The role of extracellular histones in COVID-19.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) had spread from China and, within 2 months, became a global pandemic. The infection from this disease can cause a diversity of symptoms ranging from asymptomatic to severe acute respiratory distress syndrome with an increased risk of vascular hyperpermeability, pulmonary inflammation, extensive lung damage, and thrombosis. One of the host defense systems against coronavirus disease 2019 (COVID-19) is the formation of neutrophil extracellular traps (NETs). Numerous studies on this disease have revealed the presence of elevated levels of NET components, such as cell-free DNA, extracellular histones, neutrophil elastase, and myeloperoxidase, in plasma, serum, and tracheal aspirates of severe COVID-19 patients. Extracellular histones, a major component of NETs, are clinically very relevant as they represent promising biomarkers and drug targets, given that several studies have identified histones as key mediators in the onset and progression of various diseases, including COVID-19. However, the role of extracellular histones in COVID-19 per se remains relatively underexplored. Histones are nuclear proteins that can be released into the extracellular space via apoptosis, necrosis, or NET formation and are then regarded as cytotoxic damage-associated molecular patterns that have the potential to damage tissues and impair organ function. This review will highlight the mechanisms of extracellular histone-mediated cytotoxicity and focus on the role that histones play in COVID-19. Thereby, this paper facilitates a bench-to-bedside view of extracellular histone-mediated cytotoxicity, its role in COVID-19, and histones as potential drug targets and biomarkers for future theranostics in the clinical treatment of COVID-19 patients.

Design and characterization of novel activated protein C variants for the proteolysis of cytotoxic extracellular histone H3.

BACKGROUND: Extracellular histone H3 is implicated in several pathologies including inflammation, cell death, and organ failure. Neutralization of histone H3 is a strategy that was shown beneficial in various diseases, such as rheumatoid arthritis, myocardial infarction, and sepsis. It was shown that activated protein C (APC) can cleave histone H3, which reduces histone cytotoxicity. However, due to the anticoagulant properties of APC, the use of APC is not optimal for the treatment of histone-mediated cytotoxicity, in view of its associated bleeding side effects. OBJECTIVES: This study aimed to investigate the detailed molecular interactions between human APC and human histone H3, and subsequently use molecular docking and molecular dynamics simulation methods to identify key interacting residues that mediate the interaction between APC and histone H3 and to generate novel optimized APC variants. METHODS: After molecular simulations, the designed APC variants 3D2D-APC (Lys37-39Asp and Lys62-63Asp) and 3D2D2A-APC (Lys37-39Asp, Lys62-63Asp, and Arg74-75Ala) were recombinantly expressed and their abilities to function as anticoagulant, to bind histones, and to cleave histones were tested and correlated with their cytoprotective properties. RESULTS: Compared with wild type-APC, both the 3D2D-APC and 3D2D2A-APC variants showed a significantly decreased anticoagulant activity, increased binding to histone H3, and similar ability to proteolyze histone H3. CONCLUSIONS: Our data show that it is possible to rationally design APC variants that may be further developed into therapeutic biologicals to treat histone-mediated disease, by proteolytic reduction of histone-associated cytotoxic properties that do not induce an increased bleeding risk.

Impaired Antibody Response Is Associated with Histone-Release, Organ Dysfunction and Mortality in Critically Ill COVID-19 Patients.

PURPOSE: the pathophysiologic mechanisms explaining differences in clinical outcomes following COVID-19 are not completely described. This study aims to investigate antibody responses in critically ill patients with COVID-19 in relation to inflammation, organ failure and 30-day survival. METHODS: All patients with PCR-verified COVID-19 and gave consent, and who were admitted to a tertiary Intensive care unit (ICU) in Sweden during March-September 2020 were included. Demography, repeated blood samples and measures of organ function were collected. Analyses of anti-SARS-CoV-2 antibodies (IgM, IgA and IgG) in plasma were performed and correlated to patient outcome and biomarkers of inflammation and organ failure. RESULTS: A total of 115 patients (median age 62 years, 77% male) were included prospectively. All patients developed severe respiratory dysfunction, and 59% were treated with invasive ventilation. Thirty-day mortality was 22.6% for all included patients. Patients negative for any anti-SARS-CoV-2 antibody in plasma during ICU admission had higher 30-day mortality compared to patients positive for antibodies. Patients positive for IgM had more ICU-, ventilator-, renal replacement therapy- and vasoactive medication-free days. IgA antibody concentrations correlated negatively with both SAPS3 and maximal SOFA-score and IgM-levels correlated negatively with SAPS3. Patients with antibody levels below the detection limit had higher plasma levels of extracellular histones on day 1 and elevated levels of kidney and cardiac biomarkers, but showed no signs of increased inflammation, complement activation or cytokine release. After adjusting for age, positive IgM and IgG antibodies were still associated with increased 30-day survival, with odds ratio (OR) 7.1 (1.5-34.4) and 4.2 (1.1-15.7), respectively. CONCLUSION: In patients with severe COVID-19 requiring intensive care, a poor antibody response is associated with organ failure, systemic histone release and increased 30-day mortality.

