COVID-19 affects millions of patients worldwide with clinical presentation ranging from isolated thrombosis to acute respiratory distress syndrome (ARDS) requiring ventilator support. Neutrophil extracellular traps (NETs) originate from decondensed chromatin released to immobilize pathogens and can trigger immunothrombosis. We studied the connection between NETs and COVID-19 severity and progression. We conducted a prospective cohort study of COVID-19 patients (n=33) with age- and sex-matched controls (n=17). We measured plasma myeloperoxidase (MPO)-DNA complexes (NETs), Platelet Factor 4, RANTES, and selected cytokines. Three COVID-19 lung autopsies were examined for NETs and platelet involvement. We assessed NET formation ex vivo in COVID-19 neutrophils and in healthy neutrophils incubated with COVID-19 plasma. We also tested the ability of neonatal NET-Inhibitory Factor (nNIF) to block NET formation induced by COVID-19 plasma. Plasma MPO-DNA complexes increased in COVID-19 with intubation (P<0.0001) and death as outcome (P<0.0005). Illness severity correlated directly with plasma MPO-DNA complexes (P=0.0360), while PaO2/FiO2 correlated inversely(P=0.0340). Soluble and cellular factors triggering NETs were significantly increased in COVID-19 and pulmonary autopsies confirmed NET-containing microthrombi with neutrophil-platelet infiltration. Finally, COVID-19 neutrophils ex vivo displayed excessive NETs at baseline and COVID-19 plasma triggered NET formation which was blocked by nNIF. Thus, NETs triggering immunothrombosis may, in part, explain the prothrombotic clinical presentations in COVID-19 and NETs may represent targets for therapeutic intervention.

There is an urgent need to understand the pathogenesis of coronavirus disease 2019 (COVID-19). In particular, thrombotic complications in patients with COVID-19 are common and contribute to organ failure and mortality. Patients with severe COVID-19 present with hemostatic abnormalities that mimic disseminated intravascular coagulopathy associated with sepsis with the major difference being increased risk of thrombosis rather than bleeding. However, whether SARS-CoV-2 infection alters platelet function to contribute to the pathophysiology of COVID-19 remains unknown. In this study, we report altered platelet gene expression and functional responses in patients infected with SARS-CoV-2. RNA sequencing demonstrated distinct changes in the gene expression profile of circulating platelets of COVID-19 patients. Pathway analysis revealed differential gene expression changes in pathways associated with protein ubiquitination, antigen presentation and mitochondrial dysfunction. The receptor for SARS-CoV-2 binding, ACE2, was not detected by mRNA or protein in platelets. Surprisingly, mRNA from the SARS-CoV-2 N1 gene was detected in platelets from 2/25 COVID-19 patients, suggesting platelets may take-up SARS-COV-2 mRNA independent of ACE2. Resting platelets from COVID-19 patients had increased P-selectin expression basally and upon activation. Circulating platelet-neutrophil, -monocyte, and -T-cell aggregates were all significantly elevated in COVID-19 patients compared to healthy donors. Furthermore, platelets from COVID-19 patients aggregated faster and showed increased spreading on both fibrinogen and collagen. The increase in platelet activation and aggregation could partially be attributed to increased MAPK pathway activation and thromboxane generation. These findings demonstrate that SARS-CoV-2 infection is associated with platelet hyperreactivity which may contribute to COVID-19 pathophysiology.

Neutrophils and NETs form important constituents of cerebral thrombi. Targeting of NETs with DNase 1 might have prothrombolytic potential in treatment of acute ischemic stroke. Ann Neurol 2017;82:223-232.

Ischemic stroke is caused by a thromboembolic occlusion of cerebral arteries. Treatment is focused on fast and efficient removal of the occluding thrombus, either via intravenous thrombolysis or via endovascular thrombectomy. Recanalization, however, is not always successful and factors contributing to failure are not completely understood. Although the occluding thrombus is the primary target of acute treatment, little is known about its internal organization and composition. The aim of this study, therefore, was to better understand the internal organization of ischemic stroke thrombi on a molecular and cellular level. A total of 188 thrombi were collected from endovascularly treated ischemic stroke patients and analyzed histologically for fibrin, red blood cells, von Willebrand factor , platelets, leukocytes and DNA, using bright field and fluorescence microscopy. Our results show that stroke thrombi are composed of two main types of areas: red blood cell-rich areas and platelet-rich areas. Red blood cell-rich areas have limited complexity as they consist of red blood cells that are entangled in a meshwork of thin fibrin. In contrast, platelet-rich areas are characterized by dense fibrin structures aligned with von Willebrand factor and abundant amounts of leukocytes and DNA that accumulate around and in these platelet-rich areas. These findings are important to better understand why platelet-rich thrombi are resistant to thrombolysis and difficult to retrieve via thrombectomy and can guide further improvements of acute ischemic stroke therapy.