RESUMEN
Two basic carboxypeptidases, carboxypeptidase B2 (CPB2) and carboxypeptidase N (CPN) are present in plasma. CPN is constitutively active, whereas CPB2 circulates as a precursor, procarboxypeptidase B2 (proCPB2), that needs to be activated by the thrombin-thrombomodulin complex or plasmin bound to glycosaminoglycans. The substrate specificities of CPB2 and CPN are similar; they both remove C-terminal basic amino acids from bioactive peptides and proteins, thereby inactivating them. The complement cascade is a cascade of proteases and cofactors activated by pathogens or dead cells, divided into two phases, with the second phase only being triggered if sufficient C3b is present. Complement activation generates anaphylatoxins: C3a, which stimulates macrophages; and C5a, which is an activator and attractant for neutrophils. Pharmacological intervention with inhibitors has shown that CPB2 delays fibrinolysis, whereas CPN is responsible for systemic inactivation of C3a and C5a. Among mice genetically deficient in either CPB2 or CPN, in a model of hemolytic-uremic syndrome, Cpb2-/- mice had the worst disease, followed by Cpn-/- mice, with wild-type (WT) mice being the most protected. This model is driven by C5a, and shows that CPB2 is important in inactivating C5a. In contrast, when mice were challenged acutely with cobra venom factor, the reverse phenotype was observed; Cpn-/- mice had markedly worse disease than Cpb2-/- mice, and WT mice were resistant. These observations need to be confirmed in humans. Therefore, CPB2 and CPN have different roles. CPN inactivates C3a and C5a generated spontaneously, whereas proCPB2 is activated at specific sites, where it inactivates bioactive peptides that would overwhelm CPN.
RESUMEN
Essentials Two basic carboxypeptidases are present in plasma, B2 (CPB2) and N (CPN). Cpb2-/- and Cpn-/- mice were challenged in a hemolytic uremic syndrome (HUS) model vs. wild type. Cpb2-/- exacerbates HUS while Cpn-/- exacerbates cobra venom factor challenge vs. wild type mice. CPB2 and CPN have overlapping but non-redundant roles. SUMMARY: Background There are two basic carboxypeptidases in plasma. Carboxypeptidase B2 (CPB2) is activated from a circulating zymogen, proCPB2, and carboxypeptidase N (CPN) is constitutively active with both inactivating complement C3a and C5a. Aims To test the roles of CPB2 and CPN in complement-driven mouse models of cobra venom factor (CVF) challenge and hemolytic-uremic syndrome (HUS). Methods Cpb2-/- , Cpn-/- and wild-type (WT) mice were compared in an HUS model induced by Shiga toxin and lipopolysaccharide administration and following CVF administration. Results HUS was exacerbated in Cpb2-/- mice more than in Cpn-/- mice, compared with WT mice. Cpb2-/- mice developed the HUS clinical triad of microangiopathic hemolytic anemia, uremia and thrombocytopenia. Treatment with anti-C5 antibody improved survival of both Cpb2-/- and Cpn-/- mice. In contrast, when challenged acutely with CVF, the reverse phenotype was observed. Cpn-/- mice had markedly worse disease than Cpb2-/- mice, whereas the WT mice were resistant. Conclusions CPN and CPB2 play overlapping but non-redundant roles in regulating complement activation in vivo. The constitutively active CPN is key for inactivation of systemic C5a, whereas CPB2 functions as an on-demand supplementary anaphylatoxin inhibitor in inactivating excessive C5a formed locally.
Asunto(s)
Carboxipeptidasa B2/sangre , Activación de Complemento , Complemento C3/metabolismo , Complemento C5a/metabolismo , Síndrome Hemolítico-Urémico/enzimología , Lisina Carboxipeptidasa/sangre , Animales , Carboxipeptidasa B2/deficiencia , Carboxipeptidasa B2/genética , Activación de Complemento/efectos de los fármacos , Complemento C5a/antagonistas & inhibidores , Complemento C5a/inmunología , Inactivadores del Complemento/farmacología , Modelos Animales de Enfermedad , Venenos Elapídicos/toxicidad , Endotoxinas , Genotipo , Síndrome Hemolítico-Urémico/sangre , Síndrome Hemolítico-Urémico/inducido químicamente , Síndrome Hemolítico-Urémico/tratamiento farmacológico , Lisina Carboxipeptidasa/deficiencia , Lisina Carboxipeptidasa/genética , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Proteolisis , Toxina Shiga IIRESUMEN
BACKGROUND AND OBJECTIVES: Carboxypeptidase B2 (CPB2) is a basic carboxypeptidase with fibrin and complement C3a and C5a as physiological substrates. We hypothesized that in polymicrobial sepsis, CPB2-deficient mice would have sustained C5a activity, leading to disease exacerbation. METHODS: Polymicrobial sepsis was induced by cecal ligation and puncture (CLP). RESULTS: Contrary to our hypothesis, Cpb2(-/-) mice had significantly improved survival, with reduced lung edema, less liver and kidney damage, and less disseminated intravascular coagulation. Hepatic pro-CPB2 was induced by CLP, leading to increased pro-CPB2 levels. Thrombomodulin present on mesothelium supported thrombin activation of pro-CPB2. Both wild-type and Cpb2(-/-) animals treated with a C5a receptor antagonist had improved survival, demonstrating that C5a was detrimental in this model. Treatment with a fibrinolysis inhibitor, tranexamic acid, caused a decrease in survival in both genotypes; however, the Cpb2(-/-) animals retained their survival advantage. Administration of a C3a receptor antagonist exacerbated the disease in both wild-type and Cpb2(-/-) mice and eliminated the survival advantage of Cpb2(-/-) mice. C5a receptor is expressed in both peritoneal macrophages and neutrophils; in contrast, C3a receptor expression is restricted to peritoneal macrophages, and C3a induced signaling in macrophages but not neutrophils. CONCLUSIONS: While C5a exacerbates the peritonitis, resulting in a deleterious generalized inflammatory state, C3a activation of peritoneal macrophages may limit the initial infection following CLP, thereby playing a diametrically opposing protective role in this polymicrobial sepsis model.