Histone H3 Cleavage in Severe COVID-19 ICU Patients.

The severity of coronavirus disease 19 (COVID-19) is associated with neutrophil extracellular trap (NET) formation. During NET formation, cytotoxic extracellular histones are released, the presence of which is linked to the initiation and progression of several acute inflammatory diseases. Here we study the presence and evolution of extracellular histone H3 and several other neutrophil-related molecules and damage-associated molecular patterns (DAMPs) in the plasma of 117 COVID-19-positive ICU patients. We demonstrate that at ICU admission the levels of histone H3, MPO, and DNA-MPO complex were all significantly increased in COVID-19-positive patients compared to control samples. Furthermore, in a subset of 54 patients, the levels of each marker remained increased after 4+ days compared to admission. Histone H3 was found in 28% of the patients on admission to the ICU and in 50% of the patients during their stay at the ICU. Notably, in 47% of histone-positive patients, we observed proteolysis of histone in their plasma. The overall presence of histone H3 during ICU stay was associated with thromboembolic events and secondary infection, and non-cleaved histone H3 was associated with the need for vasoactive treatment, invasive ventilation, and the development of acute kidney injury. Our data support the validity of treatments that aim to reduce NET formation and additionally underscore that more targeted therapies focused on the neutralization of histones should be considered as treatment options for severe COVID-19 patients.

Evolution of NETosis markers and DAMPs have prognostic value in critically ill COVID-19 patients.

Coronavirus disease 19 (COVID-19) presents with disease severities of varying degree. In its most severe form, infection may lead to respiratory failure and multi-organ dysfunction. Here we study the levels and evolution of the damage associated molecular patterns (DAMPS) cell free DNA (cfDNA), extracellular histone H3 (H3) and neutrophil elastase (NE), and the immune modulators GAS6 and AXL in relation to clinical parameters, ICU scoring systems and mortality in patients (n = 100) with severe COVID-19. cfDNA, H3, NE, GAS6 and AXL were increased in COVID-19 patients compared to controls. These measures associated with occurrence of clinical events and intensive care unit acquired weakness (ICUAW). cfDNA and GAS6 decreased in time in patients surviving to 30 days post ICU admission. A decrease of 27.2 ng/mL cfDNA during ICU stay associated with patient survival, whereas levels of GAS6 decreasing more than 4.0 ng/mL associated with survival. The presence of H3 in plasma was a common feature of COVID-19 patients, detected in 38% of the patients at ICU admission. NETosis markers cfDNA, H3 and NE correlated well with parameters of tissue damage and neutrophil counts. Furthermore, cfDNA correlated with lowest p/f ratio and a lowering in cfDNA was observed in patients with ventilator-free days.

The Anticoagulant and Nonanticoagulant Properties of Heparin.

Heparins represent one of the most frequently used pharmacotherapeutics. Discovered around 1926, routine clinical anticoagulant use of heparin was initiated only after the publication of several seminal papers in the early 1970s by the group of Kakkar. It was shown that heparin prevents venous thromboembolism and mortality from pulmonary embolism in patients after surgery. With the subsequent development of low-molecular-weight heparins and synthetic heparin derivatives, a family of related drugs was created that continues to prove its clinical value in thromboprophylaxis and in prevention of clotting in extracorporeal devices. Fundamental and applied research has revealed a complex pharmacodynamic profile of heparins that goes beyond its anticoagulant use. Recognition of the complex multifaceted beneficial effects of heparin underscores its therapeutic potential in various clinical situations. In this review we focus on the anticoagulant and nonanticoagulant activities of heparin and, where possible, discuss the underlying molecular mechanisms that explain the diversity of heparin's biological actions.