RESUMO
Interactions between platelets, leukocytes and the vessel wall provide alternative pathological routes of thrombo-inflammatory leukocyte recruitment. We found that when platelets were activated by a range of agonists in whole blood, they shed platelet-derived extracellular vesicles which rapidly and preferentially bound to blood monocytes compared to other leukocytes. Platelet-derived extracellular vesicle binding to monocytes was initiated by P-selectin-dependent adhesion and was stabilised by binding of phosphatidylserine. These interactions resulted in the progressive transfer of the platelet adhesion receptor GPIbα to monocytes. GPIbα+-monocytes tethered and rolled on immobilised von Willebrand Factor or were recruited and activated on endothelial cells treated with TGF-ß1 to induce the expression of von Willebrand Factor. In both models monocyte adhesion was ablated by a function-blocking antibody against GPIbα. Monocytes could also bind platelet-derived extracellular vesicle in mouse blood in vitro and in vivo Intratracheal instillations of diesel nanoparticles, to model chronic pulmonary inflammation, induced accumulation of GPIbα on circulating monocytes. In intravital experiments, GPIbα+-monocytes adhered to the microcirculation of the TGF-ß1-stimulated cremaster muscle, while in the ApoE-/- model of atherosclerosis, GPIbα+-monocytes adhered to the carotid arteries. In trauma patients, monocytes bore platelet markers within 1 hour of injury, the levels of which correlated with severity of trauma and resulted in monocyte clearance from the circulation. Thus, we have defined a novel thrombo-inflammatory pathway in which platelet-derived extracellular vesicles transfer a platelet adhesion receptor to monocytes, allowing their recruitment in large and small blood vessels, and which is likely to be pathogenic.
Assuntos
Plaquetas , Vesículas Extracelulares , Animais , Células Endoteliais , Humanos , Inflamação , Camundongos , Monócitos , Complexo Glicoproteico GPIb-IX de PlaquetasRESUMO
Deep vein thrombosis (DVT) with its major complication, pulmonary embolism, is a global health problem. Mechanisms of DVT remain incompletely understood. Platelets play a role in DVT, but the impact of specific platelet receptors remains unclear. Platelet C-type lectin-like receptor 2 (CLEC-2) is known to maintain the physiological state of blood vasculature under inflammatory conditions. DVT is a thromboinflammatory disorder developing largely as sterile inflammation in the vessel wall. We hypothesized therefore that CLEC-2 might play a role in DVT. Here, using a murine DVT model of inferior vena cava (IVC) stenosis, we demonstrate that mice with general inducible deletion of CLEC-2 or platelet-specific deficiency in CLEC-2 are protected against DVT. No phenotype in the complete stasis model was observed. Transfusion of wild-type platelets into platelet-specific CLEC-2 knockout mice restored thrombosis. Deficiency in CLEC-2 as well as inhibition of podoplanin, a ligand of CLEC-2, was associated with reduced platelet accumulation at the IVC wall after 6 hours of stenosis. Podoplanin was expressed in the IVC wall, where it was localized in the vicinity of the abluminal side of the endothelium. The level of podoplanin in the IVC increased after 48 hours of stenosis to a substantially higher extent in mice with a thrombus vs those without a thrombus. Treatment of animals with an anti-podoplanin neutralizing antibody resulted in development of smaller thrombi. Thus, we propose a novel mechanism of DVT, whereby CLEC-2 and upregulation of podoplanin expression in the venous wall trigger thrombus formation.
