Functional responses and molecular mechanisms involved in histone-mediated platelet activation (original) (raw)
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Blood, 2016
NETosis is a physiological process in which neutrophils release their nuclear material in the form of neutrophil extracellular traps (NETs). NETs have been reported to directly promote thrombosis in animal models. Although the effects of purified NET components including DNA, histone proteins and neutrophil enzymes on coagulation have been characterized, the mechanism by which intact NETs promote thrombosis is largely unknown. In this study, human neutrophils were stimulated to produce NETs in platelet-free plasma (PFP) or in buffer using PMA or calcium ionophore. DNA and histone proteins were also separately purified from normal human neutrophils, and used to reconstitute chromatin using a salt-gradient dialysis method. Neutrophil stimulation resulted in robust NET release. In re-calcified PFP, purified DNA triggered contact-dependent thrombin generation and amplified thrombin generation initiated by low concentrations of tissue factor. Similarly, in a buffer milieu, DNA initiated ...
Mechanical Stability and Fibrinolytic Resistance of Clots Containing Fibrin, DNA, and Histones
Journal of Biological Chemistry, 2013
Background: Neutrophil extracellular traps (NETs) composed of DNA and proteins form a scaffold in thrombi, supplementing the fibrin matrix. Results: DNA and histones modify the structure of fibrin and render it resistant to mechanical and enzymatic destruction. Conclusion: NET components are essential factors in thrombus stability. Significance: Therapeutic strategies could be optimized to enhance fibrinolysis in clots containing DNA and histones. Neutrophil extracellular traps are networks of DNA and associated proteins produced by nucleosome release from activated neutrophils in response to infection stimuli and have recently been identified as key mediators between innate immunity, inflammation, and hemostasis. The interaction of DNA and histones with a number of hemostatic factors has been shown to promote clotting and is associated with increased thrombosis, but little is known about the effects of DNA and histones on the regulation of fibrin stability and fibrinolysis. Here we demonstrate that the addition of histone-DNA complexes to fibrin results in thicker fibers (increase in median diameter from 84 to 123 nm according to scanning electron microscopy data) accompanied by improved stability and rigidity (the critical shear stress causing loss of fibrin viscosity increases from 150 to 376 Pa whereas the storage modulus of the gel increases from 62 to 82 pascals according to oscillation rheometric data). The effects of DNA and histones alone are subtle and suggest that histones affect clot structure whereas DNA changes the way clots are lysed. The combination of histones ؉ DNA significantly prolongs clot lysis. Isothermal titration and confocal microscopy studies suggest that histones and DNA bind large fibrin degradation products with 191 and 136 nM dissociation constants, respectively, interactions that inhibit clot lysis. Heparin, which is known to interfere with the formation of neutrophil extracellular traps, appears to prolong lysis time at a concentration favoring ternary histone-DNA-heparin complex formation, and DNase effectively promotes clot lysis in combination with tissue plasminogen activator. Neutrophil extracellular traps (NETs) 2 are networks of DNA decorated with histones and other proteins, proteases, and other antimicrobial factors (1). They are produced when neutrophils, basophils, or mast cells release nucleosomes after stimulation by inflammatory cytokines or LPS for example (1) or by interaction with platelets after stimulation of platelet tolllike receptors 4 and 2 by microbial structures (2, 3). It is proposed that NET formation is the first line of defense of the innate immune system, providing an effective way of trapping pathogenic microbes and removing them from the circulation (4). The DNA and histones of NETs provide a scaffold for cell localization (including neutrophils and red blood cells), platelet aggregation, and activation that serves to promote coagulation and thrombosis (5). Thus, NETs are a focus of significant crosstalk between innate immunity, inflammation, and hemostasis, and there are many points of contact including bidirectional interactions with platelets, inflammatory cytokines (IL-8), fibrinogen, fibronectin, von Willebrand factor, tissue factor pathway inhibitor, protein C, thrombomodulin, factor XII, and neutrophil-derived serine proteases (3-6). Histones H3 and H4 have been identified as critical players and mediators of damage able to stimulate phosphatidylserine exposure and factor Va expression on platelets and interact with polyphosphates to promote clotting even in the absence of factor XII (3). Histones may come from NETs or dying cells and are implicated in the progression of sepsis. Stimulation of coagulation by NETs can result in unwanted thrombosis (7), and infection is a common event in the development of deep vein thrombosis (8, 9). Targeting the release of nucleosomes, development of NETs, and availability of circulating histones could be a strategy for pre-* This work was supported by Wellcome Trust Grant 083174, Hungarian Scientific Research Fund Grants OTKA 75430 and 83023, and German Academic Exchange Service Grant DAAD A/12/01760.
