RESUMEN
Tissue factor (TF) is the primary initiator of blood coagulation in vivo and the only blood coagulation factor for which a human genetic defect has not been described. As there are no routine clinical assays that capture the contribution of endogenous TF to coagulation initiation, the extent to which reduced TF activity contributes to unexplained bleeding is unknown. Using whole genome sequencing, we identified a heterozygous frameshift variant (p.Ser117HisfsTer10) in F3, the gene encoding TF, causing premature termination of TF (TFshort) in a woman with unexplained bleeding. Routine hematological laboratory evaluation of the proposita was normal. CRISPR-edited human induced pluripotent stem cells recapitulating the variant were differentiated into vascular smooth muscle and endothelial cells that demonstrated haploinsufficiency of TF. The variant F3 transcript is eliminated by nonsense-mediated decay. Neither overexpression nor addition of exogenous recombinant TFshort inhibited factor Xa or thrombin generation, excluding a dominant-negative mechanism. F3+/- mice provide an animal model of TF haploinsufficiency and exhibited prolonged bleeding times, impaired thrombus formation, and reduced survival following major injury. Heterozygous TF deficiency is present in at least 1 in 25,000 individuals and could limit coagulation initiation in undiagnosed individuals with abnormal bleeding but a normal routine laboratory evaluation.
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Trastornos de la Coagulación Sanguínea Heredados/sangre , Trastornos de la Coagulación Sanguínea Heredados/genética , Mutación del Sistema de Lectura , Tromboplastina/deficiencia , Tromboplastina/genética , Animales , Secuencia de Bases , Codón sin Sentido , Modelos Animales de Enfermedad , Femenino , Edición Génica , Haploinsuficiencia , Heterocigoto , Humanos , Células Madre Pluripotentes Inducidas , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Terminación de la Cadena Péptídica Traduccional , FenotipoRESUMEN
Platelets are thought to play an important role in metastasis formation, although the mechanisms involved remain incompletely understood. Here we studied the influence of platelet numbers on organ-specific metastasis to the lungs and lymph nodes using Tpo deficient mice that have low platelet counts. After tail vein injection of 4T1 breast cancer cells, the number of lung metastases was significantly lower in Tpo-/- mice compared to Tpo+/+ mice. The same was true for the bone-tropic 4T1.2 derivative. In spontaneous orthotopic metastasis assays, 4T1 and 4T1.2 primary tumor growth was not affected by the genotype of the mice. However, the number of 4T1.2 lung metastases was significantly lower in Tpo-/- mice compared to Tpo+/+ mice, whereas the number of 4T1 lung metastases was unaffected. Moreover, in mice bearing 4T1 tumors, lymph node metastases were larger in the Tpo-/- background, and lymph node metastasis frequency was higher in Tpo-/- mice bearing 4T1.2 tumors compared to that in wild-type mice. Enhanced lymph node metastasis in Tpo-/- mice was not associated with changes in peritumoral lymphatic vessel density in the primary tumors. Together, our data indicate that platelets do not affect primary tumor growth in this breast cancer model, but can differentially influence site-specific metastasis to lymph nodes and lungs.
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Plaquetas/metabolismo , Neoplasias Mamarias Experimentales/sangre , Neoplasias Mamarias Experimentales/patología , Tromboplastina/deficiencia , Animales , Plaquetas/patología , Línea Celular Tumoral , Femenino , Neoplasias Pulmonares/sangre , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundario , Metástasis Linfática , Neoplasias Mamarias Experimentales/genética , Ratones , Ratones Endogámicos BALB C , Metástasis de la Neoplasia , Tromboplastina/genéticaRESUMEN
Intravascular transplantation of tissue factor (TF)-bearing cells elicits an instant blood-mediated inflammatory reaction (IBMIR) resulting in thrombotic complications and reduced engraftment. Here we studied the hemocompatibility of commonly used human white adipose tissue (WAT), umbilical cord (UC) and bone marrow stromal cells (BMSC) and devised a possible strategy for safe and efficient stromal cell transplantation. Methods: Stromal cell identity, purity, and TF expression was tested by RTQ-PCR, flow cytometry and immunohistochemistry. Pro-coagulant activity and fibrin clot formation/stabilization was measured In Vitro by viscoelastic rotational plasma-thromboelastometry and in vivo by injecting sorted human stromal cells intravenously into rats. The impact of TF was verified in factor VII-deficient plasma and by sort-depleting TF/CD142+ BMSC. Results: We found significantly less TF expression by a subpopulation of BMSC corresponding to reduced pro-coagulant activity. UC and WAT stroma showed broad TF expression and durable clotting. Higher cell numbers significantly increased clot formation partially dependent on coagulation factor VII. Depleting the TF/CD142+ subpopulation significantly ameliorated BMSC's hemocompatibility without affecting immunomodulation. TF-deficient BMSC did not produce thromboembolism in vivo, comparing favorably to massive intravascular thrombosis induction by TF-expressing stromal cells. Conclusion: We demonstrate that plasma-based thromboelastometry provides a reliable tool to detect pro-coagulant activity of therapeutic cells. Selecting TF-deficient BMSC is a novel strategy for improving cell therapy applicability by reducing cell dose-dependent IBMIR risk. The particularly strong pro-coagulant activity of UC and WAT preparations sounds an additional note of caution regarding uncritical systemic application of stromal cells, particularly from non-hematopoietic extravascular sources.
