medRxiv preprint doi: https://doi.org/10.1101/2020.06.15.20131029; this version posted June 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license . Complement and tissue factor-enriched neutrophil extracellular traps are key drivers in COVID-19 immunothrombosis Panagiotis Skendros1,2*, Alexandros Mitsios2*, Akrivi Chrysanthopoulou2*, Dimitrios C. Mastellos3, Simeon Metallidis4, Petros Rafailidis5, Maria Ntinopoulou2, Eleni Sertaridou6, Victoria Tsironidou2, Christina Tsigalou7, Maria Tektonidou8, Theocharis Konstantinidis2, Charalampos Papagoras1,2, Ioannis Mitroulis1,2, Georgios Germanidis4, John D. Lambris9#, Konstantinos Ritis1,2#. * These authors contributed to this work equally # These authors co-supervised this work. 1First Department of Internal Medicine, University Hospital of Alexandroupolis, Democritus University of Thrace, Alexandroupolis, Greece. 2Laboratory of Molecular Hematology, Department of Medicine, Democritus University of Thrace, Alexandroupolis, Greece. 3National Center for Scientific Research „Demokritos‟, Aghia Paraskevi, Athens, Greece. 4First Department of Internal Medicine, AHEPA University Hospital, Aristotle University of Thessaloniki, Thessaloniki, Greece. 5Second Department of Internal Medicine, University Hospital of Alexandroupolis, Democritus University of Thrace, Alexandroupolis, Greece. 6Intensive Care Unit, University Hospital of Alexandroupolis, Alexandroupolis, Greece. 7Laboratory of Microbiology, University Hospital of Alexandroupolis, Democritus University of Thrace, Alexandroupolis, Greece. NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice. 1 medRxiv preprint doi: https://doi.org/10.1101/2020.06.15.20131029; this version posted June 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license . 8First Department of Propaedeutic Internal Medicine, National and Kapodistrian University of Athens, Greece 9Department of Pathology & Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. Correspondence to: Konstantinos Ritis, MD, PhD First Department of Internal Medicine, University Hospital of Alexandroupolis, Democritus University of Thrace, Alexandoupolis, Greece Tel. +00302551351103 e-mail: [email protected] Conflict of interest JDL is the founder of Amyndas Pharmaceuticals which is developing complement inhibitors for therapeutic purposes and is the inventor of patents or patent applications that describe the use of complement inhibitors for therapeutic purposes, some of which are developed by Amyndas. JDL is also the inventor of the compstatin technology licensed to Apellis Pharmaceuticals (i.e., 4(1MeW)7W/POT-4/APL-1 and PEGylated derivatives such as APL-2/pegcetacoplan and APL-9). The remaining authors have declared that no conflict of interest exists. 2 medRxiv preprint doi: https://doi.org/10.1101/2020.06.15.20131029; this version posted June 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license . Abstract Emerging data indicate that complement and neutrophils are involved in the maladaptive host immune response that fuels hyper-inflammation and thrombotic microangiopathy increasing the mortality rate in coronavirus disease 2019 (COVID- 19). Here, we investigated the interaction between complement and the platelet/neutrophil extracellular traps (NETs)/thrombin axis, using COVID-19 clinical samples, cell-based inhibition studies and NETs/human aortic endothelial cell (HAEC) co-cultures. Increased plasma levels of NETs, TF activity and sC5b-9 were detected in patients. Neutrophils yielded high tissue factor (TF) expression and released NETs carrying functionally active TF. Confirming our ex vivo findings, treatment of control neutrophils with COVID-19 platelet-rich plasma generated TF- bearing NETs that induced thrombotic activity of HAEC. Thrombin or NETosis inhibition or C5aR1 blockade attenuated platelet-mediated NET-driven thrombogenicity. Serum isolated from COVID-19 patients induces complement activation in vitro, which is consistent with high complement activity in clinical samples. Complement inhibition at the level of C3 with compstatin Cp40 disrupted TF expression in neutrophils. In conclusion, we provide a mechanistic basis that reveals the pivotal role of complement and NETs in COVID-19 immmunothrombosis. This study supports emerging strategies against SARS-CoV-2 infection that exploit complement therapeutics or NETosis inhibition. Keywords: Complement, compstatin Cp40, platelets, neutrophil extracellular traps, tissue factor, immunothrombosis, COVID-19. 3 medRxiv preprint doi: https://doi.org/10.1101/2020.06.15.20131029; this version posted June 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license . Introduction Accumulated clinical evidence during the evolving pandemic of coronavirus disease 2019 (COVID-19) clearly indicates that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can trigger thrombotic complications that affect multiple vital organs, significantly increasing the mortality burden in COVID- 19 patients (1–3). The pathological process of coagulopathy in COVID-19 is commonly characterized as an immunothrombosis, since it is related to a maladaptive host immune response that is fueled by excessive activation of innate immune pathways, deregulated inflammation, thrombosis and endothelial dysfunction (2, 4). Understanding the immunothrombotic mechanisms in COVID-19 constitutes a significant medical challenge. Increased neutrophil count has been associated with disease severity and poor clinical outcomes in COVID-19, and extensive neutrophil infiltration of pulmonary capillaries has been described in autopsy specimens (5–9). In several inflammatory disorders (10), experimental and clinical studies have already demonstrated that neutrophil extracellular traps (NETs) can exert thrombogenic activity through the expression of functionally active tissue factor (TF). NETs appear to be involved in COVID-19 as well (11). Over-activation of complement has been implicated as an early driver of the maladaptive inflammatory response in COVID-19. Complement can enhance neutrophil/monocyte activation and recruitment to the epithelium of the infected lungs, and several complement effectors, acting in concert with platelets, can fuel thromboinflammation, microvascular thrombosis and endothelial dysfunction (thrombotic microangiopathy) in patients (9, 12). Therefore, early clinical data have 4 medRxiv preprint doi: https://doi.org/10.1101/2020.06.15.20131029; this version posted June 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license . prompted the initiation of trials to evaluate various complement-inhibitory strategies in COVID-19 patients (13). The well-established cross-talk between complement and neutrophils in immunothrombosis in human disorders (14–17) has prompted us to hypothesize that the collaboration of these innate immune systems may mediate early events leading to coagulopathy in COVID-19. Here, we have investigated the role of neutrophils in the thromboinflammation associated with COVID-19 and now provide evidence that complement activation is a key mediator in the activation of the platelet/NETs/TF/thrombin axis during SARS-CoV-2 infection. Results and discussion NETs are associated with activation of the TF/thrombin axis in COVID-19 We first used ELISA to measure the levels of myeloperoxidase (MPO)/DNA complexes (Figure 1A), a well-defined surrogate marker of NETosis (10), in the plasma of control and COVID-19 patients and detected significantly increased levels in the plasma obtained from COVID-19 patients. The levels of these complexes were positively correlated with thrombin-antithrombin (TAT) activity, suggesting that activation of the TF/thrombin axis had occurred (Figure 1B,C). These findings were further confirmed ex vivo by confocal immunofluorescence microscopy in neutrophils collected from four patients with severe COVID-19 (Supplemental Table S1). We found spontaneous formation of NETs expressing TF (Figure 1D,E) that were functional, as indicated by TAT assay (Figure 1F). Moreover, the concomitant increase in TF mRNA levels (Figure 1G) further confirmed previous studies indicating that neutrophils constitute an active source of TF in thromboinflammation. Although detection of NETosis markers in plasma by similar laboratory techniques 5 medRxiv preprint doi: https://doi.org/10.1101/2020.06.15.20131029; this version posted June 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder,