Neutrophil extracellular traps and thrombosis in COVID-19

Here, we report on four patients whose hospitalizations for COVID-19 were complicated by venous thromboembolism (VTE). All demonstrated high levels of D-dimer as well as high neutrophil-to-lymphocyte ratios. For three patients, we were able to test sera for neutrophil extracellular trap (NET) remnants and found significantly elevated levels of cell-free DNA, myeloperoxidase-DNA complexes, and citrullinated histone H3. Neutrophil-derived S100A8/A9 (calprotectin) was also elevated. Given strong links between hyperactive neutrophils, NET release, and thrombosis in many inflammatory diseases, the potential relationship between NETs and VTE should be further investigated in COVID-19.


INTRODUCTION
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the disease known as coronavirus disease 2019 . It most commonly presents with influenza-like illness and viral pneumonia, but in its most severe manifestation progresses to acute respiratory distress syndrome (ARDS) and multi-organ failure 1 . To date the viral pandemic has resulted in more than two million infections worldwide (https://coronavirus.jhu.edu/map.html).
In COVID-19, elevated levels of blood neutrophils predict severe respiratory disease and unfavorable outcomes 2, 3 . Neutrophil-derived neutrophil extracellular traps (NETs) play a pathogenic role in many thrombo-inflammatory states including sepsis 4, 5 , thrombosis [6][7][8] , and respiratory failure 9,10 . NETs are extracellular webs of chromatin and microbicidal proteins that are an evolutionarily conserved aspect of innate immune host-defense 11 ; however, NETs also have potential to initiate and propagate inflammation and thrombosis 12,13 . NETs deliver a variety of oxidant enzymes to the extracellular space, including myeloperoxidase, NADPH oxidase, and nitric oxide synthase 14 , while also serving as a source of extracellular histones that carry significant cytotoxic potential 15,16 . NETs are drivers of cardiovascular disease by propagating inflammation in vessel walls 17 . Furthermore, when formed intravascularly, NETs can occlude arteries 18 , veins 19 , and microscopic vessels 20 . Early studies of COVID-19 suggest a high risk of morbid arterial events 21 , and the risk of venous thromboembolism (VTE) is increasingly revealing itself as more data become available 22 .
Descriptive and mechanistic studies to date that examine COVID-19 pathophysiology have focused on monocytes and lymphocytes more so than neutrophils and their effector productsincluding NETs. Here, we describe four cases of VTE in patients hospitalized with COVID-19 and provide evidence for neutrophil hyperactivity.

RESULTS
We identified four patients admitted to a large academic medical center with COVID-19 who also developed VTE (either deep vein thrombosis or pulmonary embolism) despite immediate initiation of prophylactic-dose heparin. Three of the patients were diagnosed with VTE within 48 hours of admission. All three had markedly elevated D-dimers and neutrophil-to-lymphocyte ratios ( Table 1). For two of the early VTE patients, we were able to access sera for measurement of NET remnants. Three different NET-associated markers (cell-free DNA, myeloperoxidase-DNA complexes, and citrullinated-histone H3) were elevated in both patients All rights reserved. No reuse allowed without permission.
was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which this version posted May 5, 2020. . https://doi.org/10.1101/2020.04. 30.20086736 doi: medRxiv preprint ( Table 1). We also detected elevated levels of the classic marker of neutrophil activation, S100A8/A9 (also known as calprotectin). Neither patient had positive testing for antiphospholipid antibodies 23 . We identified a fourth patient who developed VTE several weeks into his hospitalization. This 66-year-old man's course was complicated by respiratory failure requiring mechanical ventilation beginning on day 6. He received prophylactic-dose heparin throughout his hospitalization, but was nevertheless diagnosed on day 20 with extensive right-lower-extremity deep vein thrombosis. We were able to test serum from day 8 of his hospitalization for NET remnants ( Table 2). Interestingly, markers of neutrophil activation including NET remnants were already significantly elevated on day 8 ( Table 2); this is in contrast to D-dimer and neutrophil count, which were only mildly elevated. Both D-dimer and neutrophil count were All rights reserved. No reuse allowed without permission.
was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which this version posted May 5, 2020. . https://doi.org/10.1101/2020.04. 30.20086736 doi: medRxiv preprint elevated by the time the patient was diagnosed with deep vein thrombosis on day 20 ( Table 2).
The patient did not have elevated levels of antiphospholipid antibodies 23 .

DISCUSSION
Hyperactivity of the coagulation system is a common finding of severe COVID-19 24 . Indeed, many patients have a profile to suggest a prothrombotic diathesis including high levels of fibrin degradation products (D-dimer), elevated fibrinogen levels, and low antithrombin levels 24,25 .
Here, we report four cases of COVID-19-associated VTE. We measured three markers commonly used to detect NET remnants in blood (cell-free DNA, myeloperoxidase-DNA complexes, and citrullinated-histone H3), as well as a fourth marker, S100A8/A9 (calprotectin), which is commonly used to track neutrophil activation. All tests were elevated in patients diagnosed with VTE, including as early as 12 days prior to detection of VTE in one case. Given the known link between NETs and venous thrombosis in many inflammatory diseases, these data suggest that the role of NETs in COVID-19-associated thrombophilia warrants systematic All rights reserved. No reuse allowed without permission.
was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which this version posted May 5, 2020. . investigation. While a recent report suggested antiphospholipid antibodies may be drivers of thrombosis in some COVID-19 patients 23 , such antibodies were not detected here.
Examples of NETs as drivers of thrombosis are myriad, as intravascular NET release is responsible for initiation and accretion of thrombotic events in arteries, veins, and microvessels, where thrombotic disease can drive end-organ damage in lungs, heart, kidneys, and other organs 26,27 . Mechanistically, DNA in NETs may directly activate the extrinsic pathway of coagulation 28 , while NETs also present tissue factor to initiate the intrinsic pathway 29 . Serine proteases in NETs such as neutrophil elastase dismantle brakes on coagulation such as tissue factor pathway inhibitor 30 . Bidirectional interplay between NETs and platelets might also be critical for COVID-19-associated thrombosis as has been characterized in a variety of disease models 27,28 .
Approaches to combatting NETs 31, 32 include the dismantling of NETs with deoxyribonucleases and strategies that prevent initiation of NET release such as neutrophil elastase inhibitors and peptidylarginine deiminase 4 inhibitors. As we await definitive antiviral and immunologic solutions to the current pandemic, we posit that anti-neutrophil therapies may be part of a personalized strategy for some individuals affected by COVID-19. Furthermore, those patients with hyperactive neutrophils may be at particularly high risk for VTE and might therefore benefit from more aggressive anticoagulation while hospitalized.

METHODS
Human samples. Blood was collected into serum separator tubes by a trained hospital phlebotomist. After completion of biochemical testing ordered by the clinician, the remaining serum was stored at 4°C for up to 48 hours before it was deemed "discarded" and released to the research laboratory. Serum samples were immediately divided into small aliquots and was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which this version posted May 5, 2020. was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which this version posted May 5, 2020. .