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Ensayo de Materiales , Células Madre Mesenquimatosas/metabolismo , Tromboplastina/deficiencia , Adulto , Animales , Coagulación Sanguínea , Recuento de Células , Tamaño de la Célula , Trasplante de Células , Células Cultivadas , Femenino , Humanos , Inmunomodulación , Masculino , Persona de Mediana Edad , Ratas , Factores de Riesgo , Tromboembolia/etiología , Tromboembolia/patología , Tromboplastina/metabolismo , Adulto JovenRESUMEN
Essentials Tissue factor (TF) represents a central link between hemostasis and inflammation. We studied the roles of myeloid and airway epithelial TF in acid-caused acute lung injury (ALI). TF on myeloid cells displays a non-coagulatory role regulating the inflammatory response in ALI. Airway epithelial TF contributes to hemostatic functions, but is dispensable in ALI pathogenesis. SUMMARY: Introduction Acute lung injury (ALI) is a life-threatening condition characterized by damaged alveolar-capillary structures and activation of inflammatory and hemostatic processes. Tissue factor (TF) represents a crucial link between inflammation and coagulation, as inflammatory mediators induce myeloid TF expression, and TF initiates extrinsic coagulation. Objective As pulmonary inflammation stimulates TF expression and TF modulates immune responses, we aimed to elucidate its impact on ALI. In particular, we wanted to distinguish the contributions of TF expressed on airway epithelial cells and TF expressed on myeloid cells. Methods Mice with different cell type-specific TF deficiency and wild-type littermates were intratracheally treated with hydrochloric acid, and leukocyte recruitment, cytokine levels, thrombin-antithrombin (TAT) complexes and pulmonary protein-rich infiltrates were analyzed. Results Our data demonstrate that a lack of epithelial TF did not influence acute responses, as bronchoalveolar neutrophil accumulation 8 h after ALI induction was unaltered. However, it led to mild, prolonged inflammation, as pulmonary leukocyte and erythrocyte numbers were still increased after 24 h, whereas those in wild-type mice had returned to basal levels. In contrast, myeloid TF was primarily involved in regulating the acute phase of ALI without affecting local coagulation, as indicated by increased bronchoalveolar neutrophil infiltration, pulmonary interleukin-6 levels, and edema formation, but equal TAT complex formation, 8 h after ALI induction. This augmented inflammatory response associated with myeloid TF deficiency was confirmed in vitro, as lipopolysaccharide-stimulated TF-deficient alveolar macrophages released increased levels of chemokine (C-X-C motif) ligand 1 and tumor necrosis factor-α as compared with wild-type macrophages. Conclusion We conclude that myeloid TF dampens inflammation in acid-induced ALI.
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Lesión Pulmonar Aguda/prevención & control , Células Epiteliales/metabolismo , Ácido Clorhídrico , Pulmón/metabolismo , Macrófagos Alveolares/metabolismo , Neumonía/prevención & control , Tromboplastina/metabolismo , Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/inmunología , Lesión Pulmonar Aguda/metabolismo , Animales , Antitrombina III/metabolismo , Coagulación Sanguínea , Células Cultivadas , Quimiotaxis de Leucocito , Citocinas/metabolismo , Modelos Animales de Enfermedad , Células Epiteliales/inmunología , Predisposición Genética a la Enfermedad , Mediadores de Inflamación/metabolismo , Pulmón/inmunología , Macrófagos Alveolares/inmunología , Ratones Endogámicos C57BL , Ratones Noqueados , Infiltración Neutrófila , Péptido Hidrolasas/metabolismo , Fenotipo , Neumonía/inducido químicamente , Neumonía/inmunología , Neumonía/metabolismo , Edema Pulmonar/inducido químicamente , Edema Pulmonar/metabolismo , Edema Pulmonar/prevención & control , Tromboplastina/deficiencia , Tromboplastina/genética , Factores de TiempoRESUMEN
UNLABELLED: Essentials H1N1 Influenza A virus (IAV) infection is a hemostatic challenge for the lung. Tissue factor (TF) on lung epithelial cells maintains lung hemostasis after IAV infection. Reduced TF-dependent activation of coagulation leads to alveolar hemorrhage. Anticoagulation might increase the risk for hemorrhages into the lung during severe IAV infection. SUMMARY: Background Influenza A virus (IAV) infection is a common respiratory tract infection that causes considerable morbidity and mortality worldwide. Objective To investigate the effect of genetic deficiency of tissue factor (TF) in a mouse model of IAV infection. Methods Wild-type mice, low-TF (LTF) mice and mice with the TF gene deleted in different cell types were infected with a mouse-adapted A/Puerto Rico/8/34 H1N1 strain of IAV. TF expression was measured in the lungs, and bronchoalveolar lavage fluid (BALF) was collected to measure extracellular vesicle TF, activation of coagulation, alveolar hemorrhage, and inflammation. Results IAV infection of wild-type mice increased lung TF expression, activation of coagulation and inflammation in BALF, but also led to alveolar hemorrhage. LTF mice and mice with selective deficiency of TF in lung epithelial cells had low basal levels of TF and failed to increase TF expression after infection; these two strains of mice had more alveolar hemorrhage and death than controls. In contrast, deletion of TF in either myeloid cells or endothelial cells and hematopoietic cells did not increase alveolar hemorrhage or death after IAV infection. These results indicate that TF expression in the lung, particularly in epithelial cells, is required to maintain alveolar hemostasis after IAV infection. Conclusion Our study indicates that TF-dependent activation of coagulation is required to limit alveolar hemorrhage and death after IAV infection.
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Células Epiteliales/virología , Hemorragia/virología , Infecciones por Orthomyxoviridae/patología , Alveolos Pulmonares/metabolismo , Tromboplastina/deficiencia , Animales , Anticoagulantes/uso terapéutico , Coagulación Sanguínea , Líquido del Lavado Bronquioalveolar , Eliminación de Gen , Hemostasis , Inflamación , Subtipo H1N1 del Virus de la Influenza A , Integrasas/metabolismo , Pulmón/patología , Pulmón/virología , Masculino , Ratones , Ratones Endogámicos C57BL , Tromboplastina/metabolismoRESUMEN
Introducción: La calcifilaxis se caracteriza por calcificación de arteriolas, fibrosis y trombosis que conduce a la aparición de úlceras cutáneas isquémicas y necrosis, y afecta con mayor frecuencia a pacientes con enfermedad renal crónica terminal. El tratamiento de la calcifilaxis es variado, sin existir una modalidad terapéutica eficaz, y presenta mal pronóstico, principalmente por la sepsis secundaria a infección de las lesiones cutáneas. Objetivo: Conocer la producción científica sobre la calcifilaxis relacionada con la enfermedad renal crónica. Metodología: Se ha llevado a cabo una revisión bibliográfica con una búsqueda de bibliografía en las principales bases de datos PubMed/Medline, Scielo, Cuiden, ProQuest/Health and Medical Complete, IME y Google Académico. Resultados: Se seleccionaron 61 artículos para la realización del estudio: 19 artículos de revisión, 13 originales y 29 casos clínicos. Las variables estudiadas han sido las alteraciones en el metabolismo del fósforo, calcio y hormona paratiroidea, varios factores de riesgo, distribución de las lesiones, fallecimiento por sepsis, tratamiento médico y cuidados de enfermería. Los factores más influyentes en la aparición de calcifilaxis son las alteraciones en el metabolismo, obesidad, diabetes, hipertensión, baja albúmina sérica y toma de anticoagulantes. El tratamiento implica múltiples medidas terapéuticas, dirigidas al control de las alteraciones del metabolismo y la cura de las manifestaciones cutáneas. Conclusiones: La calcifilaxis es una entidad poco frecuente y con una elevada mortalidad. La patogénesis es desconocida y desde el punto de vista terapéutico, no existe un tratamiento específico, por lo que es fundamental un enfoque multidisciplinar, para su prevención y detección precoz (AU)
Introduction: Calciphylaxis is characterized by calcification of arterioles, fibrosis and thrombosis which leads to the onset of ischemic skin ulcers and necrosis, and most often affects in patients with end stage renal disease. Treatment of calciphylaxis is varied, without effective therapeutic modality and poor prognosis, mainly due to sepsis secondary to infection of the skin lesions. Objective: To know the scientific production on calciphylaxis related to chronic kidney disease. Methodology: A literature review was conducted with a literature search in the following databases: PubMed / Medline, Scielo, Cuiden, ProQuest / Health and Medical Complete, IME and Google Scholar. Results: Sixty-one articles were selected for the study: 19 review articles, 13 originals and 29 clinical cases. The variables studied were: alterations in the metabolism of phosphorus, calcium and parathyroid hormone, several risk factors, distribution of injuries, death due to sepsis, medical treatment and nursing care. The most influential factors in the development of calciphylaxis are alterations in metabolism, obesity, diabetes, hypertension, low serum albumin and taking anticoagulants. Treatment involves multiple therapeutic measures aimed at controlling the metabolic disorders and cure of cutaneous manifestations. Conclusion: Calciphylaxis is an uncommon event with high mortality. The pathogenesis is unknown and from a therapeutic point of view, there is no specific treatment, so that a multidisciplinary approach is essential for its prevention and early detection (AU)
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Humanos , Masculino , Femenino , Insuficiencia Renal Crónica/metabolismo , Insuficiencia Renal Crónica/patología , Tromboplastina/metabolismo , Tromboplastina/fisiología , Cirrosis Hepática/diagnóstico , Cirrosis Hepática/patología , Hormona Paratiroidea/administración & dosificación , Hormona Paratiroidea/metabolismo , Diabetes Mellitus/sangre , Diabetes Mellitus/genética , Insuficiencia Renal Crónica/complicaciones , Insuficiencia Renal Crónica/diagnóstico , Tromboplastina/administración & dosificación , Tromboplastina/deficiencia , Cirrosis Hepática/complicaciones , Cirrosis Hepática/metabolismo , Hormona Paratiroidea/fisiología , Hormona Paratiroidea/farmacología , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patologíaRESUMEN
Tissue factor (TF) initiates the extrinsic coagulation cascade in response to tissue injury, leading to local fibrin deposition. Low levels of TF in mice are associated with increased severity of acute lung injury (ALI) after intratracheal LPS administration. However, the cellular sources of the TF required for protection from LPS-induced ALI remain unknown. In the current study, transgenic mice with cell-specific deletions of TF in the lung epithelium or myeloid cells were treated with intratracheal LPS to determine the cellular sources of TF important in direct ALI. Cell-specific deletion of TF in the lung epithelium reduced total lung TF expression to 39% of wild-type (WT) levels at baseline and to 29% of WT levels after intratracheal LPS. In contrast, there was no reduction of TF with myeloid cell TF deletion. Mice lacking myeloid cell TF did not differ from WT mice in coagulation, inflammation, permeability, or hemorrhage. However, mice lacking lung epithelial TF had increased tissue injury, impaired activation of coagulation in the airspace, disrupted alveolar permeability, and increased alveolar hemorrhage after intratracheal LPS. Deletion of epithelial TF did not affect alveolar permeability in an indirect model of ALI caused by systemic LPS infusion. These studies demonstrate that the lung epithelium is the primary source of TF in the lung, contributing 60-70% of total lung TF, and that lung epithelial, but not myeloid, TF may be protective in direct ALI.
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Lesión Pulmonar Aguda/genética , Coagulación Sanguínea/genética , Permeabilidad Capilar/genética , Hemorragia/genética , Síndrome de Dificultad Respiratoria/genética , Tromboplastina/genética , Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/metabolismo , Lesión Pulmonar Aguda/patología , Animales , Modelos Animales de Enfermedad , Células Epiteliales/metabolismo , Células Epiteliales/patología , Expresión Génica , Hemorragia/inducido químicamente , Hemorragia/metabolismo , Hemorragia/patología , Lipopolisacáridos , Ratones , Ratones Noqueados , Células Mieloides/metabolismo , Células Mieloides/patología , Alveolos Pulmonares/efectos de los fármacos , Alveolos Pulmonares/metabolismo , Alveolos Pulmonares/patología , Síndrome de Dificultad Respiratoria/inducido químicamente , Síndrome de Dificultad Respiratoria/metabolismo , Síndrome de Dificultad Respiratoria/patología , Mucosa Respiratoria/efectos de los fármacos , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/patología , Tromboplastina/deficienciaRESUMEN
INTRODUCTION: Hemostasis is a rapid response by the body to stop bleeding at sites of vessel injury. Both platelets and fibrin are important for the formation of a hemostatic plug. Mice have been used to uncover the molecular mechanisms that regulate the activation of platelets and coagulation under physiologic conditions. However, measurements of hemostasis in mice are quite variable, and current methods do not quantify platelet adhesion or fibrin formation at the site of injury. METHODS: We describe a novel hemostasis model that uses intravital fluorescence microscopy to quantify platelet adhesion, fibrin formation and time to hemostatic plug formation in real time. Repeated vessel injuries of ~ 50-100 µm in diameter were induced with laser ablation technology in the saphenous vein of mice. RESULTS: Hemostasis in this model was strongly impaired in mice deficient in glycoprotein Ibα or talin-1, which are important regulators of platelet adhesiveness. In contrast, the time to hemostatic plug formation was only minimally affected in mice deficient in the extrinsic tissue factor (TF(low)) or the intrinsic factor IX coagulation pathways, even though platelet adhesion was significantly reduced. A partial reduction in platelet adhesiveness obtained with clopidogrel led to instability within the hemostatic plug, especially when combined with impaired coagulation in TF(low) mice. CONCLUSIONS: In summary, we present a novel, highly sensitive method to quantify hemostatic plug formation in mice. On the basis of its sensitivity to platelet adhesion defects and its real-time imaging capability, we propose this model as an ideal tool with which to study the efficacy and safety of antiplatelet agents.
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Tiempo de Sangría , Plaquetas/metabolismo , Hemostasis , Vena Safena/metabolismo , Lesiones del Sistema Vascular/sangre , Animales , Coagulación Sanguínea , Plaquetas/efectos de los fármacos , Clopidogrel , Modelos Animales de Enfermedad , Factor IX/genética , Factor IX/metabolismo , Fibrina/metabolismo , Hemostasis/genética , Microscopía Intravital , Terapia por Láser , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Fluorescente , Microscopía por Video , Adhesividad Plaquetaria , Inhibidores de Agregación Plaquetaria/farmacología , Complejo GPIb-IX de Glicoproteína Plaquetaria/genética , Complejo GPIb-IX de Glicoproteína Plaquetaria/metabolismo , Vena Safena/cirugía , Talina/deficiencia , Talina/genética , Tromboplastina/deficiencia , Tromboplastina/genética , Ticlopidina/análogos & derivados , Ticlopidina/farmacología , Factores de Tiempo , Lesiones del Sistema Vascular/etiología , Lesiones del Sistema Vascular/genéticaRESUMEN
Hepatocyte (HPC) apoptosis occurs in association with hepatotoxic responses and chronic liver disease, and is coupled to activation of the blood coagulation cascade. HPCs have been shown to express tissue factor (TF), the primary activator of blood coagulation, in a form that lacks procoagulant activity. In this study, we determined the effect of inducing HPC apoptosis on the procoagulant activity of TF. Treatment of primary mouse HPCs with the Fas death receptor agonist (anti-CD95 antibody, Jo2) triggered apoptosis as shown by cleavage of caspase-3, increased caspase-3 proteolytic activity, and cell surface exposure of phosphatidylserine (PS). Jo2-induced apoptosis significantly increased TF-dependent factor Xa generation by HPCs. Moreover, Jo2 treatment was associated with increased levels of microparticle-associated TF procoagulant activity in the culture medium. Pretreatment with a caspase-3 inhibitor significantly reduced Jo2-induced HPC TF activity and prevented the increase in microparticle-associated TF procoagulant activity. Application of the high-affinity PS-binding protein lactadherin inhibited TF-dependent factor Xa generation by Jo2-treated HPCs and dramatically reduced microparticle-associated TF procoagulant activity. Treatment of wild-type mice with a sublethal dose of Jo2 was associated with a robust increase in the activation of coagulation as measured by plasma thrombin-antithrombin (TAT) levels; whereas mice with liver-specific TF deficiency had significantly lower TAT levels. Overall, the results indicate that Fas-initiated, caspase-3-dependent HPC apoptosis increases TF procoagulant activity through a mechanism involving PS externalization. This suggests that activation of liver TF likely contributes to the procoagulant state associated with HPC apoptosis in liver toxicity and disease.
