RESUMEN
The physiologic activation of the plasma kallikrein-kinin system requires the assembly of its constituents on a cell membrane. High- molecular-weight kininogen (HK) and cleaved HK (HKa) both interact with at least three endothelial cell binding proteins: urokinase plasminogen activator receptor (uPAR), globular C1q receptor (gC1qR,) and cytokeratin 1 (CK1). The affinity of HK and HKa for endothelial cells are KD=7-52 nM. The contribution of each protein is unknown. We examined the direct binding of HK and HKa to the soluble extracellular form of uPAR (suPAR), gC1qR and CK1 using surface plasmon resonance. Each binding protein linked to a CM-5 chip and the association, dissociation and KD (equilibrium constant) were measured. The interaction of HK and HKa with each binding protein was zinc-dependent. The affinity for HK and HKa was gC1qR>CK1>suPAR, indicating that gC1qR is dominant for binding. The affinity for HKa compared to HK was the same for gC1qR, 2.6-fold tighter for CK1 but 53-fold tighter for suPAR. Complex between binding proteins was only observed between gC1qR and CK1 indicating that a binary CK1-gC1qR complex can form independently of kininogen. Although suPAR has the weakest affinity of the three binding proteins, it is the only one that distinguished between HK and HKa. This finding indicates that uPAR may be a key membrane binding protein for differential binding and signalling between the cleaved and uncleaved forms of kininogen. The role of CK1 and gC1qR may be to initially bind HK to the membrane surface before productive cleavage to HKa.
Asunto(s)
Queratinas/metabolismo , Quininógeno de Alto Peso Molecular/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores de Complemento/metabolismo , Receptores del Activador de Plasminógeno Tipo Uroquinasa/metabolismo , Resonancia por Plasmón de Superficie , Coagulación Sanguínea , Endotelio/metabolismo , Humanos , Queratina-1/metabolismo , Queratinas/química , Quininógeno de Alto Peso Molecular/química , Glicoproteínas de Membrana/química , Unión Proteica , Receptores de Complemento/química , Receptores del Activador de Plasminógeno Tipo Uroquinasa/química , Transducción de SeñalRESUMEN
Lipoprotein(a) is composed of low-density lipoprotein linked both covalently and noncovalently to apolipoprotein(a). The structure of lipoprotein(a) and the interactions between low-density lipoprotein and apolipoprotein(a) were investigated by electron microscopy and correlated with analytical ultracentrifugation. Electron microscopy of rotary-shadowed and unidirectionally shadowed lipoprotein(a) prepared without glycerol revealed that it is a nearly spherical particle with no large projections. After extraction of both lipoprotein(a) and low-density lipoprotein with glycerol prior to rotary shadowing, the protein components were observed to consist of a ring of density made up of nodules of different sizes, with apolipoprotein(a) and apolipoprotein B-100 closely associated with each other. However, when lipoprotein(a) was treated with a lysine analogue, 6-aminohexanoic acid, much of the apolipoprotein(a) separated from the apolipoprotein B-100. In 6-aminohexanoic acid-treated preparations without glycerol extraction, lipoprotein(a) particles had an irregular mass of density around the core. In contrast, lipoprotein(a) particles treated with 6-aminohexanoic acid in the presence of glycerol had a long tail, in which individual kringles could be distinguished, extending from the ring of apolipoprotein B-100. The length of the tail was dependent on the particular isoform of apolipoprotein(a). Dissociation of the noncovalent interactions between apolipoprotein(a) and low-density lipoprotein as a result of shear forces or changes in the microenvironment may contribute to selective retention of lipoprotein(a) in the vasculature.
