Neha Sharma1,2, Lauren Haggstrom1, Sahar Sohrabipour1,2, Dhruva J Dwivedi1,2, Patricia C Liaw1,2,3. 1. Thrombosis and Atherosclerosis Research Institute, Hamilton, ON, Canada. 2. Department of Medical Sciences, McMaster University, Hamilton, ON, Canada. 3. Department of Medicine, McMaster University, Hamilton, ON, Canada.
Abstract
BACKGROUND: Extracellular histones exert cytotoxic and procoagulant effects which contribute to immunothrombosis in vascular diseases such as sepsis. Heparin has been shown to neutralize the pathologic effects of histones in vitro and in animal models. OBJECTIVES: To compare the effectiveness of unfractionated heparin (UFH), low-molecularweight heparin (LMWH), Vasoflux (lacks anticoagulant activity), and fondaparinux in neutralizing the cytotoxic and procoagulant activities of histones METHODS: Binding affinities between heparin variants and histone subunits were determined by Bio-layer Interferometry. The ability of heparin variants to diminish the cytotoxic and procoagulant effects of histones was studied by treating endothelial cells or monocytic THP-1 cells with histones ± heparin variants. RESULTS: Unfractionated heparin, LMWH, and Vasoflux bind histone subunits with high affinities (Kd <1 pM-66.7 nM) whereas fondaparinux exhibited a low affinity (Kd of 3.06 µM-81.1 mM). UFH, LMWH, and Vasoflux neutralize histone-mediated cytotoxicity as well as monocytic procoagulant activity (as assessed by cell surface tissue factor and phosphatidylserine). In contrast, fondaparinux has no effect on these activities. All four heparin variants reverse histone-mediated impairment of APC generation in a dose-dependent manner. CONCLUSIONS: The ability of heparin to neutralize the cytotoxic and procoagulant effects of histones require heparin fragments >1.7 kDa and is independent of the antithrombin-binding pentasaccharide. In contrast, the ability of heparin to neutralize histone-mediated impairment of APC generation is independent of size and anticoagulant activity. These findings suggest that heparin variants may have differential therapeutic potential in vascular diseases associated with elevated levels of histones.
BACKGROUND: Extracellular histones exert cytotoxic and procoagulant effects which contribute to immunothrombosis in vascular diseases such as sepsis. Heparin has been shown to neutralize the pathologic effects of histones in vitro and in animal models. OBJECTIVES: To compare the effectiveness of unfractionated heparin (UFH), low-molecularweight heparin (LMWH), Vasoflux (lacks anticoagulant activity), and fondaparinux in neutralizing the cytotoxic and procoagulant activities of histones METHODS: Binding affinities between heparin variants and histone subunits were determined by Bio-layer Interferometry. The ability of heparin variants to diminish the cytotoxic and procoagulant effects of histones was studied by treating endothelial cells or monocytic THP-1 cells with histones ± heparin variants. RESULTS: Unfractionated heparin, LMWH, and Vasoflux bind histone subunits with high affinities (Kd <1 pM-66.7 nM) whereas fondaparinux exhibited a low affinity (Kd of 3.06 µM-81.1 mM). UFH, LMWH, and Vasoflux neutralize histone-mediated cytotoxicity as well as monocytic procoagulant activity (as assessed by cell surface tissue factor and phosphatidylserine). In contrast, fondaparinux has no effect on these activities. All four heparin variants reverse histone-mediated impairment of APC generation in a dose-dependent manner. CONCLUSIONS: The ability of heparin to neutralize the cytotoxic and procoagulant effects of histones require heparin fragments >1.7 kDa and is independent of the antithrombin-binding pentasaccharide. In contrast, the ability of heparin to neutralize histone-mediated impairment of APC generation is independent of size and anticoagulant activity. These findings suggest that heparin variants may have differential therapeutic potential in vascular diseases associated with elevated levels of histones.