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Apoptosis , Coagulación Sanguínea , Hepatocitos/metabolismo , Tromboplastina/metabolismo , Receptor fas/metabolismo , Animales , Anticuerpos/farmacología , Antitrombina III/metabolismo , Apoptosis/efectos de los fármacos , Coagulación Sanguínea/efectos de los fármacos , Caspasa 3/metabolismo , Inhibidores de Caspasas/farmacología , Micropartículas Derivadas de Células/metabolismo , Células Cultivadas , Hepatocitos/efectos de los fármacos , Hepatocitos/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Péptido Hidrolasas/metabolismo , Fosfatidilserinas/metabolismo , Transducción de Señal , Tromboplastina/deficiencia , Tromboplastina/genética , Factores de Tiempo , Receptor fas/antagonistas & inhibidores , Receptor fas/inmunologíaRESUMEN
BACKGROUND: Thrombosis is a marker of poor prognosis in individuals with solid tumors. The expression of tissue factor (TF) on the cell surface membrane of malignant cells is a pivotal molecular link between activation of coagulation, angiogenesis, metastasis, aggressive tumor behavior and poor survival. Interestingly, thrombosis is associated with shortened survival in solid, but not in lymphoid neoplasias. OBJECTIVES: We sought to study whether the lack of impact of thrombosis on survival in lymphoid neoplasias could be due to a lack of tumor-derived TF expression. METHODS: We analyzed TF gene (F3) expression in lymphoid (N=114), myeloid (N=49) and solid tumor (N=856) cell lines using the publicly available dataset from the Broad-Novartis Cancer Cell Line Encyclopedia (http://www.broadinstitute.org/ccle/home), and in 90 patient-derived lymphoma samples. TF protein expression was studied by immunohistochemistry (IHC). RESULTS: In sharp contrast to wide F3 expression in solid tumors (74.2%), F3 was absent in all low and high grade T- and B-cell lymphomas, and in most myeloid tumors, except for select acute myeloid leukemias with monocytic component. IHC confirmed the absence of TF protein in all indolent and high-grade B-cell (0/90) and T-cell (0/20) lymphomas, and acute leukemias (0/11). CONCLUSIONS: We show that TF in lymphomas does not derive from the malignant cells, since these do not express either F3 or TF protein. Therefore, it is unlikely that thrombosis in patients with lymphoid neoplasms is secondary to tumor-derived tissue factor.
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Linfocitos B/patología , Neoplasias Hematológicas/metabolismo , Neoplasias Hematológicas/patología , Tejido Linfoide/patología , Linfocitos T/patología , Tromboplastina/deficiencia , Trombosis/metabolismo , Perfilación de la Expresión Génica , Neoplasias Hematológicas/sangre , Neoplasias Hematológicas/genética , Humanos , Pronóstico , Tromboplastina/biosíntesis , Tromboplastina/genética , Trombosis/sangre , Trombosis/genética , Trombosis/patologíaRESUMEN
BACKGROUND: Systemic blockade of tissue factor (TF) attenuates acute lung injury (ALI) in animal models of sepsis but the effects of global TF deficiency are unknown. We used mice with complete knockout of mouse TF and low levels (â¼1%) of human TF (LTF mice) to test the hypothesis that global TF deficiency attenuates lung inflammation in direct lung injury. METHODS: LTF mice were treated with 10 µg of lipopolysaccharide (LPS) or vehicle administered by direct intratracheal injection and studied at 24 h. RESULTS: Contrary to our hypothesis, LTF mice had increased lung inflammation and injury as measured by bronchoalveolar lavage cell count (3.4×10(5) wild-type (WT) LPS vs 3.3×10(5) LTF LPS, p=0.947) and protein (493 µg/ml WT LPS vs 1014 µg/ml LTF LPS, p=0.006), proinflammatory cytokines (TNF-α, IL-10, IL-12, p<0.035 WT LPS vs LTF LPS) and histology compared with WT mice. LTF mice also had increased haemorrhage and free haemoglobin in the airspace accompanied by increased oxidant stress as measured by lipid peroxidation products (F(2) isoprostanes and isofurans). CONCLUSIONS: These findings indicate that global TF deficiency does not confer protection in a direct lung injury model. Rather, TF deficiency causes increased intra-alveolar haemorrhage following LPS leading to increased lipid peroxidation. Strategies to globally inhibit TF may be deleterious in patients with ALI.