Asunto(s)
Lipoproteína(a)/química , LDL-Colesterol/química , LDL-Colesterol/ultraestructura , Ligandos , Lipoproteína(a)/ultraestructura , Lisina/química , Unión Proteica , Conformación Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/ultraestructura , UltracentrifugaciónRESUMEN
Inflammation may contribute to the pathogenesis of atherosclerosis. On the basis of previous reports that human atherosclerotic lesions contain alpha-defensins, a class of cationic proteins released by activated neutrophils, the study was designed to ask whether defensins modulate the binding and catabolism of low-density lipoprotein (LDL) by human vascular cells. The results of the study demonstrated that defensin stimulated the binding of (125)I-LDL to cultured human umbilical vein endothelial cells, smooth muscle cells, and fibroblasts approximately 5-fold in a dose-dependent and saturable manner. Defensin and LDL formed stable complexes in solution and on cell surfaces. Stimulation of LDL binding by defensin was not inhibited by antibodies against the LDL-receptor (LDL-R), or by recombinant receptor-associated protein, which blocks binding of ligands to the alpha(2)-macroglobulin receptor/LDL-R-related protein and other LDL-R family members. Furthermore, defensin stimulated the binding, endocytosis, and degradation of LDL by fibroblasts lacking LDL-R. Stimulation of LDL degradation by defensin was inhibited approximately 75% by low concentrations of heparin (0.2 units/mL) and was similarly reduced in CHO cells lacking heparan-sulfate-containing proteoglycans. The effect of defensin was substantially increased in cells overexpressing the core protein of the syndecan-1 heparan sulfate proteoglycan. The alpha-defensins released from activated neutrophils may provide a link between inflammation and atherosclerosis by changing the pattern of LDL catabolism from LDL-R to the less efficient LDL-R-independent, proteoglycan-dependent pathway. (Blood. 2000;96:1393-1398)
Asunto(s)
Endotelio Vascular/metabolismo , Lipoproteínas LDL/metabolismo , Músculo Liso Vascular/metabolismo , Proteínas/farmacología , Proteoglicanos/metabolismo , Receptores de LDL/metabolismo , Animales , Arteriosclerosis/etiología , Arteriosclerosis/metabolismo , Proteínas Sanguíneas/metabolismo , Células CHO , Cricetinae , Defensinas , Relación Dosis-Respuesta a Droga , Humanos , Neutrófilos/metabolismo , Proteínas/metabolismoRESUMEN
The role of urokinase-type plasminogen activator (uPA) and its receptor (uPAR) in fibrinolysis remains unsettled. The contribution of uPA may depend on the vascular location, the physical properties of the clot, and its impact on tissue function. To study the contribution of urokinase within the pulmonary microvasculature, a model of pulmonary microembolism in the mouse was developed. Iodine 125 ((125)I)-labeled fibrin microparticles injected intravenously through the tail vein lodged preferentially in the lung, distributing homogeneously throughout the lobes. Clearance of (125)I-microemboli in wild type mice was rapid and essentially complete by 5 hours. In contrast, uPA(-/-) and tissue-type plasminogen activator tPA(-/-) mice, but not uPAR(-/-) mice, showed a marked impairment in pulmonary fibrinolysis throughout the experimental period. The phenotype in the uPA(-/-) mouse was rescued completely by infusion of single chain uPA (scuPA). The increment in clot lysis was 4-fold greater in uPA(-/-) mice infused with the same concentration of scuPA complexed with soluble recombinant uPAR. These data indicate that uPA contributes to endogenous fibrinolysis in the pulmonary vasculature to the same extent as tPA in this model system. Binding of scuPA to its receptor promotes fibrinolytic activity in vivo as well as in vitro. The physical properties of fibrin clots, including size, age, and cellular composition, as well as heterogeneity in endothelial cell function, may modify the participation of uPA in endogenous fibrinolysis. (Blood. 2000;96:1820-1826)
Asunto(s)
