Circulating Heparan Sulfate Fragments Attenuate Histone-Induced Lung Injury Independently of Histone Binding

Abstract

Extracellular histones are cationic damage-associated molecular pattern molecules capable of directly inducing cellular injury via charge-mediated interactions with plasma membranes. Accordingly, histones released into the plasma during critical illness are known to contribute to the onset and propagation of lung injury. Vascular injury (with consequent degradation of the endothelial glycocalyx) simultaneously releases anionic heparan sulfate fragments (hexa- to octasaccharides in size) into the plasma. It is unknown whether this endogenous release of heparan sulfate fragments modulates charge-dependent histone cytotoxicity, or if exogenous heparan sulfate fragments could therapeutically attenuate histone-induced lung injury. Using isothermic calorimetry, we found that extracellular histones only bind to heparan sulfate fragments ≥ 10 saccharides in size, suggesting that glycocalyx-derived heparan sulfate hexa/octasaccharides are incapable of intercepting/neutralizing circulating histones. However, we found that even heparan sulfate fragments incapable of histone binding (e.g. tetrasaccharides) attenuated histone-induced lung injury in vivo, suggesting a direct, size-independent protective effect of heparan sulfate. We found that histones had no effect on human neutrophils ex vivo but exerted toll-like receptor-independent cytotoxicity on human pulmonary microvascular endothelial cells in vitro. This cytotoxicity could be prevented by either the addition of negatively-charged (i.e. highly-sulfated) heparan sulfate tetrasaccharides (incapable of binding histones) or decasaccharides (capable of binding histones). Taken together, our findings suggest that heparan sulfate oligosaccharides may directly exert pulmonary endothelial-protective effects that attenuate histone-mediated lung injury.

Heparin as a therapy for COVID-19: current evidence and future possibilities

“…As noted, unfractionated heparin is composed of a mixture of distinct biologically derived (i.e., porcine intestinal mucosae) heparan sulfate polysaccharides, which on balance not only is enriched in a pentasaccharide sequence necessary for AT3 activation (and thus anticoagulation) but also displays a wide variety of other sulfation sequences. These non-anticoagulant sulfation sequences allow heparin binding to various growth factors, potentially exerting both organ-protective ( 46 , 49 ) and organ-harmful ( 14 , 20 ) effects.…”

Beneficial non-anticoagulant mechanisms underlying heparin treatment of COVID-19 patients

“…Recent studies have reported that NETs are present in tissues and the circulation of COVID-19 patients [75] , suggesting that their cytotoxicity contributes to disease. Negatively charged heparin and, desulphated heparin oligosaccharides has been demonstrated to neutralise the cytotoxic effect of positively charged histones, thereby potentially reducing organ damage [79] , [80] , [81] . In summary, heparin/LMWH may neutralise the inflammatory and cytotoxic effects of extracellular histones in COVID-19.…”

Circulating heparan sulfate fragments mediate septic cognitive dysfunction

“…These disaccharides may be sulfated at specific sites (including the 2-O position of iduronic acid and/or the 6-O or N position of glucosamine), imparting a domain patterning of negative charge. During sepsis, the glycocalyx is fragmented, releasing heparan sulfate hexa-and octasaccharides into the bloodstream (14)(15)(16). These fragments have the capacity to interact with soluble proteins (such as growth factors) with remarkable specificity through sulfation-based electrostatic interactions, influencing a variety of homeostatic and/or pathologic signaling pathways (17).…”