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Lesión Pulmonar Aguda/metabolismo , Hemorragia/metabolismo , Estrés Oxidativo , Alveolos Pulmonares , Tromboplastina/deficiencia , Análisis de Varianza , Animales , Western Blotting , Lavado Broncoalveolar , Citocinas/metabolismo , Electroforesis en Gel de Poliacrilamida , Furanos/metabolismo , Hemoglobinas/metabolismo , Inflamación/metabolismo , Isoprostanos/metabolismo , Lipopolisacáridos/administración & dosificación , Ratones , Ratones Noqueados , Reacción en Cadena en Tiempo Real de la Polimerasa , Estadísticas no ParamétricasRESUMEN
For a long time, blood coagulation and innate immunity have been viewed as interrelated responses. Recently, the presence of leukocytes at the sites of vessel injury has been described. Here we analyzed interaction of neutrophils, monocytes, and platelets in thrombus formation after a laser-induced injury in vivo. Neutrophils immediately adhered to injured vessels, preceding platelets, by binding to the activated endothelium via leukocyte function antigen-1-ICAM-1 interactions. Monocytes rolled on a thrombus 3 to 5 minutes postinjury. The kinetics of thrombus formation and fibrin generation were drastically reduced in low tissue factor (TF) mice whereas the absence of factor XII had no effect. In vitro, TF was detected in neutrophils. In vivo, the inhibition of neutrophil binding to the vessel wall reduced the presence of TF and diminished the generation of fibrin and platelet accumulation. Injection of wild-type neutrophils into low TF mice partially restored the activation of the blood coagulation cascade and accumulation of platelets. Our results show that the interaction of neutrophils with endothelial cells is a critical step preceding platelet accumulation for initiating arterial thrombosis in injured vessels. Targeting neutrophils interacting with endothelial cells may constitute an efficient strategy to reduce thrombosis.
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Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Neutrófilos/metabolismo , Tromboplastina/genética , Trombosis/metabolismo , Animales , Coagulación Sanguínea , Plaquetas/citología , Vasos Sanguíneos/lesiones , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/patología , Adhesión Celular , Comunicación Celular , Recuento de Células , Movimiento Celular , Células Endoteliales/patología , Endotelio Vascular/patología , Factor XII/metabolismo , Deficiencia del Factor XII/genética , Deficiencia del Factor XII/metabolismo , Fibrina/metabolismo , Molécula 1 de Adhesión Intercelular/metabolismo , Rayos Láser , Ratones , Monocitos/citología , Neutrófilos/citología , Neutrófilos/trasplante , Recuento de Plaquetas , Tromboplastina/deficienciaRESUMEN
Nonalcoholic fatty liver disease (NAFLD) is the hepatic manifestation of obesity and metabolic syndrome and contributes to increased risk of cardiovascular disease and liver-related morbidity and mortality. Indeed, obese patients with metabolic syndrome generate greater amounts of thrombin, an indication of coagulation cascade activation. However, the role of the coagulation cascade in Western diet-induced NAFLD has not been investigated. Using an established mouse model of Western diet-induced NAFLD, we tested whether the thrombin receptor protease-activated receptor 1 (PAR-1) and hematopoietic cell-derived tissue factor (TF) contribute to hepatic steatosis. In association with hepatic steatosis, plasma thrombin-antithrombin levels and hepatic fibrin deposition increased significantly in C57Bl/6J mice fed a Western diet for 3 months. PAR-1 deficiency reduced hepatic inflammation, particularly monocyte chemoattractant protein-1 expression and macrophage accumulation. In addition, PAR-1 deficiency was associated with reduced steatosis in mice fed a Western diet, including reduced liver triglyceride accumulation and CD36 expression. Similar to PAR-1 deficiency, hematopoietic cell TF deficiency was associated with reduced inflammation and reduced steatosis in livers of low-density lipoprotein receptor-deficient mice fed a Western diet. Moreover, hematopoietic cell TF deficiency reduced hepatic fibrin deposition. These studies indicate that PAR-1 and hematopoietic cell TF are required for liver inflammation and steatosis in mice fed a Western diet.
Asunto(s)
Coagulación Sanguínea/fisiología , Dieta/efectos adversos , Hígado Graso/etiología , Receptor PAR-1/deficiencia , Tromboplastina/deficiencia , Animales , Trasplante de Médula Ósea , Hígado Graso/sangre , Células Madre Hematopoyéticas/metabolismo , Hepatitis/fisiopatología , Lipogénesis/genética , Ratones , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico , ARN Mensajero/metabolismo , Transducción de Señal , Factor de Necrosis Tumoral alfa/metabolismo , Aumento de Peso/fisiologíaRESUMEN
Tissue factor (TF) is traditionally known as the initiator of blood coagulation, but TF also plays an important role in inflammatory processes. Considering the pivotal role of coagulation in inflammatory bowel disease, we assessed whether genetic ablation of TF limits experimental colitis. To this end, wild-type and TF-deficient (TFlow) mice were treated with 1.5% dextran sulfate sodium (DSS) for 7 d, and effects on disease severity, cytokine production and leukocyte recruitment were examined. Clinical and histological parameters showed that the severity of colitis was reduced in both heterozygous and homozygous TFlow mice compared with controls. Most notably, edema, granulocyte numbers at the site of inflammation and cytokine levels were reduced in TFlow mice. Although anticoagulant treatment with dalteparin of wild-type mice reduced local fibrin production and cytokine levels to a similar extent as in TFlow mice, it did not affect clinical and histological parameters of experimental colitis. Mechanistic studies revealed that TF expression did not influence the intrinsic capacity of granulocytes to migrate. Instead, TF enhanced granulocyte migration into the colon by inducing high levels of the granulocyte chemoattractant keratinocyte-derived chemokine (KC). Taken together, our data indicate that TF plays a detrimental role in experimental colitis by signal transduction-dependent KC production in colon epithelial cells, thereby provoking granulocyte influx with subsequent inflammation and organ damage.
