Abstract
Extracellular histones in neutrophil extracellular traps (NETs) or in chromatin from injured tissues are highly pathological, particularly when liberated by DNases. We report the development of small polyanions (SPAs) (~0.9–1.4 kDa) that interact electrostatically with histones, neutralizing their pathological effects. In vitro, SPAs inhibited the cytotoxic, platelet-activating and erythrocyte-damaging effects of histones, mechanistic studies revealing that SPAs block disruption of lipid-bilayers by histones. In vivo, SPAs significantly inhibited sepsis, deep-vein thrombosis, and cardiac and tissue-flap models of ischemia-reperfusion injury (IRI), but appeared to differ in their capacity to neutralize NET-bound versus free histones. Analysis of sera from sepsis and cardiac IRI patients supported these differential findings. Further investigations revealed this effect was likely due to the ability of certain SPAs to displace histones from NETs, thus destabilising the structure. Finally, based on our work, a non-toxic SPA that inhibits both NET-bound and free histone mediated pathologies was identified for clinical development.
Highlights
Extracellular histones in neutrophil extracellular traps (NETs) or in chromatin from injured tissues are highly pathological, when liberated by DNases
Dead cells are detected by propidium iodide (PI) uptake and viable cells by calcein-AM retention (Supplementary Fig. 1a), with histones being cytotoxic for human microvascular endothelial cell-1 (HMEC-1) and human umbilical vein endothelial cells (HUVECs) in a concentration-dependent manner (Supplementary Fig. 1b)
We describe the development of small polyanionic molecules, such as cellobiose per-O-sulfate (CBS), methyl β-cellobioside per-O-sulfate (mCBS), and maltotriose per-Osulfate (MTS), that are very effective inhibitors of a number of pathological processes mediated by DNA-free histones, such as cytotoxicity, erythrocyte fragility/deformability, and platelet activation in vitro and in vivo
Summary
Extracellular histones in neutrophil extracellular traps (NETs) or in chromatin from injured tissues are highly pathological, when liberated by DNases. Analysis of sera from sepsis and cardiac IRI patients supported these differential findings Further investigations revealed this effect was likely due to the ability of certain SPAs to displace histones from NETs, destabilising the structure. DNAfree histones attack glomerular basement membranes[8], initiate coagulation by activating platelets[9,10], and bind erythrocytes, inducing phosphatidylserine exposure and enhancing erythrocyte fragility and rigidity resulting in splenic retention and anemia[11,12] These effects lead to the development of microthrombi and extensive tissue and organ damage resembling the pathology seen in sepsis. A recent study in COVID-19 patients correlated serum NET levels with the severity of respiratory disease[27] It appears that NETs and associated histones play a role in autoimmune diseases[28,29,30] and even in the development of gallstones[31]. We undertook a drug discovery program based on the hypothesis that, since histones are highly cationic, small polyanionic molecules that generally have a favorable safety profile, should interact electrostatically with free histones and NETs and neutralize their pathological effects
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