Asunto(s)
Carboxipeptidasa B2/deficiencia , Complemento C3a/metabolismo , Complemento C5a/metabolismo , Peritonitis/enzimología , Sepsis/enzimología , Animales , Antifibrinolíticos/farmacología , Trastornos de la Coagulación Sanguínea/enzimología , Trastornos de la Coagulación Sanguínea/genética , Trastornos de la Coagulación Sanguínea/inmunología , Trastornos de la Coagulación Sanguínea/microbiología , Carboxipeptidasa B2/genética , Ciego/microbiología , Ciego/cirugía , Células Cultivadas , Complemento C3a/antagonistas & inhibidores , Complemento C3a/inmunología , Complemento C5a/antagonistas & inhibidores , Complemento C5a/inmunología , Modelos Animales de Enfermedad , Activación Enzimática , Fibrina/metabolismo , Mediadores de Inflamación/sangre , Leucopenia/enzimología , Leucopenia/genética , Leucopenia/inmunología , Leucopenia/microbiología , Ligadura , Hígado/enzimología , Hígado/inmunología , Hígado/microbiología , Activación de Macrófagos , Macrófagos Peritoneales/enzimología , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/microbiología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Peritonitis/genética , Peritonitis/inmunología , Peritonitis/microbiología , Factores Protectores , Punciones , Factores de Riesgo , Sepsis/genética , Sepsis/inmunología , Sepsis/microbiología , Trombina/metabolismo , Trombomodulina/metabolismo , Factores de TiempoRESUMEN
BACKGROUND: Apart from its role in the coagulation system, thrombin plays an important role in the inflammatory response through its protease-activated receptors (PARs). However, the role of thrombin in the immune response is not clear. OBJECTIVE: To evaluate whether thrombin has a modulatory role in allergic bronchial asthma. METHODS: Bronchial asthma was induced in mice by intraperitoneal sensitization and inhalation challenge with ovalbumin. Thrombin or its inhibitors were administered by inhalation before each allergen challenge. RESULTS: Mice with low but sustained coagulation activation had reduced allergic inflammation, and allergic asthma was inhibited by low doses of thrombin but worsened by high doses. Allergic asthma was worsened by antithrombin, argatroban, hirudin, and anti-thrombomodulin antibody. Mice with a higher level of an inhibitor of both thrombin and activated protein C had worse disease. Heterozygous PAR-1 mice had less allergic inflammation, but PAR-1 agonist worsened it. Allergic bronchial inflammation was worsened in mice that received adoptive transfer of PAR-1 agonist-treated Th2 cells as compared with controls. Low levels of thrombin suppressed the maturation and secretion of cytokines in dendritic cells, but high levels enhanced this. CONCLUSIONS: The effects of thrombin on allergic asthma are dose-dependent, with detrimental effects at high doses and protective effects at low doses. These data demonstrate that thrombin modulates the outcome in allergic bronchial asthma.
Asunto(s)
Asma/etiología , Hipersensibilidad/etiología , Trombina/farmacología , Animales , Asma/inmunología , Asma/prevención & control , Líquido del Lavado Bronquioalveolar , Relación Dosis-Respuesta a Droga , Femenino , Hipersensibilidad/inmunología , Hipersensibilidad/prevención & control , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptor PAR-1/agonistas , Células Th2/inmunología , Trombina/fisiologíaRESUMEN
BACKGROUND: Infectious complications frequently occur after major trauma, leading to increased morbidity and mortality. Thrombin-activatable fibrinolysis inhibitor (TAFI), a procarboxypeptidase in plasma, plays a dual role in regulating both coagulation and inflammation. Activated TAFI (TAFIa) has broad anti-inflammatory properties due to its inactivation of active inflammatory mediators (anaphylatoxins C3a and C5a, bradykinin, osteopontin). OBJECTIVES: The purpose of this study was to determine if TAFI plays a role in the development of inflammatory complications after major trauma. PATIENTS/METHODS: Upon arrival at the emergency department (ED), plasma levels of TAFI and TAFIa were measured in 26 multiple traumatized patients for 10 consecutive days. Systemic levels of inflammatory mediators, including interleukin-6 (IL-6), procalcitonin (PCT), C-reactive protein (CRP) and leukocytes were determined. RESULTS: Fifteen patients developed pneumonia and/or sepsis (compl) and 11 had no complications (wo compl). Overall injury severity and age were comparable in both groups. Complications occurred approximately 5 days after trauma. IL-6 increased on day 5, whereas CRP, PCT and leukocytes started to increase on day 6 in the compl-group. Upon arrival at the ED and on days 1 and 4, TAFI levels were significantly lower in the compl-group compared to the wo compl-group (p=0.0215). Similarly, TAFIa was significantly lower on day 4 in the compl-group than in the wo compl-group (p=0.049). CONCLUSIONS: This pilot study shows that TAFI levels are inversely correlated with inflammation-associated development of complications after major trauma.