Asunto(s)
Quimiocinas/metabolismo , Colitis/metabolismo , Colon/metabolismo , Tromboplastina/fisiología , Animales , Movimiento Celular , Colitis/inducido químicamente , Colitis/genética , Colon/patología , Sulfato de Dextran , Edema/metabolismo , Edema/patología , Ensayo de Inmunoadsorción Enzimática , Femenino , Granulocitos/metabolismo , Granulocitos/patología , Humanos , Inmunohistoquímica , Mediadores de Inflamación/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Índice de Severidad de la Enfermedad , Tromboplastina/deficiencia , Tromboplastina/genética , Técnicas de Cultivo de TejidosRESUMEN
Following the molecular cloning of human Tissue Factor (TF) in mid-1980's, great strides have been made in the understanding of TF biology, TF's crucial roles in the initiation of blood coagulation and embryonic development, and TF's contribution to the pathobiology of various disease states. The 21st century brought about a rather unexpected turn in the "TF journey"--a few years back it was reported that the TF gene produces not one, but two proteins with distinct structural and functional characteristics. The so-called "full-length TF" (flTF) - a much-studied integral membrane glycoprotein long presumed to be, and experimentally handled as "the TF" in hundreds of laboratories around the world - is now known to be one of the two TF forms naturally occurring in humans as well as mice. The other, recently discovered form is termed alternatively spliced TF (asTF) which, unlike flTF, lacks a transmembrane domain and can thus be secreted. In this review, we summarize the literature on asTF by discussing asTF's biologic roles as they are currently understood, tackling a number of questions pertaining to asTF's evident and proposed biologic properties, and briefly covering the emerging field of regulated TF pre-mRNA processing.
Asunto(s)
Tromboplastina/genética , Empalme Alternativo , Animales , Enfermedades Cardiovasculares/sangre , Enfermedades Cardiovasculares/genética , Endotelio Vascular/fisiología , Ingeniería Genética , Humanos , Ratones , Ratones Noqueados , Ratones Transgénicos , Monocitos/fisiología , Neoplasias/sangre , Neoplasias/genética , Neovascularización Fisiológica/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Tromboplastina/química , Tromboplastina/deficiencia , Tromboplastina/metabolismoRESUMEN
BACKGROUND/AIMS: Tissue factor (TF) is a transmembrane protein that is essential for coagulation. TF is expressed on podocytes and its cytoplasmic domain has cell signalling functions in epithelial cells. METHODS: Mice lacking the cytoplasmic domain of TF (TF(CT-/-) mice) were used to study its role in physiological albuminuria and pathological proteinuria following induction of glomerulonephritis (GN). RESULTS: Absence of the cytoplasmic domain of TF was associated with increased albuminuria, podocyte effacement, reduced podocyte numbers and increased spontaneous glomerular tumour necrosis factor α(TNFα) production under physiological conditions. In mice developing GN, absence of the cytoplasmic domain of TF resulted in increased proteinuria and enhanced renal TNFα production without altering other parameters of renal inflammation and injury. Studies in TF(CT-/-) chimeric mice (created by bone marrow transplantation) showed increased proteinuria and renal TNFα mRNA in GN was associated with absence of the cytoplasmic domain of TF in the kidney and was independent of the leucocyte phenotype. CONCLUSION: These studies demonstrate that the cytoplasmic domain of TF contributes to renal albumin retention and its renal expression protects against proteinuria in leucocyte-mediated renal inflammation. Increased glomerular production of TNFα in the absence of cytoplasmic domain of TF may contribute to podocyte injury resulting in albuminuria and proteinuria.
Asunto(s)
Albuminuria/fisiopatología , Glomerulonefritis/fisiopatología , Proteinuria/fisiopatología , Tromboplastina/fisiología , Albuminuria/patología , Animales , Citoplasma/metabolismo , Glomerulonefritis/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Glomérulos Renales/metabolismo , Proteínas de la Membrana/biosíntesis , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Podocitos/metabolismo , Podocitos/patología , Estructura Terciaria de Proteína/fisiología , Proteinuria/metabolismo , Tromboplastina/deficiencia , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
Tissue factor (TF) is the primary activator of the coagulation cascade. During endotoxemia, TF expression leads to disseminated intravascular coagulation. However, the relative contribution of TF expression by different cell types to the activation of coagulation has not been defined. In this study, we investigated the effect of either a selective inhibition of TF expression or cell type-specific deletion of the TF gene (F3) on activation of coagulation in a mouse model of endotoxemia. We found that inhibition of TF on either hematopoietic or nonhematopoietic cells reduced plasma thrombin-antithrombin (TAT) levels 8 hours after administration of bacterial lipopolysaccharide (LPS). In addition, plasma TAT levels were significantly reduced in endotoxemic mice lacking the TF gene in either myeloid cells (TF(flox/flox),LysM(Cre) mice) or in both endothelial cells (ECs) and hematopoietic cells (TF(flox/flox),Tie-2(Cre) mice). However, deletion of the TF gene in ECs alone had no effect on LPS-induced plasma TAT levels. Similar results were observed in mice lacking TF in vascular smooth muscle cells. Finally, we found that mouse platelets do not express TF pre-mRNA or mRNA. Our data demonstrate that in a mouse model of endotoxemia activation of the coagulation cascade is initiated by TF expressed by myeloid cells and an unidentified nonhematopoietic cell type(s).