Assuntos
Plaquetas/metabolismo , Lectinas Tipo C/metabolismo , Trombose Venosa/metabolismo , Animais , Plaquetas/patologia , Constrição Patológica , Modelos Animais de Doenças , Regulação da Expressão Gênica , Lectinas Tipo C/genética , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Veia Cava Inferior/metabolismo , Veia Cava Inferior/patologia , Trombose Venosa/genética , Trombose Venosa/patologia , Trombose Venosa/prevenção & controleRESUMO
Ca2+ entry via Orai1 store-operated Ca2+ channels in the plasma membrane is critical to cell function, and Orai1 loss causes severe immunodeficiency and developmental defects. The tetraspanins are a superfamily of transmembrane proteins that interact with specific 'partner proteins' and regulate their trafficking and clustering. The aim of this study was to functionally characterize tetraspanin Tspan18. We show that Tspan18 is expressed by endothelial cells at several-fold higher levels than most other cell types analyzed. Tspan18-knockdown primary human umbilical vein endothelial cells have 55-70% decreased Ca2+ mobilization upon stimulation with the inflammatory mediators thrombin or histamine, similar to Orai1-knockdown. Tspan18 interacts with Orai1, and Orai1 cell surface localization is reduced by 70% in Tspan18-knockdown endothelial cells. Tspan18 overexpression in lymphocyte model cell lines induces 20-fold activation of Ca2+ -responsive nuclear factor of activated T cell (NFAT) signaling, in an Orai1-dependent manner. Tspan18-knockout mice are viable. They lose on average 6-fold more blood in a tail-bleed assay. This is due to Tspan18 deficiency in non-hematopoietic cells, as assessed using chimeric mice. Tspan18-knockout mice have 60% reduced thrombus size in a deep vein thrombosis model, and 50% reduced platelet deposition in the microcirculation following myocardial ischemia-reperfusion injury. Histamine- or thrombin-induced von Willebrand factor release from endothelial cells is reduced by 90% following Tspan18-knockdown, and histamine-induced increase of plasma von Willebrand factor is reduced by 45% in Tspan18-knockout mice. These findings identify Tspan18 as a novel regulator of endothelial cell Orai1/Ca2+ signaling and von Willebrand factor release in response to inflammatory stimuli.
Assuntos
Cálcio/metabolismo , Traumatismo por Reperfusão Miocárdica/genética , Proteína ORAI1/genética , Tetraspaninas/genética , Trombose Venosa/genética , Fator de von Willebrand/genética , Animais , Linfócitos B/citologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Galinhas , Modelos Animais de Doenças , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Histamina/farmacologia , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Transporte de Íons/efeitos dos fármacos , Células Jurkat , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/metabolismo , Proteína ORAI1/metabolismo , Transdução de Sinais , Tetraspaninas/metabolismo , Trombina/farmacologia , Trombose Venosa/metabolismo , Trombose Venosa/patologia , Fator de von Willebrand/metabolismoRESUMO
RATIONALE: Deep vein thrombosis (DVT) and its complication pulmonary embolism have high morbidity reducing quality of life and leading to death. Cellular mechanisms of DVT initiation remain poorly understood. OBJECTIVE: We sought to determine the role of mast cells (MCs) in DVT initiation and validate MCs as a potential target for DVT prevention. METHODS AND RESULTS: In a mouse model, DVT was induced by partial ligation (stenosis) of the inferior vena cava. We demonstrated that 2 strains of mice deficient for MCs were completely protected from DVT. Adoptive transfer of in vitro differentiated MCs restored thrombosis. MCs were present in the venous wall, and the number of granule-containing MCs decreased with thrombosis. Pharmacological depletion of MCs granules or prevention of MC degranulation also reduced DVT. Basal plasma levels of von Willebrand factor and recruitment of platelets to the inferior vena cava wall after DVT induction were reduced in MC-deficient mice. Stenosis application increased plasma levels of soluble P-selectin in wild-type but not in MC-deficient mice. MC releasate elevated ICAM-1 (intercellular adhesion molecule-1) expression on HUVEC (human umbilical vein endothelial cells) in vitro. Topical application of compound 48/80, an MC secretagogue, or histamine, a Weibel-Palade body secretagogue from MCs, potentiated DVT in wild-type mice, and histamine restored thrombosis in MC-deficient animals. CONCLUSIONS: MCs exacerbate DVT likely through endothelial activation and Weibel-Palade body release, which is, at least in part, mediated by histamine. Because MCs do not directly contribute to normal hemostasis, they can be considered potential targets for prevention of DVT in humans.
