RESUMEN
UNLABELLED: Essentials Microembolic signal (MES) is an independent predictor of stroke risk in patients. A rabbit model of cerebral microembolic signals was established. Therapeutic efficacy was demonstrated for aspirin and clopidogrel on microembolic signals. Potential translational value of this preclinical model of MES was demonstrated. SUMMARY: Objectives Cerebral microembolic signals (MESs) detected by transcranial Doppler (TCD) ultrasound constitute an independent predictor of stroke risk and prognosis. The aim of this study was to develop a novel preclinical model of MESs to facilitate translational research. Methods A clinical TCD ultrasound machine was used to detect MESs in the cerebral circulation of New Zealand White rabbits. Technical feasibility was assessed for the measurement of MESs in the middle cerebral artery (MCA) by TCD. FeCl3 -induced carotid arterial thrombosis was optimized for the generation of endogenous microemboli. Ascending doses of two antithrombotic agents (aspirin and clopidogrel) were evaluated individually and in combination for their effects on both arterial thrombosis and MESs in a 30% FeCl3 -induced carotid arterial thrombosis model, along with ex vivo functional assays. Results Dose-dependent FeCl3 -induced arterial thrombosis studies showed that 30% FeCl3 resulted in the most consistent and reproducible MESs in the MCA (3.3 ± 0.7 MESs h(-1) ). Ascending-dose studies showed that the effective doses for 50% inhibition (ED50 ) of thrombus formation, based on integrated blood flow and thrombus weight, respectively, were 3.1 mg kg(-1) and 4.2 mg kg(-1) orally for aspirin, and 0.3 mg kg(-1) and 0.28 mg kg(-1) orally for clopidogrel. The ED50 values for MES incidence were 12.7 mg kg(-1) orally for aspirin, and 0.25 mg kg(-1) orally for clopidogrel. Dual treatment with aspirin (5 mg kg(-1) ) and clopidogel (0.3 mg kg(-1) ) resulted in significant reductions in cerebral MESs (P < 0.05) as compared with monotherapy with either agent. Conclusions Our study demonstrated the successful establishment of the MES model in rabbits, and it may provide translational value for MESs and ischemic stroke research.
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Aspirina/uso terapéutico , Embolia Intracraneal/tratamiento farmacológico , Accidente Cerebrovascular/tratamiento farmacológico , Ticlopidina/análogos & derivados , Animales , Trombosis de las Arterias Carótidas/inducido químicamente , Trombosis de las Arterias Carótidas/tratamiento farmacológico , Cloruros , Clopidogrel , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Compuestos Férricos , Fibrinolíticos/uso terapéutico , Embolia Intracraneal/fisiopatología , Masculino , Arteria Cerebral Media/fisiopatología , Agregación Plaquetaria , Conejos , Accidente Cerebrovascular/complicaciones , Ticlopidina/uso terapéutico , Investigación Biomédica Traslacional , Ultrasonografía , Ultrasonografía DopplerRESUMEN
BACKGROUND: Increased plasma levels of coagulation factor (F) XI are a risk factor for venous thrombosis. OBJECTIVE: To further explore the relationship between FXI and venous thrombosis, we evaluated FXI-deficient and wild-type mice in a ferric chloride (FeCl(3))-induced vena cava thrombosis model. METHODS AND RESULTS: Thrombosis was induced by 3-min topical application of filter papers containing increasing concentrations of FeCl(3) and the thrombus was measured at 30 min. In contrast to wild-type mice, FXI-deficient mice failed to form a thrombus with 5% FeCl(3,) and were partially protected against 7.5% and 10% FeCl(3,) respectively. The protective effect was substantially stronger than a high dose of heparin (1,000 units kg(-1), i.v.), clopidogrel (30 mg kg(-1), p.o.) or argatroban (30 mg kg(-1), i.p.). These antithrombotic agents resulted in off-scale bleeding in a tail bleeding time assay, whereas the bleeding time of FXI-deficient mice was unchanged compared to wild-type mice. In addition to its known effect on the coagulation cascade, enhanced clot lysis was demonstrated in FXI-deficient mouse and human plasma compared to those supplemented with FXIa. CONCLUSION: Given the strong antithrombotic efficacy (possibly contributed by strong anticoagulant activity associated with increased fibrinolytic activity) and mild bleeding diathesis associated with FXI deficiency, therapeutic inhibition of FXI may be a reasonable therapeutic strategy to treat or prevent venous thrombosis.
Asunto(s)
Deficiencia del Factor XI/complicaciones , Compuestos Férricos/farmacología , Venas Cavas/patología , Trombosis de la Vena/prevención & control , Animales , Cloruros , Modelos Animales de Enfermedad , Fibrinólisis , Fibrinolíticos/farmacología , Ratones , Trombosis de la Vena/inducido químicamenteRESUMEN
Factor XI (FXI) and factor IX (FIX) are zymogens of plasma serine proteases required for normal hemostasis. The purpose of this work was to evaluate FXI and FIX as potential therapeutic targets by means of a refined ferric chloride (FeCl(3))-induced arterial injury model in factor-deficient mice. Various concentrations of FeCl(3) were used to establish the arterial thrombosis model in C57BL/6 mice. Carotid artery blood flow was completely blocked within 10 min in C57BL/6 mice by application of 3.5% FeCl(3). In contrast, FXI- and FIX-deficient mice were fully protected from occlusion induced by 5% FeCl(3), and were partially protected against the effect of 7.5% FeCl(3). The protective effect was comparable to very high doses of heparin (1000 units kg(-1)) and substantially more effective than aspirin. While FXI and FIX deficiencies were indistinguishable in the carotid artery injury model, there was a marked difference in a tail-bleeding-time assay. FXI-deficient and wild-type mice have similar bleeding times, while FIX deficiency was associated with severely prolonged bleeding times (>5.8-fold increase, P < 0.01). Given the relatively mild bleeding diathesis associated with FXI deficiency, therapeutic inhibition of FXI may be a reasonable strategy for treating or preventing thrombus formation.
Asunto(s)
Arterias Carótidas/efectos de los fármacos , Factor IX/fisiología , Deficiencia del Factor XI/patología , Factor XI/fisiología , Compuestos Férricos/farmacología , Hemofilia B/patología , Animales , Arterias/efectos de los fármacos , Arterias/lesiones , Aspirina/farmacología , Tiempo de Sangría , Velocidad del Flujo Sanguíneo , Enfermedades de las Arterias Carótidas/patología , Cloruros , Relación Dosis-Respuesta a Droga , Genotipo , Heparina/química , Heparina/farmacología , Homocigoto , Ratones , Ratones Endogámicos C57BL , Agregación Plaquetaria , Flujo Sanguíneo Regional/efectos de los fármacos , Trombosis/patología , Trombosis/terapia , Factores de TiempoRESUMEN
Alzheimer's disease is the most common form of dementia and a major public health problem. The amyloid hypothesis suggests that Alzheimer's disease is due to the abnormal accumulation of amyloid-beta protein (A beta) in affected brain regions. Rational therapies aimed at reducing amyloid burden in brain are currently being pursued in preclinical and early clinical development. This review summarizes recent progress in understanding the beta- and gamma-secretase activities required for the formation of A beta peptide and discusses therapeutic strategies aimed at inhibiting these activities. Recent progress in the identification of small molecule inhibitors of these secretases is also reviewed.
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Precursor de Proteína beta-Amiloide/antagonistas & inhibidores , Precursor de Proteína beta-Amiloide/metabolismo , Inhibidores Enzimáticos/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Precursor de Proteína beta-Amiloide/biosíntesis , Animales , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/uso terapéutico , HumanosRESUMEN
Platelets activated by alpha-thrombin express surface procoagulant activity (PCA) that accelerates the conversion of prothrombin to alpha-thrombin. Following activation with 10 nM alpha-thrombin, the PCA of normal platelets was approximately five-fold higher than that of Bernard-Soulier platelets (lacking GPIb). Normal platelet PCA was inhibited approximately 50% by activation in the presence of the anti-GPIb MoAbs LJIb10 or TM60. Moreover, normal platelet PCA was completely abrogated in the presence of a combination of both LJIb10 and c7E3, a MoAb directed against alphaIIbbeta3 (GPIIb/IIIa). In contrast. PCA expressed by Bernard Soulier or Glanzmann platelets was not inhibited by either LJIb10 or c7E3 MoAb. The platelet activating peptide SFLLRN at 10 microM, a concentration which fully activates platelet aggregation and Ca2+ mobilization, generated PCA activity one fifth of that generated by alpha-thrombin at 10 nM but anti-PAR1 antibodies did not affect thrombin-induced PCA expression. These results demonstrate that GPIb mediates, at least in part, the thrombin-induced activation of platelets that leads to PCA, and that alphaIIbbeta3 is also involved in PCA generation, but these results do not support a major role for PAR1 in this activation.
