Neutrophil extracellular traps and thrombosis in COVID-19.

Studies of patients with COVID-19 have demonstrated markedly dysregulated coagulation and a high risk of morbid arterial and venous thrombotic events. Elevated levels of blood neutrophils and neutrophil extracellular traps (NETs) have recently been described in patients with COVID-19. However, their potential role in COVID-19-associated thrombosis remains incompletely understood. In order to elucidate the potential role of hyperactive neutrophils and NET release in COVID-19-associated thrombosis, we conducted a case-control study of patients hospitalized with COVID-19 who developed thrombosis, as compared with gender- and age-matched COVID-19 patients without clinical thrombosis. We found that remnants of NETs (cell-free DNA, myeloperoxidase-DNA complexes, and citrullinated histone H3) and neutrophil-derived S100A8/A9 (calprotectin) in patient sera were associated with higher risk of morbid thrombotic events in spite of prophylactic anticoagulation. These observations underscore the need for urgent investigation into the potential relationship between NETs and unrelenting thrombosis in COVID-19, as well as novel approaches for thrombosis prevention.

May the Odds Be Ever in Your Favor: Non-deterministic Mechanisms Diversifying Cell Surface Molecule Expression.

How does the information in the genome program the functions of the wide variety of cells in the body? While the development of biological organisms appears to follow an explicit set of genomic instructions to generate the same outcome each time, many biological mechanisms harness molecular noise to produce variable outcomes. Non-deterministic variation is frequently observed in the diversification of cell surface molecules that give cells their functional properties, and is observed across eukaryotic clades, from single-celled protozoans to mammals. This is particularly evident in immune systems, where random recombination produces millions of antibodies from only a few genes; in nervous systems, where stochastic mechanisms vary the sensory receptors and synaptic matching molecules produced by different neurons; and in microbial antigenic variation. These systems employ overlapping molecular strategies including allelic exclusion, gene silencing by constitutive heterochromatin, targeted double-strand breaks, and competition for limiting enhancers. Here, we describe and compare five stochastic molecular mechanisms that produce variety in pathogen coat proteins and in the cell surface receptors of animal immune and neuronal cells, with an emphasis on the utility of non-deterministic variation.

Neutrophil extracellular traps and thrombosis in COVID-19.

BACKGROUND: Early studies of patients with COVID-19 have demonstrated markedly dysregulated coagulation and a high risk of morbid arterial and venous thrombotic events. While elevated levels of blood neutrophils and neutrophil extracellular traps (NETs) have been described in patients with COVID-19, their potential role in COVID-19-associated thrombosis remains unknown. OBJECTIVES: To elucidate the potential role of hyperactive neutrophils and NET release in COVID-19-associated thrombosis. PATIENTS/METHODS: This is a retrospective, case-control study of patients hospitalized with COVID-19 who developed thrombosis (n=11), as compared with gender- and age-matched COVID-19 patients without clinical thrombosis (n=33). In addition to capturing clinical data, we measured remnants of NETs (cell-free DNA, myeloperoxidase-DNA complexes, and citrullinated histone H3) and neutrophil-derived S100A8/A9 (calprotectin) in patient sera. RESULTS: The majority of patients (9/11) were receiving at least prophylactic doses of heparinoids at the time thrombosis was diagnosed. As compared with controls, patients with COVID-19-associated thrombosis had significantly higher blood levels of markers of NETs (cell-free DNA, myeloperoxidase-DNA complexes, citrullinated histone H3) and neutrophil activation (calprotectin). The thrombosis group also had higher levels of D-dimer, CRP, ferritin, and platelets, but not troponin or neutrophils. Finally, there were strong associations between markers of hyperactive neutrophils (calprotectin and cell-free DNA) and D-dimer. CONCLUSION: Elevated levels of neutrophil activation and NET formation in patients hospitalized with COVID-19 are associated with higher risk of morbid thrombotic complications. These observations underscore the need for urgent investigation into the potential relationship between NETs and unrelenting thrombosis in COVID-19.

Neutrophil calprotectin identifies severe pulmonary disease in COVID-19.