Asunto(s)
Carboxipeptidasa B2/sangre , Traumatismo Múltiple/sangre , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Proteína C-Reactiva/inmunología , Proteína C-Reactiva/metabolismo , Calcitonina/sangre , Calcitonina/inmunología , Péptido Relacionado con Gen de Calcitonina , Carboxipeptidasa B2/inmunología , Femenino , Humanos , Inflamación/sangre , Inflamación/inmunología , Mediadores de Inflamación/sangre , Mediadores de Inflamación/inmunología , Interleucina-6/sangre , Interleucina-6/inmunología , Recuento de Leucocitos , Masculino , Persona de Mediana Edad , Traumatismo Múltiple/complicaciones , Traumatismo Múltiple/inmunología , Proyectos Piloto , Neumonía/sangre , Neumonía/etiología , Neumonía/inmunología , Precursores de Proteínas/sangre , Precursores de Proteínas/inmunología , Sepsis/sangre , Sepsis/etiología , Sepsis/inmunología , Factores de TiempoRESUMEN
INTRODUCTION: The thrombin-activatable fibrinolysis inhibitor (TAFI) is a potent inhibitor of fibrinolysis. However, the time course of TAFI and its activated form (TAFIa) following trauma, in particular in patients suffering trauma-induced coagulopathy, has been poorly examined. METHODS: A total of 26 severely injured trauma patients were prospectively enrolled. TAFI and TAFIa levels were measured upon arrival and through hospital days one to 10. Trauma-induced coagulopathy was defined as elevated international normalized ratio (INR), and/or prolonged activated partial thromboplastin time (aPTT) and/or thrombocytopenia within one day of admission. RESULTS: TAFIa and TAFI levels showed the largest decrease on days one and two, respectively, with a progressive increase thereafter. Overall, 11 patients developed coagulopathy. No statistically significant differences were found for TAFI levels between the two groups. For TAFIa, however, coagulopathic patients experienced significantly lower levels on admission and on days six to eight (all p<0.05). Statistically significant correlations were found between TAFIa level on admission and the amount of packed red blood cells (p=0.011; Spearman's correlation coefficient=-0.5) and fresh frozen plasma (p=0.044; Spearman's correlation coefficient=-0.405) transfused within the initial 24hours. CONCLUSION: Depletion of TAFIa may contribute to the development of trauma-induced coagulopathy.
Asunto(s)
Trastornos de la Coagulación Sanguínea/etiología , Carboxipeptidasa B2/sangre , Heridas y Lesiones/complicaciones , Adulto , Biomarcadores/sangre , Trastornos de la Coagulación Sanguínea/sangre , Trastornos de la Coagulación Sanguínea/diagnóstico , Trastornos de la Coagulación Sanguínea/terapia , Regulación hacia Abajo , Transfusión de Eritrocitos , Femenino , Hospitalización , Humanos , Relación Normalizada Internacional , Masculino , Persona de Mediana Edad , Tiempo de Tromboplastina Parcial , Estudios Prospectivos , Factores de Riesgo , Trombocitopenia/sangre , Trombocitopenia/etiología , Factores de Tiempo , Índices de Gravedad del Trauma , Heridas y Lesiones/sangre , Heridas y Lesiones/diagnósticoRESUMEN
BACKGROUND: Activated protein C (APC) can regulate immune and inflammatory responses and apoptosis. Protein C transgenic mice develop less diabetic nephropathy but whether exogenous administration of APC suppresses established diabetic nephropathy is unknown. OBJECTIVES: We investigated the therapeutic potential of APC in mice with streptozotocin-induced diabetic nephropathy. METHODS: Diabetes was induced in unilaterally nephrectomized C57/Bl6 mice using intraperitoneal (i.p.) injection of streptozotocin. Four weeks later, the mice were treated with i.p. exogenous APC every other day for 1 month. RESULTS: APC-treated mice had a significantly improved blood nitrogen urea-to-creatinine ratio, urine total protein to creatinine ratio and proteinuria, and had significantly less renal fibrosis as measured by the levels of collagen and hydroxyproline. The renal tissue concentration of monocyte chemoattractant protein-1 (MCP-1), vascular endothelial growth factor (VEGF) and the RNA expression of platelet-derived growth factor (PDGF), transforming growth factor-ß1 and connective tissue growth factor (CTGF) were significantly lower in APC-treated mice than in untreated animals. The percentage of apoptotic cells was reduced and the expression of podocin, nephrin and WT-1 in the glomeruli was significantly improved in mice treated with APC compared with untreated mice. The levels of coagulation markers were not affected by APC treatment. CONCLUSION: Exogenous APC improves renal function and mitigates pathological changes in mice with diabetic nephropathy by suppressing the expression of fibrogenic cytokines, growth factors and apoptosis, suggesting its potential usefulness for the therapy of this disease.