Platelet activation and atherothrombosis
New England Journal of Medicine, 2007
P latelets are essential for primary hemostasis and repair of the endothelium, but they also play a key role in the development of acute coronary syndromes and contribute to cerebrovascular events. In addition, they participate in the process of forming and extending atherosclerotic plaques. Atherosclerosis is a chronic inflammatory process, 1 and inflammation is an important component of acute coronary syndromes. 2 The relation between chronic and acute vascular inflammation is unclear, but platelets are a source of inflammatory mediators, 3 and the activation of platelets by inflammatory triggers may be a critical component of atherothrombosis. 4 This review article describes the role of platelets in atherothrombosis by integrating our knowledge of basic mechanisms with the results of mechanistic studies in humans and clinical trials of inhibitors of platelet function.
Platelets: Physiology and Biochemistry
Seminars in Thrombosis and Hemostasis, 2005
Platelets are specialized blood cells that play central roles in physiologic and pathologic processes of hemostasis, inflammation, tumor metastasis, wound healing, and host defense. Activation of platelets is crucial for platelet function that includes a complex interplay of adhesion and signaling molecules. This article gives an overview of the activation processes involved in primary and secondary hemostasis, for example, platelet adhesion, platelet secretion, platelet aggregation, microvesicle formation, and clot retraction/stabilization. In addition, activated platelets are predominantly involved in cross talk to other blood and vascular cells. Stimulated ''sticky'' platelets enable recruitment of leukocytes at sites of vascular injury under high shear conditions. Platelet-derived microparticles as well as soluble adhesion molecules, sP-selectin and sCD40L, shed from the surface of activated platelets, are capable of activating, in turn, leukocytes and endothelial cells. This article focuses further on the new view of receptor-mediated thrombin generation of human platelets, necessary for the formation of a stable platelet-fibrin clot during secondary hemostasis. Finally, special emphasis is placed on important stimulatory and inhibitory signaling pathways that modulate platelet function.
Journal of biomedical research, 2015
Platelets are small anucleate cells generated from megakaryocytes in the bone marrow. Although platelet generation, maturation, and clearance are still not fully understood, significant progress has been made in the last 1-2 decades. In blood circulation, platelets can quickly adhere and aggregate at sites of vascular injury, forming the platelet plug (i.e. the first wave of hemostasis). Activated platelets can also provide negatively charged phosphatidylserinerich membrane surface that enhances cell-based thrombin generation, which facilitates blood coagulation (i.e. the second wave of hemostasis). Platelets therefore play central roles in hemostasis. However, the same process of hemostasis may also cause thrombosis and vessel occlusion, which are the most common mechanisms leading to heart attack and stroke following ruptured atherosclerotic lesions. In this review, we will introduce the classical mechanisms and newly discovered pathways of platelets in hemostasis and thrombosis, ...
Arteriosclerosis, thrombosis, and vascular biology, 2014
Activation of neutrophils by microbial or inflammatory stimuli results in the release of neutrophil extracellular traps (NETs) that are composed of DNA, histones, and antimicrobial proteins. In purified systems, cell-free DNA (CFDNA) activates the intrinsic pathway of coagulation, whereas histones promote thrombin generation through platelet-dependent mechanisms. However, the overall procoagulant effects of CFDNA/histone complexes as part of intact NETs are unknown. In this study, we examined the procoagulant potential of intact NETs released from activated neutrophils. We also determined the relative contribution of CFDNA and histones to thrombin generation in plasmas from patients with sepsis. NETs released from phorbyl myristate-activated neutrophils enhance thrombin generation in platelet-poor plasma. This effect was DNA dependent (confirmed by DNase treatment) and occurred via the intrinsic pathway of coagulation (confirmed with coagulation factor XII- and coagulation factor XI...