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Factores de Coagulación Sanguínea/biosíntesis , Plaquetas/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/fisiología , Complejo GPIb-IX de Glicoproteína Plaquetaria/fisiología , Trombina/farmacología , Adulto , Síndrome de Bernard-Soulier/sangre , Factores de Coagulación Sanguínea/genética , Plaquetas/metabolismo , Señalización del Calcio/efectos de los fármacos , Femenino , Humanos , Masculino , Fragmentos de Péptidos/farmacología , Activación Plaquetaria/efectos de los fármacos , Receptor PAR-1 , Receptores de Trombina/efectos de los fármacos , Receptores de Trombina/inmunología , Receptores de Trombina/metabolismo , Trombastenia/sangre , Tromboplastina/metabolismoRESUMEN
Plasminogen activator inhibitor type-1 (PAI-1) is bound to vitronectin (VN) in plasma and in the extracellular matrix. We previously employed a domain-swapping approach to show that the high-affinity binding site for PAI-1 in VN is contained within residues 12-30 in the amino-terminal somatomedin B (SMB) domain. In this study, we attempt to further delineate the location of this site by employing a novel approach that is based on the use of monoclonal antibodies (Mabs) together with site-directed mutagenesis. Six separate Mabs were identified that bound to the SMB domain and competed with PAI-1 for binding to VN. The relative affinity of each of the Mabs, and of PAI-1 itself, for binding to individual variants of SMB (prepared by alanine scanning mutagenesis), was then determined and compared in competitive binding experiments. Three separate, partially overlapping Mab epitopes within SMB were defined by these studies, and the PAI-1 binding site was localized to the region between residues 24 and 37. When considered together with the domain swapping data, these studies suggest that the PAI-1 binding site is contained within a common seven-residue region (i.e., residues 24-30) in the SMB domain.
Asunto(s)
Inhibidor 1 de Activador Plasminogénico/metabolismo , Somatomedinas/metabolismo , Vitronectina/metabolismo , Alanina/genética , Secuencias de Aminoácidos , Anticuerpos Monoclonales/inmunología , Sitios de Unión , Mutagénesis , Estructura Terciaria de Proteína , Somatomedinas/genética , Somatomedinas/inmunología , Vitronectina/química , Vitronectina/genéticaRESUMEN
The integrity of cerebral microvessels requires the close apposition of the endothelium to the astrocyte endfeet. Integrins alpha1beta1 and alpha6beta4 are cellular matrix receptors that may contribute to cerebral microvascular integrity. It has been hypothesized that focal ischemia alters integrin expression in a characteristic time-dependent manner consistent with neuron injury. The effects of middle cerebral artery occlusion (MCAO) and various periods of reperfusion on microvasclar integrin alpha1beta1 and alpha6beta4 expression were examined in the basal ganglia of 17 primates. Integrin subunits alpha1 and beta1 colocalized with the endothelial cell antigen CD31 in nonischemic microvessels and with glial fibrillary acidic protein on astrocyte fibers. Rapid, simultaneous, and significant disappearance of both integrin alpha1 and beta1 subunits and integrin alpha6beta4 occurred by 2 hours MCAO, which was greatest in the region of neuron injury (ischemic core, Ic), and progressively less in the peripheral (Ip) and nonischemic regions (N). Transcription of subunit beta1 mRNA on microvessels increased significantly in the Ic/Ip border and in multiple circular subregions within Ic. Microvascular integrin alpha1beta1 and integrin alpha6beta4 expression are rapidly and coordinately lost in Ic after MCAO. With loss of integrin alpha1beta1, multiple regions of microvascular beta1 mRNA up-regulation within Ic suggest that microvessel responses to focal ischemia are dynamic, and that multiple cores, not a single core, are generated. These changes imply that microvascular integrity is modified in a heterogeneous, but ordered pattern.