Asunto(s)
Diabetes Mellitus Experimental/tratamiento farmacológico , Nefropatías Diabéticas/prevención & control , Riñón/efectos de los fármacos , Proteína C/farmacología , Animales , Apoptosis/efectos de los fármacos , Biomarcadores/sangre , Presión Sanguínea/efectos de los fármacos , Quimiocinas/metabolismo , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Experimental/complicaciones , Nefropatías Diabéticas/sangre , Nefropatías Diabéticas/etiología , Nefropatías Diabéticas/patología , Nefropatías Diabéticas/fisiopatología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Esquema de Medicación , Fibrosis , Humanos , Inyecciones Intraperitoneales , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Riñón/metabolismo , Riñón/patología , Riñón/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Nefrectomía , Nefroesclerosis/etiología , Nefroesclerosis/prevención & control , Tamaño de los Órganos/efectos de los fármacos , Proteína C/administración & dosificación , Estreptozocina , Factores de TiempoRESUMEN
BACKGROUND: Activation of the complement system has been implicated in tumor growth. The antifibrinolytic protein, activated thrombin-activatable fibrinolysis inhibitor (TAFIa), can modulate the activation of the complement system by inactivating the anaphylatoxins C3a and C5a. The apolipoprotein-E (ApoE) genotype has been associated with carcinogenesis. OBJECTIVE: The aim of this study was to evaluate whether TAFIa can affect the development of cancer in the ApoE-deficient mouse model. METHODS: TAFI and ApoE double-knockout mice were generated. A group of mice was treated with the diabetogenic and carcinogenic compound streptozotocin (stz). Mice treated with saline, single knockout mice and wild-type (wt) mice served as controls. RESULTS: Six months after treatment with stz, mice were sacrificed. Hepatic tumors were found in male double-knockout mice treated with stz but none was found in control animals that were not treated with stz or in single knockout of ApoE or wt animals. There was no significant difference in coagulation system activation between the groups of mice. The plasma concentrations of C5a, factor D and transforming growth factor-ß1 were increased in TAFI/ApoE double-deficient mice treated with stz compared with the mice of the same genotype treated with saline. CONCLUSION: Apo-E deficiency alone was not associated with tumors but the lack of TAFI appears to make the mice permissive for tumor formation in ApoE mice.
Asunto(s)
Apolipoproteínas E/genética , Carboxipeptidasa B2/genética , Diabetes Mellitus Experimental/genética , Neoplasias Experimentales/genética , Animales , Activación de Complemento , Fibrinólisis , Genotipo , Homocigoto , Riñón/metabolismo , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Estreptozocina/farmacología , Factores de TiempoRESUMEN
SUMMARY: Thrombin-activatable fibrinolysis inhibitor (TAFI) is a circulating zymogen that is activated physiologically by the thrombin/thrombomodulin complex to activated TAFI (TAFIa) which is a basic carboxypeptidase. Substrates include fibrin, leading to a reduction in rate of plasmin generation, and several proinflammatory mediators such as bradykinin, thrombin-cleaved osteopontin and complement factor C5a. TAFI-deficient mice have no phenotype without being challenged and TAFIa appears to play a limited role in physiological fibrinolysis in vivo. In several disease models, the TAFI-deficient mice have different outcomes from the wild type (WT), but whether the difference is beneficial or an exacerbation of the disease depends on the model. The consequences of TAFI deficiency include increased plasmin as a result of enhanced incorporation of plasminogen and tissue plasminogen activator into the fibrin clot, but also loss of its ability to degrade other substrates, with the resultant up-regulation of several proinflammatory mediators, including C5a. Criteria are recommended to demonstrate that a substrate is a physiological substrate of TAFIa.
Asunto(s)
Carboxipeptidasa B2/genética , Animales , Fibrinólisis , Ratones , Modelos Animales , Fenotipo , Trombosis , Cicatrización de HeridasRESUMEN
BACKGROUND: The fibrinolytic system has been implicated in the pathogenesis of pulmonary hypertension (PH). Thrombin-activatable fibrinolysis inhibitor (TAFI) inhibits fibrinolysis and therefore its absence would be expected to increase fibrinolysis and ameliorate PH. OBJECTIVE: The objective of the present study was to evaluate the effect of TAFI deficiency on pulmonary hypertension in the mouse. METHODS AND RESULTS: PH was induced in C57/Bl6 wild-type (WT) or TAFI-deficient (KO) mice by weekly subcutaneous treatment with 600 mg kg(-1) monocrotaline (MCT) for 8 weeks. PH was inferred from right heart hypertrophy measured using the ratio of right ventricle-to-left ventricle-plus-septum weight [RV/(LV+S)]. Pulmonary vascular remodeling was analyzed by morphometry. TAFI-deficient MCT-treated and wild-type MCT-treated mice suffered similar weight loss. TAFI-deficient MCT-treated mice had reduced levels of total protein and tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), transforming growth factor-beta (TGF-beta) and monocyte chemoattractant protein-1 (MCP-1) in bronchial alveolar lavage compared with wild-type MCT-treated mice. The ratio of RV to (LV+S) weight was significantly higher in WT/MCT than in KO/MCT mice. The pulmonary artery wall area and vascular stenosis were both greater in MCT-treated WT mice compared with MCT-treated TAFI-deficient mice. CONCLUSIONS: TAFI-deficient MCT-treated mice had less pulmonary hypertension, vascular remodeling and reduced levels of cytokines compared with MCT-treated WT animals, possibly as a result of reduced coagulation activation.