Severe cases of coronavirus disease 2019 (COVID-19) are regularly complicated by respiratory failure. While it has been suggested that elevated levels of blood neutrophils associate with worsening oxygenation in COVID-19, it is unknown whether neutrophils are drivers of the thrombo-inflammatory storm or simple bystanders. To better understand the potential role of neutrophils in COVID-19, we measured levels of the neutrophil activation marker S100A8/A9 (calprotectin) in hospitalized patients and determined its relationship to severity of illness and respiratory status. Patients with COVID-19 (n=172) had markedly elevated levels of calprotectin in their blood. Calprotectin tracked with other acute phase reactants including C-reactive protein, ferritin, lactate dehydrogenase, and absolute neutrophil count, but was superior in identifying patients requiring mechanical ventilation. In longitudinal samples, calprotectin rose as oxygenation worsened. When tested on day 1 or 2 of hospitalization (n=94 patients), calprotectin levels were significantly higher in patients who progressed to severe COVID-19 requiring mechanical ventilation (8039 +/- 7031 ng/ml, n=32) as compared to those who remained free of intubation (3365 +/- 3146, p<0.0001). In summary, serum calprotectin levels track closely with current and future COVID-19 severity, implicating neutrophils as potential perpetuators of inflammation and respiratory compromise in COVID-19.

Prothrombotic autoantibodies in serum from patients hospitalized with COVID-19.

Patients with COVID-19 are at high risk for thrombotic arterial and venous occlusions. Lung histopathology often reveals fibrin-based blockages in the small blood vessels of patients who succumb to the disease. Antiphospholipid syndrome is an acquired and potentially life-threatening thrombophilia in which patients develop pathogenic autoantibodies targeting phospholipids and phospholipid-binding proteins (aPL antibodies). Case series have recently detected aPL antibodies in patients with COVID-19. Here, we measured eight types of aPL antibodies in serum samples from 172 patients hospitalized with COVID-19. These aPL antibodies included anticardiolipin IgG, IgM, and IgA; anti-ß2 glycoprotein I IgG, IgM, and IgA; and anti-phosphatidylserine/prothrombin (aPS/PT) IgG and IgM. We detected aPS/PT IgG in 24% of serum samples, anticardiolipin IgM in 23% of samples, and aPS/PT IgM in 18% of samples. Antiphospholipid autoantibodies were present in 52% of serum samples using the manufacturer's threshold and in 30% using a more stringent cutoff (≥40 ELISA-specific units). Higher titers of aPL antibodies were associated with neutrophil hyperactivity, including the release of neutrophil extracellular traps (NETs), higher platelet counts, more severe respiratory disease, and lower clinical estimated glomerular filtration rate. Similar to IgG from patients with antiphospholipid syndrome, IgG fractions isolated from patients with COVID-19 promoted NET release from neutrophils isolated from healthy individuals. Furthermore, injection of IgG purified from COVID-19 patient serum into mice accelerated venous thrombosis in two mouse models. These findings suggest that half of patients hospitalized with COVID-19 become at least transiently positive for aPL antibodies and that these autoantibodies are potentially pathogenic.

Prothrombotic antiphospholipid antibodies in COVID-19.

Patients with coronavirus disease 19 (COVID-19) are at high risk for thrombotic arterial and venous occlusions. At the same time, lung histopathology often reveals fibrin-based occlusion in the small vessels of patients who succumb to the disease. Antiphospholipid syndrome (APS) is an acquired and potentially life-threatening thrombophilia in which patients develop pathogenic autoantibodies (aPL) targeting phospholipids and phospholipid-binding proteins. Case series have recently detected aPL in patients with COVID-19. Here, we measured eight types of aPL [anticardiolipin IgG/IgM/IgA, anti-beta-2 glycoprotein I IgG/IgM/IgA, and anti- phosphatidylserine/prothrombin (aPS/PT) IgG/IgM] in the sera of 172 patients hospitalized with COVID-19. We detected aPS/PT IgG in 24%, anticardiolipin IgM in 23%, and aPS/PT IgM in 18%. Any aPL was present in 52% of patients using the manufacturer's threshold and in 30% using a more stringent cutoff (≥40 units). Higher levels of aPL were associated with neutrophil hyperactivity (including the release of neutrophil extracellular traps/NETs), higher platelet count, more severe respiratory disease, and lower glomerular filtration rate. Similar to patients with longstanding APS, IgG fractions isolated from patients with COVID-19 promoted NET release from control neutrophils. Furthermore, injection of these COVID-19 IgG fractions into mice accelerated venous thrombosis. Taken together, these studies suggest that a significant percentage of patients with COVID-19 become at least transiently positive for aPL and that these aPL are potentially pathogenic.