Fibrinólisis/fisiología , Circulación Pulmonar/fisiología , Activador de Plasminógeno de Tipo Uroquinasa/fisiología , Animales , Modelos Animales de Enfermedad , Fibrina/análisis , Fibrinógeno/administración & dosificación , Fibrinógeno/farmacocinética , Fibrinólisis/efectos de los fármacos , Humanos , Inmunohistoquímica , Pulmón/química , Pulmón/efectos de los fármacos , Pulmón/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Mutantes , Microesferas , Tamaño de la Partícula , Circulación Pulmonar/efectos de los fármacos , Embolia Pulmonar/metabolismo , Embolia Pulmonar/fisiopatología , Embolia Pulmonar/prevención & control , Receptores de Superficie Celular/genética , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Distribución Tisular , Activador de Plasminógeno de Tipo Uroquinasa/genéticaRESUMEN
We examined the effect of urokinase (uPA) and its fragments on vascular smooth muscle cell contraction. Single-chain uPA inhibits phenylepherine (PE) -induced contraction of rat aortic rings, whereas two-chain uPA exerts the opposite effect. Two independent epitopes mediating these opposing activities were identified. A6, a capped peptide corresponding to amino acids 136-143 (KPSSPPEE) of uPA, increased the EC(50) of PE-induced vascular contraction sevenfold by inhibiting the release of calcium from intracellular stores. A6 activity was abolished by deleting the carboxyl-terminal Glu or by mutating the Ser corresponding to position 138 in uPA to Glu. A single-chain uPA variant lacking amino acids 136-143 did not induce vasorelaxation. A second epitope within the kringle of uPA potentiated PE-induced vasoconstriction. This epitope was exposed when single-chain uPA was converted to a two-chain molecule by plasmin. The isolated uPA kringle augmented vasoconstriction, whereas uPA variant lacking the kringle had no procontractile activity. These studies reveal previously undescribed vasoactive domains within urokinase and its naturally derived fragments.
Asunto(s)
Músculo Liso Vascular/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Activador de Plasminógeno de Tipo Uroquinasa/farmacología , Vasoconstricción/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Aorta Torácica/efectos de los fármacos , Secuencia de Bases , Sitios de Unión , Calcio/metabolismo , Cartilla de ADN/genética , Técnicas In Vitro , Masculino , Contracción Muscular/efectos de los fármacos , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fenilefrina/farmacología , Ratas , Ratas Sprague-Dawley , Activador de Plasminógeno de Tipo Uroquinasa/química , Activador de Plasminógeno de Tipo Uroquinasa/genéticaRESUMEN
The urokinase receptor is composed of three homologous domains based on disulfide spacing. The contribution of each domain to the binding and activation of single chain urokinase (scuPA) remains poorly understood. In the present paper we examined the role of domain II (DII) in these processes. Repositioning DII to the amino or carboxyl terminus of the molecule abolished binding of scuPA as did deleting the domain entirely. By using alanine-scanning mutagenesis, we identified a 9-amino acid continuous sequence in DII (Arg(137)-Arg(145)) required for both activities. Competition-inhibition and surface plasmon resonance studies demonstrated that mutation of Lys(139) and His(143) to alanine in soluble receptor (suPAR) reduced the affinity for scuPA approximately 5-fold due to an increase in the "off rate." Mutation of Arg(137), Arg(142), and Arg(145), each to alanine, leads to an approximately 100-fold decrease in affinity attributable to a 10-fold decrease in the apparent "on rate" and a 6-fold increase in off rate. These differences were confirmed on cells expressing variant urokinase receptor. suPAR-K139A/H143A displayed a 50% reduction in scuPA-mediated plasminogen activation activity, whereas the 3-arginine variant was unable to stimulate scuPA activity at all. Mutation of the three arginines did not affect binding of a decamer peptide antagonist of scuPA known to interact with DI and DIII. However, this mutation abolished both the binding of soluble DI to DII-III in the presence of scuPA and the synergistic activation of scuPA mediated by DI and wild type DII-DIII. These data show that DII is required for high affinity binding of scuPA and its activation. DII does not serve merely as a spacer function but appears to be required for interdomain cooperativity.