Asunto(s)
Carboxipeptidasa B2/deficiencia , Fibrinólisis , Hipertensión Pulmonar/prevención & control , Pulmón/metabolismo , Animales , Biomarcadores/metabolismo , Líquido del Lavado Bronquioalveolar/inmunología , Permeabilidad Capilar , Carboxipeptidasa B2/genética , Quimiocina CCL2/metabolismo , Modelos Animales de Enfermedad , Fibrinólisis/genética , Hipertensión Pulmonar/sangre , Hipertensión Pulmonar/inducido químicamente , Hipertensión Pulmonar/genética , Hipertensión Pulmonar/inmunología , Hipertensión Pulmonar/patología , Hipertrofia Ventricular Derecha/sangre , Hipertrofia Ventricular Derecha/prevención & control , Mediadores de Inflamación/metabolismo , Interleucina-6/metabolismo , Pulmón/irrigación sanguínea , Pulmón/inmunología , Pulmón/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Monocrotalina , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Arteria Pulmonar/metabolismo , Arteria Pulmonar/patología , Factor de Crecimiento Transformador beta1/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Pérdida de PesoRESUMEN
OBJECTIVE: Protein S may exert an anticoagulant activity by enhancing the anticoagulant activity of activated protein C and/or by directly inhibiting the prothrombinase complex. Protein S itself may also directly regulate inflammatory responses and apoptosis. The role of protein S in acute lung injury (ALI) was unknown. This study evaluated the effect of protein S on ALI in the mouse. METHODS: Animal ALI was induced in C57/BL6 mice by intratracheal instillation of lipopolysaccharide (LPS). Mice were treated with protein S or saline by intraperitoneal injection 1 h before LPS instillation. RESULTS: Activated protein or protein S alone and combined activated protein C + protein S therapy decreased inflammatory markers and cytokines in mice with acute lung injury. In LPS-treated mice compared with controls ALI was induced as shown by significantly increased levels of total protein, tumor necrosis factor-alpha, interleukin-6 and monocyte chemoattractant protein-1 in the bronchoalveolar lavage fluid. Mice with ALI treated with protein S had significantly decreased concentrations of tumor necrosis factor-alpha and interleukin-6 in the lung compared with untreated animals. Thrombin-antithrombin III, a marker of the activity of the coagulation cascade, was unchanged. Protein S inhibited the expression of cytokines in vitro and increased activation of the Axl tyrosine kinase pathway in A549 epithelial cells. CONCLUSION: Protein S protects against LPS-induced ALI, possibly by directly inhibiting the local expression of inflammatory cytokines without affecting coagulation.
Asunto(s)
Lesión Pulmonar Aguda/tratamiento farmacológico , Proteína S/farmacología , Lesión Pulmonar Aguda/inducido químicamente , Animales , Biomarcadores/análisis , Citocinas/análisis , Quimioterapia Combinada , Inflamación , Interleucina-6/análisis , Lipopolisacáridos/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Proteína C/farmacología , Proteína C/uso terapéutico , Proteína S/uso terapéutico , Resultado del Tratamiento , Factor de Necrosis Tumoral alfa/análisisRESUMEN
BACKGROUND: Thrombin-activatable fibrinolysis inhibitor (TAFI) has been reported to affect wound healing and fibrotic processes, but its role in renal tubulointerstitial fibrosis remains unknown. OBJECTIVE: To study its potential role, we compared TAFI-deficient and wild-type mice for the degree of renal fibrosis caused by unilateral ureteral obstruction (UUO). METHODS: The grade of tubulointerstitial fibrosis, the activity of plasmin, MMP-2 and MMP-9 were evaluated on days 4 and 9 after UUO. RESULTS: The renal content of hydroxyproline and the activity of plasmin, MMP-2 and MMP-9 were significantly increased in kidneys with UUO from TAFI-deficient mice compared with those from wild-type mice. These differences disappeared when animals with UUO from both groups were treated with the plasmin inhibitor tranexamic acid. The renal concentrations of fibrogenic cytokines were also significantly elevated in kidneys with UUO from TAFI-deficient mice compared with those from wild-type mice. CONCLUSION: The results of this study suggest that increased renal activity of plasmin in TAFI-deficient mice causes increased renal interstitial fibrosis in obstructive nephropathy.