Asunto(s)
Antígenos de Superficie/genética , Expresión Génica , Integrinas/genética , Ataque Isquémico Transitorio/metabolismo , Animales , Antígenos de Superficie/análisis , Astrocitos/química , Proteína Ácida Fibrilar de la Glía/análisis , Inmunohistoquímica , Hibridación in Situ , Integrina alfa1beta1 , Integrina alfa6beta4 , Integrinas/análisis , Ataque Isquémico Transitorio/patología , Masculino , Microcirculación/química , Microscopía Confocal , Arteria Cerebral Media , Neuronas/patología , Papio , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/análisis , ARN Mensajero/análisisRESUMEN
In this report, a novel positive-negative epitope tagging approach was developed to study the cellular processing of beta amyloid precursor protein (beta APP). Amino acids centered around the alpha-secretase cleavage site within the A beta sequence were replaced with residues comprising an epitope for which high-affinity monoclonal antibodies are commercially available. The resulting mutant beta APP cDNAs were expressed in human embryonic kidney cells (HEK 293). Cleavage of labeled beta APP by beta- and gamma-secretase(s) results in the release of an epitope-tagged A beta peptide, whereas cleavage by alpha-secretase results in destruction of the epitope. Highly sensitive and specific immunoassays were developed to study processing of this labeled beta APP via the amyloidogenic pathway. Secretion of epitope-tagged A beta was prevented by MDL 28170, a previously described gamma-secretase inhibitor. Confocal microscopic studies revealed that processing and cellular trafficking of epitope-tagged beta APP was not different from wild-type beta APP. These results suggest that positive-negative epitope-tagged beta APP is normally processed within the cell and may be used to identify secretase inhibitors as therapeutics for Alzheimer's disease.
Asunto(s)
Precursor de Proteína beta-Amiloide/metabolismo , Endopeptidasas/metabolismo , Epítopos/metabolismo , Inhibidores de Proteasas/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Recombinantes de Fusión/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Secretasas de la Proteína Precursora del Amiloide , Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/inmunología , Anticuerpos Monoclonales/inmunología , Ácido Aspártico Endopeptidasas , Western Blotting , Línea Celular , Medios de Cultivo Condicionados/química , Medios de Cultivo Condicionados/metabolismo , Dipéptidos/farmacología , Ensayo de Inmunoadsorción Enzimática , Epítopos/química , Epítopos/genética , Epítopos/inmunología , Humanos , Inmunohistoquímica , Isoenzimas/antagonistas & inhibidores , Isoenzimas/metabolismo , Inhibidores de Proteasas/análisis , Inhibidores de Proteasas/uso terapéutico , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Sensibilidad y Especificidad , TransfecciónRESUMEN
Presenilins are integral membrane protein involved in the production of amyloid beta-protein. Mutations of the presenilin-1 and -2 gene are associated with familial Alzheimer's disease and are thought to alter gamma-secretase cleavage of the beta-amyloid precursor protein, leading to increased production of longer and more amyloidogenic forms of A beta, the 4-kDa beta-peptide. Here, we show that radiolabeled gamma-secretase inhibitors bind to mammalian cell membranes, and a benzophenone analog specifically photocross-links three major membrane polypeptides. A positive correlation is observed among these compounds for inhibition of cellular A beta formation, inhibition of membrane binding and cross-linking. Immunological techniques establish N- and C-terminal fragments of presenilin-1 as specifically cross-linked polypeptides. Furthermore, binding of gamma-secretase inhibitors to embryonic membranes derived from presenilin-1 knockout embryos is reduced in a gene dose-dependent manner. In addition, C-terminal fragments of presenilin-2 are specifically cross-linked. Taken together, these results indicate that potent and selective gamma-secretase inhibitors block A beta formation by binding to presenilin-1 and -2.
