Extracellular histones promote thrombin generation through platelet-dependent mechanisms: involvement of platelet TLR2 and TLR4

Abstract

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...

Histone H4 Promotes Prothrombin Autoactivation

“…During response to traumatic injury, sepsis, inflammation, and cell necrosis, the levels of histones, RNA, and polyamines become elevated in the blood (8,9,11,14,15,26) and correlate with thrombosis, inflammation, and multiple organ dysfunction (27,28). We explored the possibility that some of these components express their prothrombotic function via direct interaction with prothrombin.…”

“…Histones promote activation of factor VII activating protease in vivo (8) and have been suggested to interact with fibrinogen (12,13), prothrombin (12), thrombomodulin, and protein C (14). Histones have been postulated to enhance thrombin generation indirectly by reducing the thrombomodulin-dependent activation of protein C anticoagulant pathway (14) or directly through platelet dependent mechanisms involving the Toll-like receptors TLR2 and TLR4 (15). Here, we show that histone H4 promotes prothrombin autoactivation by interacting directly with the zymogen, and we elucidate the underlying mechanism.…”

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

“…As core histones oligomerize readily with each other in solution131 and will surely rapidly do so upon histone release from any cell type, it is difficult to dissociate toxicity of individual histones from each other in biological systems. When used in in vitro studies, recombinant histones H2A and H2B were also able to induce cellular currents126 or activate thrombin 122…”

“…Histones act synergistically to produce a profound pro‐coagulant drive. Histones are able to induce platelet aggregation and factor V/Va expression and prothrombinase activity, leading to thrombin activation independent of the intrinsic coagulation pathway 122. Histones also inhibit thrombomodulin and protein C activation,138 an effect most pronounced with histones H3 and H4, thus reducing a natural thrombin inhibitor system.…”

Changing world of neutrophils

“…They were shown to activate dendritic cells via TLR9 receptors [38,61], linking thereby innate and adaptive immune processes. Activation of platelets via TLR2 and TLR4 receptors [101] and degradation of coagulation inhibitory proteins by proteases on the surface of NETs both promote blood clotting which also impairs bacterial dissemination [51,69]. On the other hand, formation of NETs was suggested to contribute to the viscosity of bronchial fluid in cystic fibrosis patients [67], to formation of autoantibodies in systemic lupus erythematosus [44] and to the pathogenesis of autoimmune vasculitis [54].…”