Blood, 2011
Histone deacetylase inhibitor (HDACI)–induced thrombocytopenia (TCP) is a major dose-limiting toxicity of this new class of drugs. Using preclinical models to study the molecular and biologic events that underpin this effect of HDACI, we found that C57BL/6 mice treated with both the HDAC1/2-selective HDACI romidepsin and the pan-HDACI panobinostat developed significant TCP. HDACI-induced TCP was not due to myelosuppression or reduced platelet lifespan, but to decreased platelet release from megakaryocytes. Cultured primary murine megakaryocytes showed reductions in proplatelet extensions after HDACI exposure and a dose-dependent increase in the phosphorylation of myosin light chain 2 (MLC2). Phosphorylation of MLC to phospho-MLC (pMLC) and subsequent proplatelet formation in megakaryocytes is regulated by the Rho-GTPase proteins Rac1, CDC42, and RhoA. Primary mouse megakaryocytes and the human megakaryoblastic cell line Meg-01 showed reductions in Rac1, CDC42, and RhoA protein level...
2020
Inflammation, Chronic Diseases and Cancer-Cell and Molecular Biology, Immunology and Clinical Bases 98 inflammatory diseases such as sepsis, rheumatoid arthritis, and acute lung injury set the stage for modification of the thespian paradigm. One that helps us complete the continuum from coagulation to inflammation and back to coagulation again. Here we propose to name this field of study "Immunohemostasis". No better model to appreciate the crosstalk between coagulation and inflammation than atherosclerosis. 2. Platelets have a role early in the development of atherosclerosis Although platelets are not solely responsible for the development of atherosclerosis, their contribution to the inception of the vascular lesion, up until to atherothrombosis-its most critical consequence-is conceptually best understood as a model of inflammation. This is somewhat amusingly explained by Rudolf Virchow on a footnote in, Cellular Pathology (1865), "Suppose three people were sitting quietly on a bench, and suddenly a stone came and injured one of them, the others would be excited, not only by the sudden appearance of the stone, but also by the injury done to their companion, to whose help they would feel bound to hasten. Here the stone would be the irritant, the injury the irritament, the help an expression of the irritation called forth in the bystanders". Following Dr. Virchow's thought process, modern science not only has documented many different stones but also acknowledges that at times, these bystanders can hasten the irritament (inflammatory stimulus), therefore as we will understand an overzealous and excited bystander could prove to be, vessel hardening. If we look at atherosclerosis as a model of inflammatory disease, platelet adhesion could similarly be regarded as a model of platelet induced disease (Langer & Gawaz, 2008). Atherogenesis is influenced by platelets that adhere to activated vascular endothelial cells and feed chemotactic mediators to adjoining cells. Although the underlying mechanism of atherosclerosis is attributed to endothelial impairment due to insults from genetic and environmental factors (Lusis, 2000), it needs platelet firm adhesion to the endothelium for inception of the atheromatous plaque (Spagnoli et al., 2007). Genetic and environmental factors that trigger injurious events, which include the formation of reactive oxygen species, reduce the bioavailability of nitric oxide (Lowenstein et al., 2005). Then the nondenuded, but aggravated endothelium fails to inhibit control over Weibel-Palade body exocytosis translocating P-selectin and von Willebrand Factor (vWF) from within the granules to the outer cellular surface (Wagner & Frenette, 2008). These two proteins allow the adhesion of platelets to the vascular endothelium in a multistep process. First platelets are tethered to the vascular wall with assistance by endothelial selectins. Platelets then roll on the vascular endothelial cells (Polgar et al., 2005). Depending on further activation of the endothelial cell and expression of endothelial integrins, the platelet adheres firmly to the vascular wall (May et al., 2008), or in the absence of further endothelial activation, the platelet, disengages from the vessel wall and returns to circulation (White, 2007). Remarkably this can occur due to the fact that platelet activation is not required for platelet rolling (Harrison, 2005). In contrast, experimental models of mice infused with activated platelets also stimulate Weibel-Palade body exocytosis, promoting the development of atherosclerosis which is attributed to platelet P-selectin-mediated delivery of platelet-derived proinflammatory factors to monocytes/leukocytes and the vessel wall (Delvaeye & Conway, 2009).
