ABSTRACT
Background: Coagulopathy and inflammation are hallmarks of COVID-19 and are associated with increased mortality. Clinical and experimental data have revealed a role for neutrophil extracellular traps (NETs) in COVID-19. Mechanisms that drive thrombo-inflammation in COVID-19 are poorly understood. Aim(s): In this study, we aimed to investigate a possible role of NETs-driven coagulation factor XII (FXII) activation in COVID-19- related thrombo-inflammation. Method(s): We performed comprehensive proteomics and immunostaining of postmortem lung tissues from COVID-19 patients and patients with other lung pathologies. We compared FXII and DNase1 activities in plasma samples from COVID-19 patients and healthy control donors and determined NET-induced FXII activation using a chromogenic substrate assay. Result(s): FXII expression and activity were increased in the lung parenchyma, within the pulmonary vasculature and in fibrin-rich alveolar spaces of postmortem lung tissues from COVID-19 patients over controls. Active FXII (FXIIa) was increased in plasma of COVID-19 patients. Furthermore, FXIIa colocalized with NETs in COVID-19 lung tissue indicating that NETs accumulation leads to FXII activation in COVID-19. Accumulation of NETs in COVID-19 was at least in parts due to impaired DNA clearance by extracellular DNases. In plasma from COVID-19 patients, DNase1 substitution improved NET dissolution and reduced FXII activation in vitro. Conclusion(s): Collectively, our study shows that the NETs/FXIIa axis contributes to procoagulant and proinflammatory reactions in COVID-19. Targeting NETs and FXIIa may offer a potential therapeutic strategy for interfering with the COVID-19 lung pathology.
ABSTRACT
Background: SARS-CoV-2 vaccine ChAdOx1 nCov-19 rarely causes vaccine-induced immune thrombotic thrombocytopenia (VITT) that-like autoimmune heparin-induced thrombocytopenia-is mediated by platelet-activating anti-platelet factor 4 (PF4) antibodies. Aims: To understand how the SARS-CoV-2 vaccine ChAdOx1 nCov-19 can induce anti-PF4 antibodies and how these antibodies induce thrombosis Methods: We investigated vaccine, PF4, and VITT patient-derived anti-PF4 antibody interactions using 3D-super-resolution microscopy, dynamic light scattering, and transmission electron microscopy. Vaccine composition was analyzed by mass spectrometry. Experimental vascular leakage models assessed early post-vaccine reactions. We evaluated VITT antibody-mediated platelet activation and formation of procoagulant DNA-containing neutrophil extracellular traps (NETs), including within VITT patient cerebral venous thrombi. Results: Biophysical analyses showed HEK cell line proteins and free virus proteins in the vaccine, formation of complexes between PF4 and vaccine constituents (including viral proteins) that were recognized by VITT antibodies. In a murine model, EDTA (vaccine constituent) increased microvascular leakage with dissemination of virus-and cell culture-derived human proteins. Free viral proteins and preexisting antibodies in normal human sera reacting with vaccine-containing constituents, likely contribute to commonly observed acute ChAdOx1 nCov-19 post-vaccination inflammatory reactions. PF4-binding polyanions (polyphosphates, DNA) enhanced PF4-dependent platelet activation by VITT antibodies. In the presence of platelets, PF4 enhanced VITT antibody-driven NETs formation;further evidence for NETosis included elevated NETs biomarkers and low DNase activity in VITT sera, and NETs/ neutrophil-rich cerebral vein thrombi extracted from VITT patients. Conclusions: ChAdOx1 nCoV-19 vaccine constituents form antigenic complexes with PF4, and EDTA increases microvascular permeability, enhancing risk of early post-vaccination acute inflammatory reactions;PF4/polyanion antigen formation in a proinflammatory milieu offers an explanation for anti-PF4 antibody production. Resulting high-titer anti-PF4 antibodies activate platelets and induce neutrophil activation with NETosis, further fueling the VITT prothrombotic response.