e.g., arterial stiffness, hypertension) primes neutrophils for NETosis via calpain-PI3K/FAK pathways, linking hemodynamic forces to thrombosis 10 Flow shear stress increases the intracellular calcium level by activating Piezo1 ion channel, promotes NETosis, and enhances platelet adhesion to NETs¹¹
4. Migrasomes: Recently identified neutrophil-derived vesicles adsorb coagulation factors (e.g., prothrombin, FX) and localize to injury sites, acting as platforms for fibrin generation.¹²

Other disorders and NETs:

NETs in AMI and stroke are more likely caused by arterial thrombosis. Numerous neutrophils have been found in tissue samples in patients with AMI long before the discovery of NETs. NETs were discovered in thrombus samples and plaques in these patients and the content positively correlated with the extent of MI and degree of ST-segment elevation as well as the severity of outcomes within 2 years providing evidence of NETs as a prognostic indication in MI.¹

Stroke is caused by thrombus resulting in a high morbidity and mortality rate. Neutrophils are recruited in the brain after stroke and contribute to brain injury due to multiple mechanisms. NETs trap bacteria, degrading pathogenic factors but they can also exacerbate certain non-infectious diseases by activating autoimmune or inflammatory responses and play a role in the pathological process of stroke. They promote the coagulation cascade and interact with platelets resulting in thrombosis. The higher the NETs level the worse the neurological function. NETs aggravate stroke by mediating inflammation, atherosclerosis and vascular injury. They may serve as a biomarker of stroke and may be a target for stroke treatment.¹³ In a study of stroke thrombosis, NETs were found to be more abundant in cardiogenic and structurally mature old thrombi, and the content was positively correlated with the duration of thrombectomy and the number of surgical instruments used, which prompted the local microenvironment to affect NETosis.¹

Sepsis is an inflammatory response to infection and can result in activation of the coagulation system which may lead to DIC, microvascular thrombosis, hypoperfusion and multiple organ dysfunction and death. In sepsis patients, a large amount of lipopolysaccharide is shed by bacteria which activated neutrophils to produce NETS. Neutrophils isolated from the blood of patients with sepsis can release tissue factor (TF) through NETs, and this form of TF can induce thrombin generation in vitro and play a key role in the activation of the coagulation system in sepsis1. NETosis amplifies platelet activation, fibrin deposition and systemic inflammation which exacerbates organ failure. This has been seen in ARDS, blocking NETs can reduce disease progression. Elevated NET markers (histones and MPO) are associated with microvascular endothelial injury and hypercoagulability. In MIS-C in pediatric patients NETs may perpetuate cytokine storms despite the resolution of the virus. However, studies have shown that the thrombosis mechanism for these disorders is different in adults and children. Adults' mechanism is driven by fibrin-mediated red blood cell aggregation, while in children, it is more likely to be associated with excessive cytokine release, suggesting that NETs may lead to thrombotic inflammation through different pathways in different age groups.³

Therapies:

The formation of NETs results in a complicated outcome of thrombosis. There are several therapies that interfere with the production of NETs reducing the risk of thrombosis.

1. DNase-1 (deoxyribonuclease I) breaks down the DNA network of NETs and disrupts its structure reducing thrombus formation.³
2. PF4 can bind to NETs and enhance resistance to DNase, stabilizing NETs and reducing the release of NET degradation products (cell-free DNA and histones) which are factors in thrombus formation.³
3. Inhibiting PAD4 enzyme which is key in NETs formation can reduce the release of NETs. Inhibiting the activation of neutrophils can also reduce NET formation.³
4. Anti-platelet drugs may prevent platelet adhesion by targeting GPIIb/IIIa receptors on the surface of platelets.⁴
5. Complement inhibitors (such as anti-C5 monoclonal antibodies) may reduce the prothrombotic effects of NETs by inhibiting the activation of the complement system.

Conclusions:

Understanding the process that activated neutrophils become NETs and contribute to thrombus formation is an important process. Clinicians need to be aware of the outcomes and treatments that can aid in controlling outcomes.

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References

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8. Darbousset R., Thomas G.M., Mezouar S., Frère C., Bonier R., Mackman N., Renné T., Dignat-George F., Dubois C., Panicot-Dubois L. Tissue factor–positive neutrophils bind to injured endothelial wall and initiate thrombus formation. Blood. 2012;120:2133–2143
9. de Los Reyes-García A.M., Aroca A., Arroyo A.B., García-Barbera N., Vicente V., González-Conejero R., Martínez C. Neutrophil extracellular trap components increase the expression of coagulation factors. Biomed. Rep. 2019;10:195–20
10. Khanmohammadi M., Danish H., Sekar N.C., Suarez S.A., Chheang C., Peter K., Khoshmanesh K., Baratchi S. Cyclic stretch enhances neutrophil extracellular trap formation. BMC Biol. 2024;22:209.
11. Baratchi S., Danish H., Chheang C., Zhou Y., Huang A., Lai A., Khanmohammadi M., Quinn K.M., Khoshmanesh K., Peter K. Piezo1 expression in neutrophils regulates shear-induced NETosis. Nat. Commun. 2024;15:7023.
12. Jiang D., Jiao L., Li Q., Xie R., Jia H., Wang S., Chen Y., Liu S., Huang D., Zheng J., et al. Neutrophil-derived migrasomes are an essential part of the coagulation system. Nat. Cell Biol. 2024;26:1110–1123.
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