key: cord-0967833-cnp2odtg
authors: Busch, Matthias H.; Timmermans, Sjoerd A.M.E.G.; Nagy, Magdolna; Visser, Mayken; Huckriede, Joram; Aendekerk, Joop P.; de Vries, Femke; Potjewijd, Judith; Jallah, Borefore; Ysermans, Renée; Oude Lashof, Astrid M.L.; Breedveld, Paul H.; van de Poll, Marcel C.G.; van de Horst, Iwan C.C.; van Bussel, Bas C.T.; Theunissen, Ruud O.M.F.I.H.; Spronk, Henri M.H.; Damoiseaux, Jan G.M.C.; ten Cate, Hugo; Nicolaes, Gerry A.F.; Reutelingsperger, Chris P.; van Paassen, Pieter
title: Neutrophils and Contact Activation of Coagulation as Potential Drivers of COVID-19
date: 2020-09-18
journal: Circulation
DOI: 10.1161/circulationaha.120.050656
sha: 6445b033e29fdfa15e88c606cce01c4cf79130bf
doc_id: 967833
cord_uid: cnp2odtg

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CORRESPONDENCE severity, linking the pulmonary vasculature and platelets to hypercoagulability. Next, we questioned whether the overwhelming immune response contributes to hypercoagulability. We hypothesized that neutrophils, and more specific NET formation with release of histones and DNA fragments, trigger contact activation. 2 First, we assessed the presence of extracellular histone H3 (H3) in plasma and sputum by Western blot using an antihuman H3 polyclonal antibodies as described. 3 Extracellular H3 was detected in patients with moderate and progressive/severe CO-VID-19 but not in those with mild disease. Extracellular H3 was citrullinated in 38 (73%) out of 52 cases, as detected by anticitrulline monoclonal antibodies, indicating NETs as its origin. The presence of citrullinated H3 in sputum from 9 patients requiring invasive ventilation linked neutrophil extracellular trap-osis (NETosis) to the lungs. Thereafter, we assessed whether patients with COVID-19 have circulating factors that induce NETosis by incubating neutrophils from healthy donors with patient serum diluted in medium for 4 hours at 37°C. Serum samples from all 9 patients with progressive/ severe COVID-19 showed abundant formation of NETs that stain positive for DNA, citrullinated H3, neutrophil elastase, and myeloperoxidase on immunofluorescence microscopy, whereas serum from patients with mild and those with moderate COVID-19 did not; 2 (40%) out of 5 samples from patients with moderate disease only induced swelling of nuclei without NETosis. NETs have been shown to colocalize with thrombi in CO-VID-19. 4 NETs with release of histones and DNA fragments, which trigger contact activation, 2 may thereby contribute to COVID-19-associated coagulopathy.

It has been appreciated that neutrophils, especially NETs, engage with coagulation and complement in a triangular relationship to produce the immunothrombotic response to invading pathogens. The complement system plays a critical role in binding SARS-CoV-2's N-protein, leading to the generation of the potent anaphylatoxin C5a. The subsequent attraction, priming, and activation of neutrophils causes an amplification loop of complement and neutrophil activation with more generation of C5a. 5 C5a, quantified by enzyme immunoassay, was elevated in COVID-19 and increased significantly with disease severity.

In conclusion, our analysis of a large cohort of patients with COVID-19 presents strong indications that hypercoagulability and thrombotic events are driven by NETosis, contact activation, and complement. The triangular relationship with its multiple amplifying feedback loops urges therapeutic multiple-target strategies to dampen the immunothrombotic response effectively. Likely candidates are blockers of C5a, emerging inhibitors of plasma kallikrein and FXIa, and agents that neutralize extracellular histones. The challenge is to identify high-risk patients in the earliest possible stage of disease.

The data that support the findings of this study are available from the corresponding author upon reasonable request.

ten Cate H. The plasma kallikrein-kinin system and risk of cardiovascular disease in men

In vitro activation of coagulation by human neutrophil DNA and histone proteins but not neutrophil extracellular traps

CORRESPONDENCE Nonanticoagulant heparin prevents histone-mediated cytotoxicity in vitro and improves survival in sepsis

Neutrophil extracellular traps contribute to immunothrombosis in COVID-19 acute respiratory distress syndrome

Complement alternative pathway acts as a positive feedback amplification of neutrophil activation