Inflammation, Chronic Diseases and Cancer - Cell and Molecular Biology, Immunology and Clinical Bases, 2012
Inflammation, Chronic Diseases and Cancer-Cell and Molecular Biology, Immunology and Clinical Bases 98 inflammatory diseases such as sepsis, rheumatoid arthritis, and acute lung injury set the stage for modification of the thespian paradigm. One that helps us complete the continuum from coagulation to inflammation and back to coagulation again. Here we propose to name this field of study "Immunohemostasis". No better model to appreciate the crosstalk between coagulation and inflammation than atherosclerosis. 2. Platelets have a role early in the development of atherosclerosis Although platelets are not solely responsible for the development of atherosclerosis, their contribution to the inception of the vascular lesion, up until to atherothrombosis-its most critical consequence-is conceptually best understood as a model of inflammation. This is somewhat amusingly explained by Rudolf Virchow on a footnote in, Cellular Pathology (1865), "Suppose three people were sitting quietly on a bench, and suddenly a stone came and injured one of them, the others would be excited, not only by the sudden appearance of the stone, but also by the injury done to their companion, to whose help they would feel bound to hasten. Here the stone would be the irritant, the injury the irritament, the help an expression of the irritation called forth in the bystanders". Following Dr. Virchow's thought process, modern science not only has documented many different stones but also acknowledges that at times, these bystanders can hasten the irritament (inflammatory stimulus), therefore as we will understand an overzealous and excited bystander could prove to be, vessel hardening. If we look at atherosclerosis as a model of inflammatory disease, platelet adhesion could similarly be regarded as a model of platelet induced disease (Langer & Gawaz, 2008). Atherogenesis is influenced by platelets that adhere to activated vascular endothelial cells and feed chemotactic mediators to adjoining cells. Although the underlying mechanism of atherosclerosis is attributed to endothelial impairment due to insults from genetic and environmental factors (Lusis, 2000), it needs platelet firm adhesion to the endothelium for inception of the atheromatous plaque (Spagnoli et al., 2007). Genetic and environmental factors that trigger injurious events, which include the formation of reactive oxygen species, reduce the bioavailability of nitric oxide (Lowenstein et al., 2005). Then the nondenuded, but aggravated endothelium fails to inhibit control over Weibel-Palade body exocytosis translocating P-selectin and von Willebrand Factor (vWF) from within the granules to the outer cellular surface (Wagner & Frenette, 2008). These two proteins allow the adhesion of platelets to the vascular endothelium in a multistep process. First platelets are tethered to the vascular wall with assistance by endothelial selectins. Platelets then roll on the vascular endothelial cells (Polgar et al., 2005). Depending on further activation of the endothelial cell and expression of endothelial integrins, the platelet adheres firmly to the vascular wall (May et al., 2008), or in the absence of further endothelial activation, the platelet, disengages from the vessel wall and returns to circulation (White, 2007). Remarkably this can occur due to the fact that platelet activation is not required for platelet rolling (Harrison, 2005). In contrast, experimental models of mice infused with activated platelets also stimulate Weibel-Palade body exocytosis, promoting the development of atherosclerosis which is attributed to platelet P-selectin-mediated delivery of platelet-derived proinflammatory factors to monocytes/leukocytes and the vessel wall (Delvaeye & Conway, 2009).