Complement and tissue factor–enriched neutrophil extracellular traps are key drivers in COVID-19 immunothrombosis
Panagiotis Skendros, … , John D. Lambris, Konstantinos Ritis
Panagiotis Skendros, … , John D. Lambris, Konstantinos Ritis
Published August 6, 2020
Citation Information: J Clin Invest. 2020;130(11):6151-6157. https://doi.org/10.1172/JCI141374.
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Concise Communication Immunology

Complement and tissue factor–enriched neutrophil extracellular traps are key drivers in COVID-19 immunothrombosis

Abstract

Emerging data indicate that complement and neutrophils contribute to the maladaptive immune response that fuels hyperinflammation and thrombotic microangiopathy, thereby increasing coronavirus 2019 (COVID-19) mortality. Here, we investigated how complement interacts with the platelet/neutrophil extracellular traps (NETs)/thrombin axis, using COVID-19 specimens, cell-based inhibition studies, and NET/human aortic endothelial cell (HAEC) cocultures. Increased plasma levels of NETs, tissue factor (TF) activity, and sC5b-9 were detected in patients. Neutrophils of patients yielded high TF expression and released NETs carrying active TF. Treatment of control neutrophils with COVID-19 platelet-rich plasma generated TF-bearing NETs that induced thrombotic activity of HAECs. Thrombin or NETosis inhibition or C5aR1 blockade attenuated platelet-mediated NET-driven thrombogenicity. COVID-19 serum induced complement activation in vitro, consistent with high complement activity in clinical samples. Complement C3 inhibition with compstatin Cp40 disrupted TF expression in neutrophils. In conclusion, we provide a mechanistic basis for a pivotal role of complement and NETs in COVID-19 immunothrombosis. This study supports strategies against severe acute respiratory syndrome coronavirus 2 that exploit complement or NETosis inhibition.

Authors

Panagiotis Skendros, Alexandros Mitsios, Akrivi Chrysanthopoulou, Dimitrios C. Mastellos, Simeon Metallidis, Petros Rafailidis, Maria Ntinopoulou, Eleni Sertaridou, Victoria Tsironidou, Christina Tsigalou, Maria Tektonidou, Theocharis Konstantinidis, Charalampos Papagoras, Ioannis Mitroulis, Georgios Germanidis, John D. Lambris, Konstantinos Ritis

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Figure 2

Inhibition of PRP-neutrophil interactions in COVID-19.

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Inhibition of PRP-neutrophil interactions in COVID-19. (A) Relative fold...
(A) Relative fold expression of TF mRNA in control neutrophils treated with COVID-19–derived PRP (COV PRP) and inhibited with dabigatran (thrombin inhibitor), HCQ (autophagy inhibitor), or C5aR1 antagonist (C5aRa/PMX-53). Data are from 4 independent experiments (mean ± SD). (B) MPO-DNA complex levels in NETs isolated from control neutrophils treated with COV PRP and inhibited with dabigatran, HCQ, or C5aRa/PMX-53. Data are from 4 independent experiments (mean ± SD). (C) TAT complex levels in control plasma stimulated with NET structures isolated from control neutrophils treated with COV PRP and inhibited with dabigatran, HCQ, or C5aR. Data are from 4 independent experiments (mean ± SD). (D and E) Confocal fluorescence microscopy showing TF/neutrophil elastase (NE) staining in control neutrophils treated with COV PRP and inhibited with (F) dabigatran, (G) FLLRN (PAR1 receptor inhibitor), (H) HCQ, or (I) C5aRa/PMX-53. A representative example of 4 independent experiments is shown. Original magnification: ×600; scale bar: 5 μm. Blue: DAPI, green: TF, red: NE. All conditions were compared with control/untreated. *Statistical significance at P < 0.05. A–C: Friedman’s test. The in vitro experiments mentioned above are also summarized in Supplemental Table 2. Conditions of real-time RT-PCR are described in Supplemental Table 3.