Hyper–inflammatory responses can lead to a variety of diseases including sepsis1. We now report that extracellular histones released in response to inflammatory challenge contribute to endothelial dysfunction, organ failure and death during sepsis. They can be targeted pharmacologically by antibody to histone or by activated protein C (APC). Antibody to histone reduced the mortality of mice in lipopolysaccharide (LPS), tumor necrosis factor (TNF) or cecal ligation and puncture models of sepsis. Extracellular histones are cytotoxic toward endothelium in vitro and are lethal in mice. In vivo, histone administration resulted in neutrophil margination, vacuolated endothelium, intra–alveolar hemorrhage and macro and microvascular thrombosis. Histone was detected in the circulation of baboons challenged with E. coli and the increase in histone levels accompanied the onset of renal dysfunction. APC cleaves histones and reduces their cytotoxicity. Co–infusion of APC with E. coli in baboons or histones in mice prevented lethality. Blockade of protein C activation exacerbated sublethal LPS challenge into lethality which was reversed by antibody to histone. We conclude that extracellular histones are potential molecular targets for therapeutics for sepsis and other inflammatory diseases.

The release of histones from dying cells is associated with microvascular thrombosis and, because histones activate platelets, this could represent a possible pathogenic mechanism. In the present study, we assessed the influence of histones on the procoagulant potential of human platelets in platelet-rich plasma (PRP) and in purified systems. Histones dose-dependently enhanced thrombin generation in PRP in the absence of any trigger, as evaluated by calibrated automated thrombinography regardless of whether the contact phase was inhibited. Activation of coagulation required the presence of fully activatable platelets and was not ascribable to platelet tissue factor, whereas targeting polyphosphate with phosphatase reduced thrombin generation even when factor XII (FXII) was blocked or absent. In the presence of histones, purified polyphosphate was able to induce thrombin generation in plasma independently of FXII. In purified systems, histones induced platelet aggregation; P-selectin, phosphatidylserine, and FV/Va expression; and prothrombinase activity. Blocking platelet TLR2 and TLR4 with mAbs reduced the percentage of activated platelets and lowered the amount of thrombin generated in PRP. These data show that histone-activated platelets possess a procoagulant phenotype that drives plasma thrombin generation and suggest that TLR2 and TLR4 mediate the activation process. (Blood. 2011;118(7):1952-1961) IntroductionHistones are cationic proteins that associate with DNA in nucleosomes and are involved in chromatin remodeling and regulation of gene transcription. Despite their physiologic nuclear localization, nucleosomes have been found in the circulation of both healthy subjects and patients, where they can be released from dying cells 1 or actively secreted by activated inflammatory cells (neutrophils, basophils, and mast cells) in the form of "extracellular traps," complex structures of DNA strands, histones, and cell-specific granule proteins. 2,3 High blood levels of nucleosomes have been detected in several inflammatory, ischemic, autoimmune, and neoplastic diseases 4 ; in some cases, a correlation with disease severity has been found. 5 Whether extracellular nucleosomes are merely bystanders or active mediators in disease and which role histones play are important emerging questions. Histones are known to possess cytotoxic properties against both microorganisms 6 and eukaryotic cells. 7 Xu et al 8 reported that extracellular histones behave as late mediators of cell damage and organ dysfunction during the hyperinflammatory reaction that characterizes sepsis, as shown by the efficacy of a neutralizing antibody against histone H4 in reducing mortality in several experimental models of murine sepsis. Moreover, direct injection of histones into mice resulted in death with pathologic lesions suggestive of a massive prothrombotic response similar to that found in sepsis, including diffuse microvascular thrombosis, fibrin and platelet deposition in the lung alveoli, and intra-alveolar hemorrhage. Fuchs et al 9 rec...

Histones enhance plasma thrombin generation by reducing TM-dependent protein C activation. This mechanism might contribute to microvascular thrombosis induced by histones in vivo at sites of organ failure or severe inflammation.

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Sepsis is almost invariably associated with haemostatic abnormalities ranging from subclinical activation of blood coagulation (hypercoagulability), which may contribute to localized venous thromboembolism, to acute disseminated intravascular coagulation (DIC), characterized by massive thrombin formation and widespread microvascular thrombosis, partly responsible of the multiple organ dysfunction syndrome (MODS), and subsequent consumption of platelets and coagulation proteins causing, in most severe cases, bleeding manifestations. There is general agreement that the key event underlying this life-threatening sepsis complication is the overwhelming inflammatory host response to the infectious agent leading to the overexpression of inflammatory mediators. Mechanistically, the latter, together with the micro-organism and its derivatives, causes DIC by 1) up-regulation of procoagulant molecules, primarily tissue factor (TF), which is produced mainly by stimulated monocytes-macrophages and by specific cells in target tissues; 2) impairment of physiological anticoagulant pathways (antithrombin, protein C pathway, tissue factor pathway inhibitor), which is orchestrated mainly by dysfunctional endothelial cells (ECs); and 3) suppression of fibrinolysis due to increased plasminogen activator inhibitor-1 (PAI-1) by ECs and likely also to thrombin-mediated activation of thrombin-activatable fibrinolysis inhibitor (TAFI). Notably, clotting enzymes non only lead to microvascular thrombosis but can also elicit cellular responses that amplify the inflammatory reactions. Inflammatory mediators can also cause, directly or indirectly, cell apoptosis or necrosis and recent evidence indicates that products released from dead cells, such as nuclear proteins (particularly extracellular histones), are able to propagate further inflammation, coagulation, cell death and MODS. These insights into the pathogenetic mechanisms of DIC and MODS may have important implications for the development of new therapeutic agents that could be potentially useful particularly for the management of severe sepsis.