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.
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
Submitter: Dimitrios Mastellos | email@example.com
Authors: Dimitrios C. Mastellos and John D. Lambris
National Center for Scientific Research ‘Demokritos’, Athens, Greece; Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, USA
Published October 7, 2020
The complement and coagulation cascades are steadily perceived as two tightly intertwined, blood-borne sentinels of the innate immune response that respond swiftly to external perturbations (e.g. viral or bacterial infections) to contain pathogen spread and restore tissue homeostasis (1). Mounting clinical and experimental evidence indicates that deregulated complement and procoagulant responses lie at the heart of a hyperinflammatory vicious cycle that forces these otherwise host protective systems to plunge into a destructive ‘spiral’, negatively affecting multiple vital organs in severe COVID-19 (2-5). Indeed, persistent complement activation can have detrimental effects on endothelial barrier integrity and drive endothelial cell activation and platelet-neutrophil interactions that fuel thrombogenic responses, thus exacerbating microvascular injury and immunothrombosis in severe COVID-19 (6-8). In this respect, Skendros et al, showed that complement C3 activation is a key mechanism that interlocks neutrophil and platelet responses fueling NET-dependent thromboinflammation in COVID-19 (7). From a therapeutic standpoint, C3 inhibition with compstatin Cp40 disrupted TF expression by healthy neutrophils exposed to COVID-19 serum, thus providing early evidence that C3 activation is a key amplifier of the platelet/NETs/TF/thrombin axis in COVID-19. The NET-lowering capacity of Cp40 was demonstrated in an ex vivo assay that emulates disease pathophysiology by piecing together isolated cellular and humoral components of the thromboinflammatory milieu. Therefore, the need to corroborate this important finding in the actual clinical setting, in COVID19 patients dosed with complement C3 inhibitors, becomes highly relevant for the design of effective anti-complement therapies.
Following up on these observations, a new study provides direct, in vivo, evidence that C3 activation regulates the magnitude of NET-driven thromboinflammation in severe COVID-19. An exploratory study comparing for the first time the biological efficacy of the C5-targeting monoclonal antibody eculizumab with that of the compstatin-based C3-targeted drug candidate AMY-101 in small COVID-19 patient cohorts, supports the broader impact of C3 inhibition on thromboinflammatory pathways associated with severe COVID-19 (9). Treatment with AMY-101 is associated with a broader therapeutic profile in COVID-19 patients, marked by faster lymphocyte recovery, a steep decline of neutrophil numbers and a greater reduction of NET levels in AMY-101-treated patient plasma. These results provide first in-patient validation for the broad inhibitory effect of C3 therapeutics on NET-driven immunothrombosis. They also illuminate previously elusive aspects of complement’s pathogenic contribution in COVID-19> and reveal target-specific biological traits that may help design more tailored therapeutics for COVID-19 patients.
1. Ricklin D, Hajishengallis G, Yang K, Lambris JD. Complement: a key system for immune surveillance and homeostasis. Nat.Immunol. 2010;11(1529-2916 (Electronic)):785–797.
2. Ramlall V, Thangaraj PM, Meydan C, et al. Immune complement and coagulation dysfunction in adverse outcomes of SARS-CoV-2 infection. Nat. Med. 2020;1–7.
3. Carvelli J, Demaria O, Vély F, et al. Association of COVID-19 inflammation with activation of the C5a-C5aR1 axis. Nature. 2020;1–9.
4. Risitano AM, Mastellos DC, Huber-Lang M, et al. Complement as a target in COVID-19? Nat. Rev. Immunol. 2020. 2020;1–2.
5. Java A, Apicelli AJ, Liszewski MK, et al. The complement system in COVID-19: friend and foe? JCI Insight. 2020;5(15):e140711.
6. Magro C, Mulvey JJ, Berlin D, et al. Complement associated microvascular injury and thrombosis in the pathogenesis of severe COVID-19 infection: A report of five cases. Transl. Res. 2020; 10.1016%2Fj.trsl.2020.04.007.
7. Skendros P, Mitsios A, Chrysanthopoulou A, et al. Complement and tissue factor-enriched neutrophil extracellular traps are key drivers in COVID-19 immunothrombosis. J. Clin. Invest. 2020;
8. Ghebrehiwet B, Peerschke EI. Complement and coagulation: key triggers of COVID-19-induced multiorgan pathology. J. Clin. Invest. 2020; https://doi.org/10.1172/JCI142780.
9. Mastellos DC, Pires da Silva BGP, Fonseca BAL, et al. Complement C3 vs C5 inhibition in severe COVID-19: Early clinical findings reveal differential biological efficacy. Clin. Immunol. 2020;108598.
Submitter: Ming-Lin Liu | firstname.lastname@example.org
Authors: Ming-Lin Liu, Xing Lyu, and Victoria P. Werth
Department of Dermatology, Perelman School of Medicine, University of Pennsylvania
Published August 28, 2020
A recent interesting work by Skendros et al showed that complement- and tissue factor (TF)-containing neutrophil extracellular traps (NETs) are key drivers in COVID-19 immunothrombosis (1). However, NETotic neutrophils also release extracellular vesicles (EVs) that may also contribute to the pro-thrombotic conditions in COVID-19.