Asunto(s)
Endopeptidasas/efectos de los fármacos , Inhibidores Enzimáticos/metabolismo , Proteínas de la Membrana/metabolismo , Secretasas de la Proteína Precursora del Amiloide , Membrana Celular/metabolismo , Endopeptidasas/metabolismo , Pruebas de Precipitina , Presenilina-1 , Presenilina-2 , Especificidad por SustratoRESUMEN
The suitability of rabbit prothrombin activation fragment F 1.2 as a marker for the activation of the coagulation system was tested. Monoclonal antibodies to rabbit F 1.2 were raised, and a competitive F 1.2 ELISA was developed. Within the detection limit of the ELISA, no increase in rabbit F 1.2 was detected upon recalcification of plasma, whereas human F 1.2 increased 1500-fold. The apparent lack of F 1.2 formation in rabbit serum was confirmed by immunoblotting analysis of endogenous and biotin-labeled prothrombin. Meizothrombin and the B-chain of thrombin were the only prothrombin fragments detectable. In contrast, labeled human prothrombin formed, in addition, prethrombin 2 and F 1.2 in both human and rabbit serum. In contrast, rabbit F 1.2 formation could be demonstrated using purified rabbit prothrombin and factor Xa. These observations raise the possibility that rabbit prothrombin is less susceptible than the human counterpart to factor Xa cleavage at the 271/272 peptide bond. Thus, the primary structure of rabbit prothrombin was deduced by cDNA sequencing. While the 320/321 Xa cleavage site giving rise to meizothrombin was identical in rabbit and human prothrombin, the flanking region of the 271/272 Xa sensitive site contained a six amino acid deletion in the rabbit sequence. Taken together, these observations suggest that the observed differences between human and rabbit prothrombin activation may be due to different susceptibilities of the two Xa cleavage sites rather than plasma or serum cofactor(s).
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Coagulación Sanguínea , Fragmentos de Péptidos/fisiología , Protrombina/fisiología , Secuencia de Aminoácidos , Animales , ADN Complementario/genética , Ensayo de Inmunoadsorción Enzimática , Humanos , Datos de Secuencia Molecular , Conejos , Alineación de Secuencia , Análisis de Secuencia de ADN , Especificidad de la EspecieRESUMEN
Although extensive tissue remodeling occurs during the various phases of aortic dissection, the underlying proteinases remain to be identified. Matrix metalloproteinase-9 (MMP-9) and components of the fibrinolytic system have been implicated in numerous tissue remodeling events and were therefore analyzed in surgical specimens of acute (n = 9), subacute (n = 4), and chronic (n = 7) aortic dissection by in situ hybridization. In the acute phase, intense plasminogen activator inhibitor 1 (PAI-1) gene expression was apparent in areas interfacing the dissecting hematoma, but no tissue-type PA (t-PA), urokinase-type PA (u-PA), or MMP-9 mRNAs were detected. Although PAI-1 mRNA was still present in the subacute phase, t-PA, u-PA, and MMP-9 mRNAs were now obvious, with PA gene expression co-localizing with areas of PAI-1 gene expression. In the chronic phase, PAI-1 mRNA was demonstrated around erythrocyte extravasations and surrounding bands of medial degeneration. However, there was little expression of PAs in these areas, and no MMP-9 was detected. Thus, fibrinolytic genes and MMP-9 are differentially expressed during the progression of aortic dissections. The kinetics of expression are consistent with acute fibrinolytic shutdown in response to the initial injury, a secondary subacute phase with active proteolysis, and finally, a chronic hypofibrinolytic state. Extensive neovascularization in the chronic phase may further reduce the physical stability of the dissected wall.
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Aneurisma de la Aorta Abdominal/metabolismo , Disección Aórtica/metabolismo , Colagenasas/metabolismo , Inhibidor 1 de Activador Plasminogénico/metabolismo , Activador de Tejido Plasminógeno/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Enfermedad Aguda , Disección Aórtica/patología , Aneurisma de la Aorta Abdominal/patología , Enfermedad Crónica , Colagenasas/genética , Expresión Génica , Humanos , Inmunohistoquímica , Hibridación in Situ , Metaloproteinasa 9 de la Matriz , Inhibidor 1 de Activador Plasminogénico/genética , ARN Mensajero/metabolismo , Activador de Tejido Plasminógeno/genética , Activador de Plasminógeno de Tipo Uroquinasa/genéticaRESUMEN
Tissue homeostasis depends on spatially and temporally controlled expression of multifunctional adhesive glycoproteins and their cellular counter receptors, and on a tight regulation of proteolytic enzyme systems. The adhesive glycoprotein vitronectin (Vn) not only regulates adhesive events, but also controls a number of these proteolytic enzyme cascades, including the complement, coagulation, and fibrinolytic systems. However, understanding of the biological functions of this molecule is complicated due to it's conformationally lability and its tendency to self-associate. While plasma Vn is monomeric and lacks exposure of conformationally sensitive epitopes, platelet and tissue-associated Vn are believed to be conformationally altered and multimeric. The latter forms express a functional repertoire distinct from plasma Vn. While little Vn immunoreactivity is detectable in most normal tissues, increased depositions of Vn have been observed in areas of tissue injury and necrosis. Tissue Vn was believed to be plasma-derived, but recent studies indicate that extrahepatic cells have the biosynthetic potential to produce Vn and that its synthesis can be regulated under inflammatory conditions. Here, the constitutive and regulated expression of Vn, its locations in tissues and interaction with other matrix molecules are reviewed and their implications for the functions of this molecule are discussed.
