Extracellular Histones Bind Vascular Glycosaminoglycans and Inhibit the Anti-Inflammatory Function of Antithrombin.

BACKGROUND/AIMS: Binding of histones to molecular pattern recognition receptors on endothelial cells and leukocytes provokes proinflammatory responses and promotes activation of coagulation. Histones also bind therapeutic heparins, thereby neutralizing their anticoagulant functions. The aim of this study was to test the hypothesis that histones can interact with the antithrombin (AT)-binding vascular glycosaminoglycans (GAGs) to induce inflammation and inhibit the anti-inflammatory function of AT. METHODS: We evaluated the heparin-binding function of histones by an AT-dependent protease-inhibition assay. Furthermore, we treated endothelial cells with histones in the absence and presence of AT and monitored cellular phenotypes employing established signaling assays. RESULTS: Histones neutralized AT-dependent anticoagulant function of heparin in both purified protease-inhibition and plasma-based assays. Histones also disrupted endothelial cell barrier-permeability function by a GAG-dependent mechanism as evidenced by the GAG-antagonist, surfen, abrogating their disruptive effects. Further studies revealed histones and AT compete for overlapping binding-sites on GAGs, thus increasing concentrations of one protein abrogated effects of the other. Histones elicited proapoptotic effects by inducing nuclear localization of PKC-δ in endothelial cells and barrier-disruptive effects by destabilizing VE-cadherin, which were inhibited by AT, but not by a D-helix mutant of AT incapable of interacting with GAGs. Finally, histones induced release of Weibel-Palade body contents, VWF and angiopoietin-2, and promoted expression of cell adhesion molecules on endothelial cells, which were all downregulated by AT but not by D-helix mutant of AT. CONCLUSION: We conclude that histones and AT compete for overlapping binding sites on vascular GAGs to modulate coagulation and inflammation.

Antithrombin protects against Plasmodium falciparum histidine-rich protein II-mediated inflammation and coagulation.

Plasmodium falciparum-derived histidine-rich protein II (HRPII) has been shown to inhibit heparin-dependent anticoagulant activity of antithrombin (AT) and induce inflammation in vitro and in vivo. In a recent study, we showed that HRPII interacts with the AT-binding vascular glycosaminoglycans (GAGs) not only to disrupt the barrier-permeability function of endothelial cells but also to inhibit the antiinflammatory signaling function of AT. Here we investigated the mechanisms of the proinflammatory function of HRPII and the protective activity of AT in cellular and animal models. We found that AT competitively inhibits the GAG-dependent HRPII-mediated activation of NF-κB and expression of intercellular cell adhesion molecule 1 (ICAM1) in endothelial cells. Furthermore, AT inhibits HRPII-mediated histone H3 citrullination and neutrophil extracellular trap (NET) formation in HL60 cells and freshly isolated human neutrophils. In vivo, HRPII induced Mac1 expression on blood neutrophils, MPO release in plasma, neutrophil infiltration, and histone H3 citrullination in the lung tissues. HRPII also induced endothelial cell activation as measured by increased ICAM1 expression and elevated vascular permeability in the lungs. AT effectively inhibited HRPII-mediated neutrophil infiltration, NET formation, and endothelial cell activation in vivo. AT also inhibited HRPII-meditated deposition of platelets and fibrin(ogen) in the lungs and circulating level of von Willebrand factor in the plasma. We conclude that AT exerts protective effects against pathogenic effects of P falciparum-derived HRPII in both cellular and animal models.