Neutrophils and Contact Activation of Coagulation as Potential Drivers of COVID-19
Authors: Matthias H. Busch, Sjoerd A.M.E.G. Timmermans, Magdolna Nagy, Mayken Visser, Joram Huckriede, Joop P. Aendekerk, Femke de Vries, Judith Potjewijd, Borefore Jallah, Renée Ysermans, Astrid M.L. Oude Lashof, Paul H. Breedveld, Marcel C.G. van de Poll, Iwan C.C. van de Horst, Bas C.T. van Bussel, Ruud O.M.F.I.H. Theunissen, Henri M.H. Spronk, Jan G.M.C. Damoiseaux, Hugo ten Cate, Gerry A.F. Nicolaes, Chris P. Reutelingsperger, Pieter van Paassen
Journal: Circulation (2020)

Coronavirus disease 2019 (COVID-19)-associated coagulopathy, responsible for high rates of pulmonary thrombosis, is poorly characterized.Clinical studies identified several biomarkers, such as D-dimer.Different mechanisms have been proposed, including neutrophil and complement activation, vascular damage, and tissue factor expression.The intrinsic pathway of coagulation, which not only amplifies fibrin generation but also links to inflammation, including plasma kallikrein and bradykinin, both proposed to contribute to COVID-19, has not been studied.We performed a comprehensive analysis on the intrinsic pathway to characterize its role in COVID-19.By simultaneously studying potential triggers of the intrinsic pathway, we were able to identify neutrophils, neutrophil extracellular traps (NETs), and complement activation as potential drivers of this complex immunothrombotic disease.We included 228 consecutive patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) confirmed by real-time polymerase chain reaction (Table 1).Disease severity was classified as mild in patients not admitted to the hospital, moderate in admitted patients requiring oxygen via nasal cannula, and progressive/severe in patients requiring oxygen via a face mask, or admitted to the intensive care unit for invasive ventilation or those who died ≤7 days.Thrombotic events were uncommon in mild (n/N=2/54) and moderate COVID-19 (n/N=3/68) as compared with progressive/severe disease (n/N=23/106; P=0.002).At presentation and during follow-up, blood was obtained; sputum was collected in selected cases.This study was approved by the local ethics committee (no.2020-1315), with a waiver of informed consent.To delineate activation of coagulation, we used enzyme-linked immunosorbent assays to measure activated coagulation factors in complex with their natural inhibitors as described with modifications. 1 Hypercoagulability was found in 131 (60%) out of 217 tested patients as indicated by increased levels of thrombin:antithrombin.We report for the first time that hypercoagulability was driven, at least partly, through contact activation as indicated by increased levels of plasma kallikrein:C1 esterase inhibitor (n/N=195/217), FXIa:α1 antitrypsin (n/N=206/217), FXIa:antithrombin (n/N=60/217), and FIXa:antithrombin (n/ N=145/217).The finding that these circulating complexes remained stable at day 5 (±2; n=53) and day 10 (±2; n=40) in patients with progressive/severe CO-VID-19 treated with low molecular weight heparin corroborates this conclusion as heparins exert only mild effects on contact activation.It is remarkable that our data show that contact activation is already ongoing in most patients with mild disease, but the downstream markers FXIa:antithrombin, FIXa:antithrombin, and thrombin:antithrombin are clearly linked to COVID-19 severity.This might explain the high incidence of thrombotic events in patients with progressive/ severe disease.Also,