Asunto(s)
Receptores de Superficie Celular/química , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Animales , Sitios de Unión , Células CHO , Cricetinae , Mutación , Receptores de Superficie Celular/metabolismo , Receptores del Activador de Plasminógeno Tipo UroquinasaRESUMEN
Urokinase-type plasminogen activator (uPA) binds to its receptor (uPAR) with a K(d) of about 1 nm. The catalytic activity of the complex is apparent at uPA concentrations close to K(d). Other functions of the complex, such as signal transduction, are apparent at much higher concentrations (35-60 nm). In the present study, we show that uPA and recombinant soluble uPAR (suPAR), at concentrations that exceed the K(d) and the theoretical saturation levels (10-80 nm), establish novel interactions that lead to a further increase in the activity of the single-chain uPA (scuPA)/suPAR and two-chain uPA (tcuPA)/suPAR complexes. Experiments performed using dynamic light scattering, gel filtration, and electron microscopy techniques indicate that suPAR forms dimers and oligomers. The three techniques provide evidence that the addition of an equimolar concentration of scuPA leads to the dissociation of these dimers and oligomers. Biacore data show that suPAR dimers and oligomers bind scuPA with decreased affinity when compared with monomers. We postulate that uPAR is present in equilibrium between oligomer/dimer/monomer forms. The binding of uPA to suPAR dimers and oligomers occurs with lower affinity than the binding to monomer. These novel interactions regulate the activity of the resultant complexes and may be involved in uPA/uPAR mediated signal transduction.
Asunto(s)
Receptores de Superficie Celular/química , Receptores de Superficie Celular/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/química , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Fibrinólisis , Humanos , Cinética , Luz , Microscopía Electrónica , Plasminógeno/metabolismo , Receptores de Superficie Celular/ultraestructura , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestructura , Dispersión de Radiación , Trombina/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/ultraestructuraRESUMEN
Retention of lipoproteins within the vasculature is a central event in the pathogenesis of atherosclerosis. However, the signals that mediate this process are only partially understood. Prompted by putative links between inflammation and atherosclerosis, we previously reported that alpha-defensins released by neutrophils are present in human atherosclerotic lesions and promote the binding of lipoprotein(a) [Lp(a)] to vascular cells without a concomitant increase in degradation. We have now tested the hypothesis that this accumulation results from the propensity of defensin to form stable complexes with Lp(a) that divert the lipoprotein from its normal cellular degradative pathways to the extracellular matrix (ECM). In accord with this hypothesis, defensin stimulated the binding of Lp(a) to vascular matrices approximately 40-fold and binding of the reactants to the matrix was essentially irreversible. Defensin formed stable, multivalent complexes with Lp(a) and with its components, apoprotein (a) and low-density lipoprotein (LDL), as assessed by optical biosensor analysis, gel filtration, and immunoelectron microscopy. Binding of defensin/Lp(a) complexes to matrix was inhibited (>90%) by heparin and by antibodies to fibronectin (>70%), but not by antibodies to vitronectin or thrombospondin. Defensin increased the binding of Lp(a) (10 nmol/L) to purified fibronectin more than 30-fold. Whereas defensin and Lp(a) readily traversed the endothelial cell membranes individually, defensin/Lp(a) complexes lodged on the cell surface. These studies demonstrate that alpha-defensins released from activated or senescent neutrophils stimulate the binding of an atherogenic lipoprotein to the ECM of endothelial cells, a process that may contribute to lipoprotein accumulation in atherosclerotic lesions.