Asunto(s)
Coagulación Sanguínea/fisiología , Endotoxemia/fisiopatología , Células Mieloides/metabolismo , Tromboplastina/fisiología , Animales , Antitrombina III/análisis , Plaquetas/metabolismo , Células Cultivadas , Coagulación Intravascular Diseminada/etiología , Coagulación Intravascular Diseminada/fisiopatología , Células Endoteliales/metabolismo , Endotoxemia/sangre , Eliminación de Gen , Leucocitos/metabolismo , Lipopolisacáridos/toxicidad , Macrófagos Peritoneales/metabolismo , Ratones , Ratones Endogámicos C57BL , Péptido Hidrolasas/análisis , Precursores del ARN/biosíntesis , Precursores del ARN/sangre , ARN Mensajero/biosíntesis , ARN Mensajero/sangre , Quimera por Radiación , Especificidad de la Especie , Tromboplastina/antagonistas & inhibidores , Tromboplastina/deficiencia , Tromboplastina/genéticaRESUMEN
Altered hepatic lipid homeostasis, hepatocellular injury, and inflammation are features of nonalcoholic steatohepatitis, which contributes significantly to liver-related morbidity and mortality in the Western population. A collection of inflammatory mediators have been implicated in the pathogenesis of steatohepatitis in mouse models. However, the pathways essential for coordination and amplification of hepatic inflammation and injury caused by steatosis are not completely understood. We tested the hypothesis that tissue factor (TF)-dependent thrombin generation and the thrombin receptor protease activated receptor-1 (PAR-1) contribute to liver inflammation induced by steatosis in mice. Wild-type C57Bl/6J mice fed a diet deficient in methionine and choline for 2 weeks manifested steatohepatitis characterized by increased serum alanine aminotransferase activity, macrovesicular hepatic steatosis, hepatic inflammatory gene expression, and lobular inflammation. Steatohepatitis progression was associated with thrombin generation and hepatic fibrin deposition. Coagulation cascade activation was significantly reduced in low TF mice, which express 1% of normal TF levels. Hepatic triglyceride accumulation was not affected in low TF mice or PAR-1-deficient mice. In contrast, biomarkers of hepatocellular injury, inflammatory gene induction, and hepatic accumulation of macrophages and neutrophils were greatly reduced by TF-deficiency and PAR-1-deficiency. The results suggest that TF-dependent thrombin generation and activation of PAR-1 amplify hepatic inflammation and injury during the pathogenesis of steatohepatitis.
Asunto(s)
Dieta/efectos adversos , Hígado Graso/complicaciones , Hígado Graso/patología , Inflamación/complicaciones , Inflamación/patología , Receptor PAR-1/deficiencia , Tromboplastina/deficiencia , Alanina Transaminasa/sangre , Animales , Coagulación Sanguínea , Factores de Coagulación Sanguínea/metabolismo , Quimiocina CCL2/sangre , Colina/metabolismo , Hígado Graso/sangre , Hígado Graso/enzimología , Regulación de la Expresión Génica , Inflamación/genética , Hígado/metabolismo , Hígado/patología , Metionina/deficiencia , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Receptor PAR-1/metabolismo , Trombina/biosíntesis , Tromboplastina/metabolismo , Triglicéridos/metabolismoAsunto(s)
Neoplasias Experimentales/irrigación sanguínea , Neovascularización Patológica/metabolismo , Tromboplastina/metabolismo , Animales , Antineoplásicos/uso terapéutico , Supervivencia Celular , Células Madre Embrionarias/metabolismo , Células Endoteliales/metabolismo , Humanos , Metástasis de la Neoplasia , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Células Madre Neoplásicas/metabolismo , Vesículas Secretoras/metabolismo , Tromboplastina/deficienciaRESUMEN
OBJECTIVE: The role of host-derived tissue factor (TF) in tumor growth, angiogenesis, and metastasis has hitherto been unclear and was investigated in this study. METHODS AND RESULTS: We compared tumor growth, vascularity, and responses to cyclophosphamide (CTX) of tumors in wild-type (wt) mice, or in animals with TF levels reduced by 99% (low-TF mice). Global growth rate of 3 different types of transplantable tumors (LLC, B16F1, and ES teratoma) or metastasis were unchanged in low-TF mice. However, several unexpected tumor/context-specific alterations were observed in these mice, including: (1) reduced tumor blood vessel size in B16F1 tumors; (2) larger spleen size and greater tolerance to CTX toxicity in the LLC model; (3) aborted tumor growth after inoculation of TF-deficient tumor cells (ES TF(-/-)) in low-TF mice. TF-deficient tumor cells grew readily in mice with normal TF levels and attracted exclusively host-related blood vessels (without vasculogenic mimicry). We postulate that this complementarity may result from tumor-vascular transfer of TF-containing microvesicles, as we observed such transfer using human cancer cells (A431) and mouse endothelial cells, both in vitro and in vivo. CONCLUSIONS: Our study points to an important but context-dependent role of host TF in tumor formation, angiogenesis and therapy.