Asunto(s)
Vitronectina/metabolismo , Animales , Regulación de la Expresión Génica , Humanos , Distribución Tisular , Vitronectina/análisis , Vitronectina/química , Vitronectina/genéticaRESUMEN
BACKGROUND AND PURPOSE: Species- and model-dependent differences in cell response to focal brain ischemia may underlie differences in adhesion receptor expression. The aim of this study was to quantitatively evaluate the spatial and temporal distribution of dUTP incorporation into damaged DNA, as an indicator of ischemic injury, in the corpus striatum. METHODS: Cerebral ischemia was produced in 16 nonhuman primates and 19 rats by occluding the middle cerebral artery (MCA:O) with reperfusion for various periods. In situ dUTP was incorporated into cells with DNA damage by terminal deoxynucleotidyl transferase (TdT), DNA polymerase I, or the Klenow fragment of DNA polymerase. Dual immunolabeling experiments with immunoprobes against neuronal, vascular, or glial marker proteins were performed. RESULTS: Significant topographical differences in dUTP between the two species were seen. In both models the TdT and polymerase I regions changed characteristically during focal ischemia. The number and density of dUTP-labeled cells increased with time from MCA:O and were dramatically different between the species (2P < .001). By 2 hours of ischemia, the density of dUTP label was 48.8 +/- 10.3 cells/mm2 in the primate and 2.4 +/- 0.8 cells/mm2 in the rat (2P < .05), but these values became nearly identical by 24 hours of reperfusion. In the primate, 80.0 +/- 6.6% of labeled cells displayed microtubule-associated protein-2 antigen (at 2-hour MCA:O), while 1.8 +/- 0.5% were associated with microvessels at 24 hours of reperfusion. CONCLUSIONS: In situ detection of DNA damage, accomplished by three methods, reveals distinct temporal, topographical, and density differences in ischemic injury to cells in the primate and the rat corpus striatum as a result of MCA:O.
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Isquemia Encefálica/genética , Daño del ADN , Animales , Cuerpo Estriado/metabolismo , ADN Nucleotidilexotransferasa/metabolismo , ADN Polimerasa I/metabolismo , Nucleótidos de Desoxiuracil/metabolismo , Masculino , Papio , Ratas , Ratas Wistar , Reperfusión , Especificidad de la EspecieRESUMEN
Vitronectin (Vn) is a major adhesive glycoprotein in blood. However, many of the functions of Vn are regulated by its conformational state and degree of multimerization. Here, the ability of native and denatured Vn to bind to integrin adhesion receptors was compared. Three lines of evidence suggest that the native, plasma form of Vn is not an adhesive glycoprotein. (i) Antibodies that bind in close proximity to the cell adhesion domain of Vn fail to bind to native Vn present in unfractionated plasma. (ii) Denatured Vn binds to both glycoprotein IIb/IIIa and alphavbeta3 in a dose-dependent manner. In contrast, native Vn is unable to bind either integrin. (iii) Thermal denaturation of native Vn, or its complexation with type 1 plasminogen activator inhibitor, exposed the cell adhesion domain of Vn. Thus, while plasma Vn is unable to bind integrins and is not an adhesive glycoprotein, the conformationally altered from of the protein binds avidly to both alphavbeta3 and glycoprotein IIb/IIIa. The data presented here indicate that such conformational changes in Vn are likely to occur in areas of tissue injury and thrombosis.