Asunto(s)
Carboxipeptidasa B2/fisiología , Fibrinolisina/análisis , Fibrosis/etiología , Enfermedades Renales/etiología , Obstrucción Ureteral/complicaciones , Animales , Carboxipeptidasa B2/deficiencia , Citocinas/análisis , Hidroxiprolina/análisis , Enfermedades Renales/patología , Metaloproteinasa 2 de la Matriz/análisis , Metaloproteinasa 9 de la Matriz/análisis , Ratones , Ratones NoqueadosRESUMEN
BACKGROUND/OBJECTIVE: The efficacy of a direct factor (F)Xa inhibitor, ZK-807834, was compared with indirect inhibition by enoxaparin for inhibition and deaggregation of acute platelet-rich thrombi in a well-characterized porcine carotid injury model. METHODS: A crush injury was performed on a randomly chosen carotid artery and the thrombus allowed to propagate for 30 min. Pigs then received intravenous drug for 35 min: ZK-807834-Dose 1 (40 microg kg(-1) bolus + 1.5 microg kg(-1) min(-1) infusion, n=6); ZK-807834-Dose 2 (20 microg kg(-1) bolus + 0.75 microg kg(-1) min(-1) infusion; n=6); enoxaparin (1 mg kg(-1) bolus; n=6); or saline (n=6). Five minutes after drug initiation, the contralateral artery was injured. Thrombus size was monitored by scintillation detection of autologous 111In-platelets. RESULTS: The prothrombin time ratio was 2.2 +/- 0.1; 1.4 +/- 0.3; 1.2 +/- 0.9 and 1.1 +/- 0.2, respectively. ZK-807834-Dose 1 significantly inhibited carotid platelet deposition (525 +/- 226 x 10(6) cm(-2); P = 0.008), whereas ZK-807834-Dose 2 (2325 +/- 768) and enoxaparin (1236 +/- 383) were not different from saline (2776 +/- 642). Thrombus deaggregation was greatest for animals receiving ZK-807834-Dose 1 (473 +/- 185). Neither ZK-807834-Dose 2 (1588 +/- 480) nor enoxaparin (1618 +/- 686) was different from saline control (2222 +/- 598). CONCLUSIONS: Direct FXa inhibition with ZK-807834, at a prothrombin time ratio of 2.2, effectively inhibits thrombosis and promptly deaggregates thrombi induced by arterial injury. In contrast, indirect FXa inhibition with enoxaparin was ineffective.
Asunto(s)
Plaquetas/metabolismo , Arterias Carótidas/patología , Trombosis de las Arterias Carótidas/tratamiento farmacológico , Trombosis de las Arterias Carótidas/prevención & control , Amidinas/farmacología , Animales , Anticoagulantes/farmacología , Relación Dosis-Respuesta a Droga , Enoxaparina/farmacología , Inhibidores del Factor Xa , Femenino , Heparina/metabolismo , Concentración 50 Inhibidora , Perfusión , Tiempo de Protrombina , Piridinas/farmacología , Porcinos , Trombosis/tratamiento farmacológico , Trombosis/prevención & control , Factores de TiempoRESUMEN
Inhibition of factor Xa with the small molecule inhibitor ZK-807834 (Mr 527 Da, Ki 0.11 nM) attenuates progression of thrombosis, but the ED50 is substantially lower for venous compared with arterial thrombosis in experimental animals. To determine whether this reflects differences in the extent of vascular injury, we compared the dose-response of ZK-807834 for inhibition of venous thrombosis induced with a cotton thread and copper wire device in the presence and absence of balloon catheter-induced injury to the vena cava in rabbits. ZK-807834 administration over 2 h (total dosages of 0.0023-2.3 micro mol kg-1, n = 6/group) resulted in dose-dependent reductions in clot weight compared with vehicle controls, but the ED50 was 0.03 micro mol kg-1 for non-injured veins and 0.42 micro mol kg-1 for injured veins. We conclude that vascular injury invokes a tissue factor-mediated response that increases the dose requirements for inhibition of venous thrombosis with ZK-807834.
Asunto(s)
Amidinas/farmacología , Endotelio Vascular/lesiones , Piridinas/farmacología , Trombosis de la Vena/tratamiento farmacológico , Trombosis de la Vena/etiología , Animales , Pruebas de Coagulación Sanguínea , Cateterismo/efectos adversos , Relación Dosis-Respuesta a Droga , Inhibidores del Factor Xa , Conejos , Terapia Trombolítica , Venas Cavas/patologíaRESUMEN
Factor Xa is a serine protease positioned at the convergence point of the intrinsic and extrinsic coagulation pathways and is therefore an attractive target in the development of novel anticoagulant drugs. The objective of this study was to evaluate the efficacy of CI-1031 (N-[2-[5-amidino-2-hydroxyphenoxy]-6-[3-(1-methyl-1H-imidazolin-2-yl)-phenoxy]-3,5-difluoropyrid), a potent and selective inhibitor of Factor Xa, in a canine electrolytic injury model of arterial and venous thrombosis. Enoxaparin (enoxaparin sodium), a low molecular weight heparin currently approved for treatment and prevention of deep vein thrombosis and unstable angina, was also tested for efficacy in this model. CI-1031 was administered intravenously to anesthetized dogs at three doses: 1.25, 2.5 and 5 microg/kg/min (n=5 for each group) as a continuous infusion for 5.5 h. The control group (n=5) received a continuous infusion of vehicle (3.69 mmol citric acid and 0.9% sodium chloride solution) at a rate of 1 ml/kg/h. Ninety minutes after administration of CI-1031 prothrombin times increased 1.2-, 1.6- and 2.0-fold over baseline values in the 1.25, 2.5 and 5 microg/kg/min groups, respectively. The time to formation of an occlusive thrombus in the femoral arteries averaged 69+/-5 min in the control group compared to 127+/-19, 192+/-33 and 219+/-15 min in the low-, mid- and high-dose CI-1031 groups. In the femoral veins, occlusion time in the controls averaged 56+/-11 min compared to 153+/-22, 137+/-30 and 214+/-26 min in the three treatment groups. Thrombus weights in the control arteries averaged 51+/-4 mg compared to 45+/-5, 28+/-10 and 15+/-3 mg in the CI-1031 treated groups. On the venous