Carboxypeptidase B blocks ex vivo activation of the anaphylatoxin-neutrophil extracellular trap axis in neutrophils from COVID-19 patients

Abstract

Background Thrombosis and coagulopathy are highly prevalent in critically ill patients with COVID-19 and increase the risk of death. Immunothrombosis has recently been demonstrated to contribute to the thrombotic events in COVID-19 patients with coagulopathy. As the primary components of immunothrombosis, neutrophil extracellular traps (NETs) could be induced by complement cascade components and other proinflammatory mediators. We aimed to explore the clinical roles of NETs and the regulation of complement on the NET formation in COVID-19. Methods We recruited 135 COVID-19 patients and measured plasma levels of C5, C3, cell-free DNA and myeloperoxidase (MPO)-DNA. Besides, the formation of NETs was detected by immunofluorescent staining and the cytotoxicity to vascular endothelial HUVEC cells was evaluated by CCK-8 assay. Results We found that the plasma levels of complements C3 and MPO-DNA were positively related to coagulation indicator fibrin(-ogen) degradation products (C3: r = 0.300, p = 0.005; MPO-DNA: r = 0.316, p = 0.002) in COVID-19 patients. Besides, C3 was positively related to direct bilirubin (r = 0.303, p = 0.004) and total bilirubin (r = 0.304, p = 0.005), MPO-DNA was positively related to lactate dehydrogenase (r = 0.306, p = 0.003) and creatine kinase (r = 0.308, p = 0.004). By using anti-C3a and anti-C5a antibodies, we revealed that the complement component anaphylatoxins in the plasma of COVID-19 patients strongly induced NET formation. The pathological effect of the anaphylatoxin-NET axis on the damage of vascular endothelial cells could be relieved by recombinant carboxypeptidase B (CPB), a stable homolog of enzyme CPB2 which can degrade anaphylatoxins to inactive products. Conclusions Over-activation in anaphylatoxin-NET axis plays a pathological role in COVID-19. Early intervention in anaphylatoxins might help prevent thrombosis and disease progression in COVID-19 patients.

ICODE: the international COVID-19 thrombosis biomarkers colloquium—novel soluble biomarkers: circulating cell-free nucleic acids and other molecules

“…In parallel to plasma, Carvelli et al [ 25 ] tested BALF samples. The association of elevated levels of complement factors with severe courses of COVID-19 could be verified by most of the groups [ 12 , 25 – 28 ].…”

“…135 COVID-19 patients and 25 healthy controls were enrolled in a study by Zhang et al [ 12 ]. Cell-free DNA and MPO/DNA complexes were higher in plasma of patients with COVID-19 than in healthy controls and were associated with severity of COVID-19.…”

“…The usefulness of complement factors (C3 [ 12 ], C5 [ 12 ], C5a [ 25 – 28 ], sC5b-9 [ 7 , 26 – 28 ]) as biomarkers for COVID-19 has been suggested by several groups. All of the studies were performed with assays using plasma samples and demonstrated increased levels in COVID-19 patients.…”

Patients with COVID-19: in the dark-NETs of neutrophils

“…Treatment options targeting the pro-inflammatory action of NETs such as PADI4 inhibitors, or antibodies that block extrusion of NETs [46], or R406, a potent SYK inhibitor and the metabolically active component of fostamatinib [177], are potential new classes of drugs to tackle NET formation and to alleviate NET toxicity and in patients with severe COVID-19 [178]. In addition, the pharmacological inhibition of CatC to counterbalance the unwanted effects of neutrophil serine proteases in severe COVID-19 is considered a potential therapeutic target [179].…”

The case of complement inhibitors

“…In addition, control neutrophils stimulated with platelet rich plasma (PRP) from COVID-19 patients generate NETs that induce tissue factor expression in human aortic endothelial cells in vitro and both effects were attenuated by C5a receptor blockade ( Skendros et al, 2020 ). Finally, plasma from severe COVID-19 patients requiring mechanical ventilation activated control neutrophils to release NETs, an effect that was restrained by an anti-C5a neutralizing antibody ( Zhang et al, 2021 ).…”