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Vitronectina/sangre , Anticuerpos Monoclonales/inmunología , Sitios de Unión/inmunología , Adhesión Celular/inmunología , Humanos , Oligopéptidos/metabolismo , Inhibidor 1 de Activador Plasminogénico/metabolismo , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Conformación Proteica , Desnaturalización Proteica , Receptores Inmunológicos/metabolismo , Receptores de Péptidos/metabolismo , Receptores de Vitronectina/metabolismo , Vitronectina/química , Vitronectina/inmunologíaRESUMEN
Plasmin, the primary fibrinolytic enzyme, has a broad substrate spectrum and participates in other biological processes dependent upon proteolytic activity. Consequently, plasmin activity is tightly regulated by plasminogen activators and protease inhibitors. In this study, we examined whether regulation of plasminogen gene expression also might provide a new mechanism for controlling this system. We examined the effects of recombinant human interleukin-6 (rhIL-6), a pleiotropic cytokine, on plasminogen mRNA expression in primary murine hepatocytes and Hep3B human hepatoma cells. In primary hepatocytes, rhIL-6 and hydrocortisone separately increased plasminogen mRNA expression, but hydrocortisone did not markedly enhance the response to rhIL-6. Hep3B hepatoma cells exhibited more modest responses to rhIL-6. We used the polymerase chain reaction to amplify a 1,067-bp fragment of the human plasminogen promoter/5' flanking region. This fragment was cloned upstream of a luciferase reporter gene. Hep3B cells transiently transfected with this construct provided approximately 100-fold higher luciferase activity compared to cells transfected with control plasmids, and luciferase activity was increased approximately 4.5-fold when these cells were treated with rhIL-6. Furthermore, mice injected with rhIL-6 exhibited increases in hepatic plasminogen mRNA. Circulating plasminogen levels were significantly higher in the mice injected with rhIL-6 compared to mice injected with saline. Mice injected with lipopolysaccharide (an inducer of IL-6 in vivo) also showed increased hepatic plasminogen mRNA. Thus, plasminogen gene expression can be modulated by rhIL-6, suggesting a new mechanism for regulating biological systems that use plasmin.
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Regulación de la Expresión Génica/efectos de los fármacos , Interleucina-6/farmacología , Plasminógeno/genética , Animales , Carcinoma Hepatocelular/patología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Lipopolisacáridos/farmacología , Neoplasias Hepáticas/patología , Ratones , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Plasminógeno/biosíntesis , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Neoplásico/biosíntesis , ARN Neoplásico/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes/farmacología , Estimulación Química , Células Tumorales CultivadasRESUMEN
Vitronectin (Vn) circulates in plasma primarily in the native, monomeric form, whereas platelet-associated Vn in conformationally altered and multimeric. Here, we report that denatured Vn specifically binds to L-Arg, whereas the L-Arg binding site is cryptic in the native form of Vn. In addition, combined treatment of disulfide-linked Vn multimers with L-Arg, urea, and reducing agent results in the formation of disperse oligomers with reduced expression of denaturation-sensitive epitopes. These results suggest that L-Arg modulates the partitioning between monomeric and multimeric Vn species and that L-Arg affinity chromatography can be employed to test for exposure of conformationally sensitive binding sites in Vn. The effects of denaturation on the exposure of conformationally sensitive epitopes in the N-terminus of Vn is controversial. Treatment of Vn with reducing agents abolished type 1 plasminogen activator inhibitor and antibody binding to the highly disulfide-linked N-terminal somatomedin B domain (amino acids 1 to 51), whereas epitopes located in the connecting region/first hemopexin-like repeat (amino acids 52 to 239) and the glycosaminoglycan binding domain (amino acids 343-379) were not affected. These observations indicate that appropriate disulfide-linkage of the N-terminal somatomedin B domain is required for ligand binding and that published differences on the effects of denaturation on the expression of binding sites are probably due to the use of reducing agents in the denaturation process.