side, control thrombus weights averaged 96+/-18 mg compared to 75+/-16, 51+/-16 and 25+/-4 mg in the low-, mid- and high-dose CI-1031 groups. A plasma CI-1031 concentration of approximately 400 ng/ml was associated with a 50% reduction in thrombus weight relative to control animals. Enoxaparin was administered intravenously at a loading dose of 50, 100 or 200 IU/kg for 1 h followed by a maintenance infusion of 25, 50 or 100 IU/kg/h for 4.5 h. The most dramatic changes in coagulation parameters were observed in thrombin time with virtually no changes in prothrombin time. Enoxaparin elicited a dose-dependent increase in time to thrombotic occlusion and a dose-dependent decrease in thrombus weight similar to that observed with CI-1031. Time to occlusion in the enoxaparin-treated groups averaged 117+/-33, 188+/-32 and 217+/-22 min in the low-, mid- and high-dose groups in the femoral arteries and 84+/-22, 171+/-31 and 133+/-33 min in the femoral veins. Thrombus weights averaged 33+/-10, 12+/-5 and 10+/-4 mg in the arteries and 32+/-9, 13+/-2 and 21+/-6 mg in the veins in the low-, mid- and high-dose groups. Blood loss with CI-1031 tended to be less than enoxaparin at doses that provided comparable efficacy. These results demonstrate that CI-1031, like enoxaparin, is an effective antithrombotic agent in an established canine model of arterial and venous thrombosis. CI-1031 provided dose-dependent efficacy with minimal changes in ex vivo coagulation parameters, suggesting it may be a safe and effective antithrombotic agent for both arterial and venous indications.
Asunto(s)
Amidinas/farmacología , Anticoagulantes/farmacología , Enoxaparina/farmacología , Piridinas/farmacología , Trombosis/prevención & control , Trombosis de la Vena/prevención & control , Amidinas/sangre , Animales , Modelos Animales de Enfermedad , Perros , Relación Dosis-Respuesta a Droga , Electrólisis/efectos adversos , Inhibidores del Factor Xa , Tiempo de Tromboplastina Parcial , Tiempo de Protrombina , Piridinas/sangre , Tiempo de Trombina , Trombosis/etiología , Factores de Tiempo , Trombosis de la Vena/etiologíaRESUMEN
Single-chain urokinase-type plasminogen activator (scu-PA) can be cleaved by thrombin into a virtually inactive form called thrombin-cleaved two-chain urokinase-type plasminogen activator (tcu-PA/T), a process accelerated by thrombomodulin, which contains six epidermal growth factor (EGF)-like domains. In this study, we identified the EGF-like domains of thrombomodulin required for the acceleration of the inactivation of scu-PA by thrombin using various forms of thrombomodulin (TM). scu-PA was treated with thrombin in the absence and presence of full-length rabbit TM (containing EGF1-6), recombinant TM comprising all of the extracellular domains including EGF1-6 (TMLEO) and recombinant TM comprising EGF4-6 plus the interconnecting region between EGF3 and EGF4 (TMEi4-6), and the tcu-PA/T generated was quantitated in each case. Rabbit TM accelerated the inactivation of scu-PA approximately 35-fold, while both recombinant forms accelerated it only threefold due to the absence of a critical chondroitin sulfate moiety. Subsequently, TME5-6 was prepared by cyanogen bromide digestion of TMEi4-6. TME5-6 bound to thrombin but did not accelerate the activation of protein C. In contrast, the inactivation of scu-PA by thrombin was accelerated to the same extent as that induced by TMLEO and TMEi4-6. This study demonstrates that, in addition to the chondroitin sulfate moiety, only EGF-like domains 5 and 6 are essential for the acceleration of the inactivation of scu-PA by thrombin. This differs from the domains that are critical for activation of protein C (EGF-like domains i4-6) and thrombin activatable fibrinolysis inhibitor (EGF-like domains 3-6).
Asunto(s)
Factor de Crecimiento Epidérmico/metabolismo , Trombina/metabolismo , Trombomodulina/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Anticuerpos Monoclonales/farmacología , Carboxipeptidasa B2/metabolismo , Bromuro de Cianógeno/química , Activación Enzimática , Glicosilación , Humanos , Proteína C/metabolismo , Estructura Terciaria de Proteína , Trombina/química , Trombomodulina/química , Trombomodulina/efectos de los fármacos , Trombomodulina/inmunología , Activador de Plasminógeno de Tipo Uroquinasa/químicaRESUMEN
Urokinase-type plasminogen activator (uPA) is increased in human abdominal aortic aneurysm (AAA). Chronic infusion of angiotensin II (Ang II) results in AAA in apolipoprotein E-deficient mice. We tested the hypothesis that Ang II infusion results in an elevation of uPA expression contributing to aneurysm formation. Ang II or vehicle was infused by osmotic pumps into apoE-KO mice. All mice treated with Ang II developed a localized expansion of the suprarenal aorta (75% increase in outer diameter), accompanied by an elevation of blood pressure (22 mmHg), compared to the vehicle-treated group. Histological examination of the dilated aortic segment revealed similarities to human AAA including focal elastin fragmentation, macrophage infiltration, and intravascular hemorrhage. Ang II treatment resulted in a 13-fold increase in the expression of uPA mRNA in the AAA segment in contrast to a twofold increase in the atherosclerotic aortic arch. Increased uPA protein was detected in the abdominal aorta as early as 10 days after Ang II infusion before significant aorta expansion. Thus, Ang II infusion results in macrophage infiltration, increased uPA activity, and aneurysm formation in the abdominal aorta of apoE-KO mice. These data are consistent with a causal role for uPA in the pathogenesis of AAA.