Assuntos
Coagulação Sanguínea , Degranulação Celular , Histamina/metabolismo , Mastócitos/metabolismo , Veia Cava Inferior/metabolismo , Trombose Venosa/metabolismo , Transferência Adotiva , Animais , Coagulação Sanguínea/efeitos dos fármacos , Plaquetas/metabolismo , Degranulação Celular/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Fibrinolíticos/farmacologia , Predisposição Genética para Doença , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Ligadura , Mastócitos/efeitos dos fármacos , Mastócitos/transplante , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Proteínas Proto-Oncogênicas c-kit/deficiência , Proteínas Proto-Oncogênicas c-kit/genética , Selenoproteína P/metabolismo , Transdução de Sinais , Veia Cava Inferior/efeitos dos fármacos , Veia Cava Inferior/cirurgia , Trombose Venosa/sangue , Trombose Venosa/genética , Trombose Venosa/prevenção & controle , Corpos de Weibel-Palade/metabolismo , Fator de von Willebrand/metabolismoRESUMO
Deep vein thrombosis (DVT) and its devastating complication, pulmonary embolism, are a severe health problem with high mortality. Mechanisms of thrombus formation in veins remain obscure. Lack of mobility (e.g., after surgery or long-haul flights) is one of the main factors leading to DVT. The pathophysiological consequence of the lack of mobility is blood flow stagnation in venous valves. Here, a model is described that mimics such flow disturbance as a thrombosis-driving factor. In this model, partial flow restriction (stenosis) in the inferior vena cava (IVC) is created. Closure of about 90% of the IVC lumen for 48 h results in development of thrombi structurally similar to those in humans. The similarities are: i) most of the thrombus volume is red, i.e., consists of red blood cells and fibrin, ii) presence of a white part (lines of Zahn), iii) non-denuded endothelial monolayer, iv) elevated plasma D-Dimer levels, and v) possibility to prevent thrombosis by low molecular weight heparin. Limitations include variable size of thrombi and the fact that a certain percentage of wild-type mice (0 - 35%) may not produce a thrombus. In addition to visual observation and measurement, thrombi may be visualized by non-invasive technologies, such as ultrasonography, which allows for monitoring the dynamics of thrombus development. At shorter time points (1 - 6 h), intravital microscopy may be applied to directly observe events (e.g., recruitment of cells to the vessel wall) preceding thrombus formation. Use of this method by several teams around the world has made it possible to uncover basic mechanisms of DVT initiation and identify potential targets that might be beneficial for its prevention.
Assuntos
Constrição Patológica/cirurgia , Veia Cava Inferior/cirurgia , Trombose Venosa/cirurgia , Animais , Constrição Patológica/patologia , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Veia Cava Inferior/patologia , Trombose Venosa/patologiaRESUMO
This corrects the article DOI: 10.1038/ncomms11208.
RESUMO
Fifty years ago, the first successful kidney transplantation was performed. Since then significant advances in transplantation have been made. Unfortunately, the number of people needing transplants far surpasses the number willing to donate. The literature addresses a plethora of variables influencing whether individuals will become donors themselves or whether family members will donate a loved one's organs. However, to date, no model has examined similarities and differences in the processes by which individuals and families go about making decisions to donate their own or another's organs. In this article, individual and family donation decisions are discussed using Janis' 5-stage model of decision making. We suggest that the weighty nature of donation decisions levels the playing field for decisions made for self and for others. Although the specifics (eg, micro-level) may vary slightly, the processes by which people go about deciding to donate their own organs or those of another individual are similar (eg, macro-level).
Assuntos
Atitude Frente a Saúde , Tomada de Decisões , Família/psicologia , Modelos Psicológicos , Doadores de Tecidos/psicologia , Altruísmo , Emoções , Retroalimentação Psicológica , Conhecimentos, Atitudes e Prática em Saúde , Humanos , Consentimento Livre e Esclarecido/psicologia , Motivação , Negativismo , Resolução de Problemas , Doadores de Tecidos/educaçãoRESUMO
The production of megakaryocytes (MKs)--the precursors of blood platelets--from human pluripotent stem cells (hPSCs) offers exciting clinical opportunities for transfusion medicine. Here we describe an original approach for the large-scale generation of MKs in chemically defined conditions using a forward programming strategy relying on the concurrent exogenous expression of three transcription factors: GATA1, FLI1 and TAL1. The forward programmed MKs proliferate and differentiate in culture for several months with MK purity over 90% reaching up to 2 × 10(5) mature MKs per input hPSC. Functional platelets are generated throughout the culture allowing the prospective collection of several transfusion units from as few as 1 million starting hPSCs. The high cell purity and yield achieved by MK forward programming, combined with efficient cryopreservation and good manufacturing practice (GMP)-compatible culture, make this approach eminently suitable to both in vitro production of platelets for transfusion and basic research in MK and platelet biology.