Asunto(s)
Arginina/metabolismo , Epítopos/metabolismo , Sustancias Reductoras/farmacología , Vitronectina/metabolismo , Anticuerpos Monoclonales/metabolismo , Sitios de Unión de Anticuerpos , Ditiotreitol/farmacología , Electroforesis en Gel de Poliacrilamida , Calor , Humanos , Inhibidor 1 de Activador Plasminogénico/metabolismo , Conformación Proteica , Desnaturalización Proteica , Somatomedinas/metabolismo , Urea/farmacología , Vitronectina/inmunologíaRESUMEN
Vitronectin (Vn) regulates proteolytic enzyme systems, as well as cell migration and tissue remodelling. These processes have been implicated in the pathogenesis of atherosclerosis. In this study, the distribution of Vn antigen in apparently normal and atherosclerotic human blood vessels was evaluated. Normal and diseased vessels showed Vn immunostaining in the lamina elastica interna and externa, and in strand-like structures in the adventitia. In most of these instances, the Vn antigen appeared to be located in the proximity of elastin. In pulmonary arteries, Vn staining was additionally detected in the media. The intima was devoid of Vn antigen in all vessels studied. In general, there was increased deposition of Vn antigen in the atherosclerotic arteries. In particular, strong Vn staining was apparent in amorphous material adjacent to cholesterol clefts and in acellular fibrous tissue, in plaques present in the carotic artery and aorta. Collagen layers and fresh fibrin depositions were devoid of Vn antigen. In spite of the abundance of Vn immunostaining throughout the normal and diseased vessel wall, the Vn transcript was not detectably by in situ hybridization. These results indicate that Vn is a constituent of the normal vessel wall and raise the possibility that increased local deposition of Vn may be related to the development of atherosclerotic vascular disease.
Asunto(s)
Arteriosclerosis/patología , Músculo Liso Vascular/química , Vitronectina/análisis , Humanos , Hibridación in Situ , Músculo Liso Vascular/patología , ARN Mensajero/metabolismo , Vitronectina/genéticaRESUMEN
The ligand binding functions of vitronectin (Vn) are regulated by its conformational state/degree of multimerization. In the native plasma form of Vn, the C-terminal glycosaminoglycan (GAG) binding domain is believed to be cryptic. Here, evidence is provided that the addition of fucoidan or dextran sulfate to unfractionated plasma results in the formation of covalently and non-covalently stabilized Vn multimers. These multimers express conformationally sensitive antibody epitopes and ligand binding sites located in the N terminus of the Vn molecule. While heparin forms complexes with monomeric plasma Vn and induces conformational changes, a reduction in ionic strength is required for induction of multimerization. In addition, heparin serves as a template for the assembly of type 1 plasminogen activator inhibitor-induced disulfide-linked Vn multimers. These results support a new model for the structure of native Vn. The C-terminal GAG binding domain is predicted to be exposed in the native conformation, whereas the N terminus is cryptic. Ligand binding to the GAG binding site unfolds the N terminus, thereby exposing cryptic ligand binding sites.
Asunto(s)
Glicosaminoglicanos/metabolismo , Somatomedinas/metabolismo , Vitronectina/metabolismo , Anticoagulantes/farmacología , Sitios de Unión , Cromatografía de Afinidad , Sulfato de Dextran/farmacología , Electroforesis en Gel de Poliacrilamida , Heparina/metabolismo , Humanos , Peso Molecular , Concentración Osmolar , Polisacáridos/farmacología , Conformación Proteica , Somatomedinas/química , Vitronectina/químicaRESUMEN
The conformation and degree of multimerization of vitronectin (Vn) appears to be of critical importance for its functions, but little is known about the underlying mechanisms that control Vn multimerization. We report that Vn secreted by cultured hepatoma cells is present as a mixture of monomeric and multimeric forms. A single protein of Mr 45,000 co-purified with hepatoma cell-derived Vn, which was immunologically identified as type 1 plasminogen activator inhibitor (PAI-1). The possibility that PAI-1 may modulate Vn multimerization was investigated. The addition of active PAI-1 to unfractionated plasma containing Vn monomers resulted in the formation of covalently and noncovalently associated Vn multimers and expression of conformationally sensitive epitopes. In contrast, inactive forms of PAI-1 did not efficiently induce Vn multimerization and conformational change. Gel filtration analysis revealed that Vn remained multimeric after dissociation from PAI-1. Vn multimers were also assembled using purified monomeric Vn and PAI-1, suggesting that a plasma cofactor was not required to induce Vn multimerization. This study provides insights into physiological mechanism responsible for the generation of homomultimeric Vn, a multimeric form of Vn that is not in complex with other proteins and which expresses a functional repertoire distinct from that of plasma Vn.