Asunto(s)
Endotelio Vascular/fisiología , Matriz Extracelular/fisiología , Lipoproteína(a)/sangre , Proteínas/fisiología , Apolipoproteínas/sangre , Apoproteína(a) , Técnicas Biosensibles , Proteínas Sanguíneas/fisiología , Proteínas Sanguíneas/ultraestructura , Células Cultivadas , Defensinas , Matriz Extracelular/ultraestructura , Humanos , Cinética , Lipoproteína(a)/ultraestructura , Lipoproteínas LDL/sangre , Microscopía Inmunoelectrónica , Unión Proteica , Proteínas/ultraestructura , Venas UmbilicalesRESUMEN
Single-chain urokinase plasminogen activator (scuPA), the unique form secreted by cells, expresses little intrinsic plasminogen activator activity. scuPA can be activated by proteolytic cleavage to form a two-chain enzyme (tcuPA), which is susceptible to inhibition by plasminogen activator inhibitor type I (PAI-1). scuPA is also activated when it binds to its cellular receptor (uPAR), in which case the protein remains as a single chain molecule with less susceptibility to PAIs. Fibrin clots are invested with PAI-1 derived from plasma and from activated platelets. Therefore, we compared the fibrinolytic activity of complexes between scuPA and recombinant soluble uPAR (suPAR) to that of scuPA, tcuPA, and tcuPA/suPAR complexes. scuPA/suPAR complexes mediated the lysis of plasma-derived fibrin clots 14-fold more extensively than did equimolar concentrations of scuPA and threefold more extensively than did tcuPA or tcuPA/suPAR, respectively. The enhanced catalytic activity of scuPA/suPAR required that all three domains of the receptor be present, correlated with its PAI-1 resistance, was not dependent on fibrin alone, and required a plasma cofactor that was identified as IgG. Human IgG bound specifically to suPAR and scuPA/suPAR as determined by using affinity chromatography and immunoprecipitation. Plasma depleted of IgG lost most of its capacity to promote the fibrinolytic activity of scuPA/suPAR, and the activity of the complex was restored by adding plasma concentrations of purified IgG. These studies indicate that scuPA/suPAR can function as a plasminogen activator in a physiological milieu.
Asunto(s)
Coagulación Sanguínea/fisiología , Fibrinólisis , Receptores de Superficie Celular/fisiología , Activador de Plasminógeno de Tipo Uroquinasa/fisiología , Secuencia de Aminoácidos , Coagulación Sanguínea/efectos de los fármacos , Coagulación Sanguínea/genética , Fibrinólisis/efectos de los fármacos , Fibrinólisis/genética , Humanos , Inmunoglobulina G/aislamiento & purificación , Inmunoglobulina G/fisiología , Cadenas Pesadas de Inmunoglobulina/aislamiento & purificación , Región Variable de Inmunoglobulina/aislamiento & purificación , Región Variable de Inmunoglobulina/fisiología , Datos de Secuencia Molecular , Receptores de Superficie Celular/genética , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Proteínas Recombinantes/farmacología , Solubilidad , Activador de Plasminógeno de Tipo Uroquinasa/genéticaRESUMEN
There is evidence to suggest that elevated plasma levels of lipoprotein (a) [Lp(a)] represent a risk factor for the development of atherosclerotic vascular disease, but the mechanism by which this lipoprotein localizes to involved vessels is only partially understood. In view of studies suggesting a link between inflammation and atherosclerosis and our previous finding that leukocyte defensin modulates the interaction of plasminogen and tissue-type plasminogen activator with cultured human endothelial cells, we examined the effect of this peptide on the binding of Lp(a) to cultured vascular endothelium and vascular smooth muscle cells. Defensin increased the binding of Lp(a) to endothelial cells approximately fourfold and to smooth muscle cells approximately sixfold. Defensin caused a comparable increase in the amount of Lp(a) internalized by each cell type, but Lp(a) internalized as a consequence of defensin being present was not degraded, resulting in a marked increase in the total amount of cell-associated lipoprotein. Abundant defensin was found in endothelium and in intimal smooth muscle cells of atherosclerotic human cerebral arteries, regions also invested with Lp(a). These studies suggest that defensin released from activated or senescent neutrophils may contribute to the localization and persistence of Lp(a) in human vessels and thereby predispose to the development of atherosclerosis.