Tissue factor (TF), a transmembrane protein, initiates the extrinsic coagulation cascade that was thought to be triggered by trauma (2). However, Giesen and colleagues detected EV-associated TF and TF-containing monocytes and neutrophils in peripheral blood. These blood-borne TF can trigger experimental thrombus formation (3). Our earlier studies found that cholesterol enrichment or tobacco smoke extract treatment of monocytes results in the generation of TF-positive EVs with potent procoagulant activity (4,5).
In patients with severe COVID-19 infection, thrombotic complications result in multi-organ damage (6). Skendros found the increased TF expression in neutrophils from COVID-19 patients, while the treatment of healthy control neutrophils with platelet-rich plasma from COVID-19 patients increased their TF expression (1). Thiam et al demonstrated that NETotic neutrophils also release EVs during an early stage of NETosis (7). Given that TF is a transmembrane protein, it can be budded and released with EVs from the cell membrane area where TF is expressed. Therefore, TF-positive EVs will bud from TF-positive neutrophils (3-5) that undergo NETosis in COVID-19 infection. Skendros et al have convincingly shown that NET-associated TF has potent procoagulant activity (1). However, the EV-associated TF also integrates with membrane phospholipids, which provide a procoagulant surface for the binding and activation of clotting factors. The synergistic effects of TF and phospholipids may explain the exaggerated TF procoagulant activity in the TF-positive EVs in our previous works (4). Therefore, the TF-positive EVs from NETotic neutrophils would be an important contributor to the prothrombotic condition in patients with COVID-19 infection.
In NETotic neutrophils, decondensed nuclear chromatin forms the backbone of extracellular NETs (8). It’s well known that extracellular DNA in NETs, including histones, contributes to the coagulation process and thrombus formation by promoting platelet aggregation, and induce endothelial damage in the vessels (9). However, our unpublished data indicate that EVs budding from early-stage NETotic neutrophils do not contain nuclear DNA. In line with our observation, Thiam and colleagues also reported that nuclear DNA cannot be detected in the EVs released from NETotic neutrophils (7). Therefore, TF-positive EVs released from NETotic neutrophils may also contribute to the pro-thrombotic conditions in patients with COVID-19 infection.
1. Skendros, P., Mitsios, A., Chrysanthopoulou, A., Mastellos, D.C., Metallidis, S., Rafailidis, P., Ntinopoulou, M., Sertaridou, E., Tsironidou, V., Tsigalou, C., et al. 2020. Complement and tissue factor-enriched neutrophil extracellular traps are key drivers in COVID-19 immunothrombosis. J Clin Invest. doi: 10.1172/JCI141374
2. Grover, S.P., and Mackman, N. 2018. Tissue Factor: An Essential Mediator of Hemostasis and Trigger of Thrombosis. Arterioscler Thromb Vasc Biol 38:709-725.
3. Giesen, P.L., Rauch, U., Bohrmann, B., Kling, D., Roque, M., Fallon, J.T., Badimon, J. J., Himber, J., Riederer, M.A., and Nemerson, Y. 1999. Blood-borne tissue factor: another view of thrombosis. Proc Natl Acad Sci U S A 96:2311-2315.
4. Li, M., Yu, D., Williams, K.J., and Liu, M.L. 2010. Tobacco smoke induces the generation of procoagulant microvesicles from human monocytes/macrophages. Arterioscler Thromb Vasc Biol 30:1818-1824.
5. Liu, M.L., Reilly, M.P., Casasanto, P., McKenzie, S.E., and Williams, K.J. 2007. Cholesterol enrichment of human monocyte/macrophages induces surface exposure of phosphatidylserine and the release of biologically-active tissue factor-positive microvesicles. Arterioscler Thromb Vasc Biol 27:430-435.
6. McFadyen, J.D., Stevens, H., and Peter, K. 2020. The Emerging Threat of (Micro)Thrombosis in COVID-19 and Its Therapeutic Implications. Circ Res 127:571-587.
7. Thiam, H.R., Wong, S.L., Qiu, R., Kittisopikul, M., Vahabikashi, A., Goldman, A.E., Goldman, R.D., Wagner, D.D., and Waterman, C.M. 2020. NETosis proceeds by cytoskeleton and endomembrane disassembly and PAD4-mediated chromatin decondensation and nuclear envelope rupture. Proc Natl Acad Sci U S A 117:7326-7337.
8. Li, Y., Li, M.H., Weigel, B., Mall, M., Werth, V.P., and Liu, M.L. 2020. Nuclear envelope rupture and NET formation is driven by PKCα-mediated lamin B disassembly. EMBO rep 21:(In press).
9. Martinod, K., and Wagner, D.D. 2014. Thrombosis: tangled up in NETs. Blood 123:2768-2776.