Recombinant Thrombomodulin Protects Mice against Histone-Induced Lethal Thromboembolism

Abstract

IntroductionRecent studies have shown that histones, the chief protein component of chromatin, are released into the extracellular space during sepsis, trauma, and ischemia-reperfusion injury, and act as major mediators of the death of an organism. This study was designed to elucidate the cellular and molecular basis of histone-induced lethality and to assess the protective effects of recombinant thrombomodulin (rTM). rTM has been approved for the treatment of disseminated intravascular coagulation (DIC) in Japan, and is currently undergoing a phase III clinical trial in the United States.MethodsHistone H3 levels in plasma of healthy volunteers and patients with sepsis and DIC were measured using enzyme-linked immunosorbent assay. Male C57BL/6 mice were injected intravenously with purified histones, and pathological examinations were performed. The protective effects of rTM against histone toxicity were analyzed both in vitro and in mice.ResultsHistone H3 was not detectable in plasma of healthy volunteers, but significant levels were observed in patients with sepsis and DIC. These levels were higher in non-survivors than in survivors. Extracellular histones triggered platelet aggregation, leading to thrombotic occlusion of pulmonary capillaries and subsequent right-sided heart failure in mice. These mice displayed symptoms of DIC, including thrombocytopenia, prolonged prothrombin time, decreased fibrinogen, fibrin deposition in capillaries, and bleeding. Platelet depletion protected mice from histone-induced death in the first 30 minutes, suggesting that vessel occlusion by platelet-rich thrombi might be responsible for death during the early phase. Furthermore, rTM bound to extracellular histones, suppressed histone-induced platelet aggregation, thrombotic occlusion of pulmonary capillaries, and dilatation of the right ventricle, and rescued mice from lethal thromboembolism.ConclusionsExtracellular histones cause massive thromboembolism associated with consumptive coagulopathy, which is diagnostically indistinguishable from DIC. rTM binds to histones and neutralizes the prothrombotic action of histones. This may contribute to the effectiveness of rTM against DIC.

Biology, role and therapeutic potential of circulating histones in acute inflammatory disorders

“…Elevated histone‐degrading activated protein C (APC) levels are associated with better outcomes in sepsis115, 149 and trauma patients137; APC therapy is being evaluated for treatment of sepsis150 and pancreatitis 151. The effects of histones and NETs on the coagulation cascade can be overcome by therapy with thrombomodulin121 or tissue plasminogen activator (tPA),143 but the clot‐stabilizing effects of DNA in NETs must be overcome, which is well illustrated by the finding that DNAse therapy in addition to tPA is more effective than either therapy alone 152…”

Histones as Mediators of Host Defense, Inflammation and Thrombosis

“…These events have been directly linked to lung injury after trauma or transfusions [22,[97][98][99]. PAD4 and neutrophils have been shown to be critical for deep-vein thrombosis in mice [100].…”

“…PAD4 and neutrophils have been shown to be critical for deep-vein thrombosis in mice [100]. IIIa [92,97,99]. Heparin has been shown to prevent binding of histones to platelets and protect against thrombocytopenia, tissue injury and death caused by histone infusion [92].…”

DNA and factor VII–activating protease protect against the cytotoxicity of histones

“…Indeed, increased levels of circulating histones and nucleosomes have been found in patients with a range of inflammatory conditions, including sepsis, [1][2][3] traumatic injury and surgery, [3][4][5][6] cerebral stroke, 7 systemic lupus erythematosus, 8 and cancer. 9,10 In 2009, Xu et al 11 demonstrated that histones released by macrophages upon inflammatory challenge were cytotoxic to endothelial cells in vitro.…”