Asunto(s)
Angiotensina II/farmacología , Aneurisma de la Aorta Abdominal/inducido químicamente , Apolipoproteínas E/deficiencia , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Animales , Aorta/metabolismo , Aorta/patología , Aneurisma de la Aorta Abdominal/diagnóstico por imagen , Aneurisma de la Aorta Abdominal/patología , Apolipoproteínas E/genética , Técnicas In Vitro , Interleucina-6/metabolismo , Ratones , Ratones Noqueados/genética , Valores de Referencia , Factores de Tiempo , UltrasonografíaRESUMEN
During thrombosis, vascular wall cells are exposed to clotting factors, including the procoagulant proteases thrombin and factor Xa (FXa), both known to induce cell signaling. FXa shows dose-dependent induction of intracellular Ca(2+) transients in vascular wall cells that is active-site-dependent, Gla-domain-independent, and enhanced by FXa assembly into the prothrombinase complex. FXa signaling is independent of prothrombin activation as shown by the lack of inhibition by argatroban, hirudin and the sulfated C-terminal peptide of hirudin (Hir(54-65)(SO3(-))). This peptide binds to both proexosite I in prothrombin and exosite I in thrombin. In contrast, signaling is completely blocked by the FXa inhibitor ZK-807834 (CI-1031). No inhibition is observed by peptides which block interaction of FXa with effector cell protease 1 receptor (EPR-1), indicating that this receptor does not mediate signaling in the cells assayed. Receptor desensitization studies with thrombin or peptide agonists (PAR-1 or PAR-2) and experiments with PAR-1-blocking antibodies indicate that signaling by FXa is mediated by both PAR-1 and PAR-2. Potential pathophysiological responses to FXa include increased cell proliferation, increased production of the proinflammatory cytokine IL-6 and increased production of prothrombotic tissue factor. These cellular responses, which may complicate vascular disease, are inhibited by ZK-807834.
Asunto(s)
Endotelio Vascular/efectos de los fármacos , Factor Xa/farmacología , Receptores de Trombina/fisiología , Transducción de Señal/efectos de los fármacos , Amidinas/farmacología , Señalización del Calcio/efectos de los fármacos , Células Cultivadas , Endotelio Vascular/citología , Factor V/farmacología , Inhibidores del Factor Xa , Humanos , Proteínas Inhibidoras de la Apoptosis , Piridinas/farmacología , Receptor PAR-1 , Receptor PAR-2 , Receptores de Superficie Celular , Inhibidores de Serina Proteinasa/farmacología , Survivin , Trombina/farmacologíaRESUMEN
TAFI (thrombin activatable fibrinolysis inhibitor) is a plasma procarboxypeptidase that upon activation inhibits the fibrinolytic process by removing the C-terminal lysines from partially degraded fibrin. The generation of activated TAFI (TAFIa) has been suggested to represent a mechanism of thrombus resistance to thrombolytic therapy. However, the ability of TAFI to inhibit fibrinolysis by pharmacological concentrations of t-PA has not been properly investigated. We used an in vitro model consisting of 125I-fibrin blood clots submerged in autologous defibrinated plasma. Upon addition of t-PA (125-5,000 ng/ml) and CaCl2 (25 mM), samples were incubated at 37 degrees C, and clot lysis was measured at intervals from the radioactivity released into solution. The role of TAFI was assessed either by neutralizing the generated TAFIa with the specific inhibitor PTI (50 microg/ml) or by enhancing TAFI activation through the addition of recombinant soluble thrombomodulin (solulin, 1 microg/ml). In our clot lysis model, activation of TAFI amounted to about 20% of inducible carboxypeptidase activity. Addition of PTI, however, produced a significant increase in the extent of lysis only at concentrations of t-PA equal to or lower than 250 ng/ml. When solulin was added to the plasma surrounding the clot, about 70% of TAFI was activated within 15 min. Under these conditions, inhibition of clot lysis was very marked in samples containing 125 or 250 ng/ml of t-PA, but negligible in those containing pharmacological concentrations of the activator (1,000 and 5,000 ng/ml). Additional experiments suggest that loss of fibrin-dependence by elevated concentrations of t-PA may be one of the mechanisms explaining the lack of effect of TAFIa. Our data indicate that, under our experimental conditions, clot lysis by pharmacological concentrations of t-PA is not influenced by TAFIa even after maximal activation of this procarboxy-peptidase.