Patients with coronavirus disease-19 (COVID-19) are at high risk for thrombotic arterial and venous occlusions. However, bleeding complications have also been observed in some patients. Understanding the balance between coagulation and fibrinolysis will help inform optimal approaches to thrombosis prophylaxis and potential utility of fibrinolytic-targeted therapies. 118 hospitalized COVID-19 patients and 30 healthy controls were included in the study. We measured plasma antigen levels of tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) and performed spontaneous clot-lysis assays. We found markedly elevated tPA and PAI-1 levels in patients hospitalized with COVID-19. Both factors demonstrated strong correlations with neutrophil counts and markers of neutrophil activation. High levels of tPA and PAI-1 were associated with worse respiratory status. High levels of tPA, in particular, were strongly correlated with mortality and a significant enhancement in spontaneous ex vivo clot-lysis. While both tPA and PAI-1 are elevated among COVID-19 patients, extremely high levels of tPA enhance spontaneous fibrinolysis and are significantly associated with mortality in some patients. These data indicate that fibrinolytic homeostasis in COVID-19 is complex with a subset of patients expressing a balance of factors that may favor fibrinolysis. Further study of tPA as a biomarker is warranted.

Background: Patients with coronavirus disease 19 (COVID-19) are at high risk for thrombotic arterial and venous occlusions, while lung histopathology often reveals fibrin-based occlusion of small vessels in patients who succumb to the disease. At the same time, bleeding complications have been observed in some patients. Better understanding the balance between coagulation and fibrinolysis will help inform optimal approaches to thrombosis prophylaxis and potential utility of fibrinolytic-targeted therapies. Objective: To evaluate fibrinolysis among a large cohort of hospitalized COVID-19 patients. Patients and methods: 118 hospitalized COVID-19 patients and 30 healthy controls were included in the study. We measured plasma antigen levels of tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) and performed spontaneous clot lysis assays. Results: We found markedly elevated levels of tPA and PAI-1 among patients hospitalized with COVID-19. Both factors demonstrated a strong correlation with neutrophil counts and markers of neutrophil activation, but not with D-dimer. High levels of tPA and PAI-1 were associated with worse respiratory status. High levels of tPA, in particular, were also strongly correlated with mortality and with a significant enhancement in spontaneous ex vivo clot lysis. Conclusion: While both tPA and PAI-1 are elevated among COVID-19 patients, extremely high levels of tPA enhance spontaneous fibrinolysis and are significantly associated with mortality in some patients. These data indicate that fibrinolytic homeostasis in COVID-19 is complex with a subset of patients expressing a balance of factors that may favor fibrinolysis and suggests that further study of tPA as a potential biomarker is warranted.

The release of neutrophil extracellular traps (NETs) by hyperactive neutrophils is recognized to play an important role in the thromboinflammatory milieu inherent to severe presentations of COVID-19. At the same time, a variety of functional autoantibodies have been observed in individuals with severe COVID-19 where they likely contribute to immunopathology. Here, we aimed to determine the extent to which autoantibodies might target NETs in COVID-19 and, if detected, to elucidate their potential functions and clinical associations. We measured anti-NET antibodies in 328 individuals hospitalized with COVID-19 alongside 48 healthy controls. We found high anti-NET activity in the IgG and IgM fractions of 27% and 60% of patients, respectively. There was a strong correlation between anti-NET IgG and anti-NET IgM (r=0.4, p<0.0001). Both anti-NET IgG and IgM tracked with high levels of circulating NETs, impaired oxygenation efficiency, and high circulating D-dimer. Furthermore, patients who required mechanical ventilation had a greater burden of anti-NET antibodies than did those not requiring oxygen supplementation. Levels of anti-NET IgG (and to a lesser extent anti-NET IgM) demonstrated an inverse correlation with the efficiency of NET degradation by COVID sera. Furthermore, purified IgG from COVID sera with high levels of anti-NET antibodies impaired the ability of healthy control serum to degrade NETs. In summary, many individuals hospitalized with COVID-19 have anti-NET antibodies, which likely impair NET clearance and may potentiate SARS-CoV-2-mediated thromboinflammation.

Objective While endothelial dysfunction has been implicated in the widespread thrombo‐inflammatory complications of coronavirus disease‐19 ( COVID‐19 ), the upstream mediators of endotheliopathy remain for the most part cryptic. Our aim was to identify circulating factors contributing to endothelial cell activation and dysfunction in COVID‐19. Methods Human endothelial cells were cultured in the presence of serum or plasma from 244 patients hospitalized with COVID‐19 and plasma from 100 patients with non‐COVID sepsis. Cell adhesion molecules (E‐selectin, VCAM‐1, and ICAM‐1) were quantified by in‐cell ELISA. Results Serum and plasma from patients with COVID‐19 increased surface expression of cell adhesion molecules. Furthermore, levels of soluble ICAM‐1 and E‐selectin were elevated in patient serum and tracked with disease severity. The presence of circulating antiphospholipid antibodies was a strong marker of the ability of COVID‐19 serum to activate endothelium. Depletion of total IgG from antiphospholipid antibody‐positive serum markedly restrained upregulation of cell adhesion molecules. Conversely, supplementation of control serum with patient IgG was sufficient to trigger endothelial activation. Conclusion These data are the first to suggest that some patients with COVID‐19 have potentially diverse antibodies that drive endotheliopathy, adding important context regarding thrombo‐inflammatory effects of autoantibodies in severe COVID‐19.

Patients with coronavirus disease 19 (COVID-19) are at high risk for fibrin-based occlusion of vascular beds of all sizes. Although endothelial cell activation has regularly been described as part of the COVID-19 thrombo-inflammatory storm, the upstream mediators of this activation have yet to be fully elucidated. Here, we began by pursuing the hypothesis that circulating factors such as neutrophil extracellular trap (NET) remnants, D-dimer, or C-reactive protein might predict the COVID-19 serum samples (n=118) that most robustly activated cultured endothelial cells. Indeed, we found modest correlations between serum NET remnants (cell-free DNA, myeloperoxidase-DNA complexes, citrullinated histone H3) and upregulation of surface E-selectin, VCAM-1, and ICAM-1 on endothelial cells. However, a more robust predictor of the ability of COVID-19 serum to activate endothelial cells was the presence of circulating antiphospholipid antibodies, specifically anticardiolipin IgG and IgM and anti-phosphatidlyserine/prothrombin (anti-PS/PT) IgG and IgM. Depletion of total IgG from anticardiolipin-high and anti-PS/PT-high samples markedly restrained upregulation of E-selectin, VCAM-1, and ICAM-1. At the same time, supplementation of control serum with patient IgG was sufficient to trigger endothelial cell activation. These data are the first to reveal that patient antibodies are a driver of endothelial cell activation and add important context regarding thrombo-inflammatory effects of COVID-19 autoantibodies in severe COVID-19.