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PubMed1993

Immunohistochemical localization of C3d fragment of complement and S-protein (vitronectin) in normal and diseased human kidneys: association with the C5b-9 complex and vitronectin receptor.

The localization of C3d, a fragment produced by C3 activation and S-protein (vitronectin), a regulatory factor of C5b-9, was studied immunohistochemically in normal human kidney and renal biopsies from patients with several types of glomerulonephritis. Immunofluorescent staining of the normal kidneys showed that C3d was present along the glomerular basement membrane (GBM), tubular basement membrane (TBM) and arterioles, and that S-protein was present in the GBM, mesangium, TBM, and arterioles. Immunoelectron microscopy of isolated basement membranes showed that C3d was localized exclusively on the epithelial side of the GBM, and that S-protein was present along both the epithelial and endothelial sides. In nephritic tissues, glomerular staining of C3d, C5b-9, and S-protein was increased when compared with that in normal tissues. S-protein, frequently co-localized with C3d and C5b-9 neoantigen, was intensely positive in the immune deposits of glomerular capillaries and the mesangial area, overlapping the background staining of GBM and mesangial matrix. S-protein and its receptor were occasionally co-localized in the glomeruli. These findings indicate that C3d and S-protein are normally present in the glomeruli. Co-staining of C3d, C5b-9 neoantigen, and S-protein within the immune deposits of nephritic kidneys suggests in situ binding of S-protein to locally-formed C5b-9 complex, or merely co-distribution of S-protein with the complex, rather than trapping of large molecular SC5b-9 complex from the circulation.

Complement C3d↗
PubMed1995

Hepatocellular localisation of biosynthesis of vitronectin. Characterisation of the primary structure of rat vitronectin.

To characterize the primary structure of rat Vn, a cDNA library constructed from freshly isolated rat hepatocytes, was screened with a human Vn cDNA probe. Comparison of the sequence of the obtained rat cDNA clone with the sequences of human, mouse and rabbit Vn cDNA's showed predominantly consensus in the somatomedin B domain, the RGD-sequence and its flanking regions, in the first hemopexin type domain and at the carboxyl terminal part of Vn, the heparin binding site. To specify the liver cell type involved in the biosynthesis of Vn, we used a competitive PCR-assay to discriminate between expression levels. We found that expression of Vn in hepatocytes is at least 1000-fold higher than in Kupffer cells and 3000-fold higher than in endothelial liver cells.

Amino Acid Sequence↗
PubMed1991

Vitronectin: effects on keratinocyte motility and inhibition of collagen-induced motility.

Epibolin, a plasma protein, was initially purified on the basis of its ability to enhance spreading of keratinocytes. It is now known that epibolin is identical to serum spreading factor, S protein, and vitronectin, and the current name for the protein is vitronectin. Studies of vitronectin on cultured keratinocytes showed that it caused spreading and epiboly but not cellular adhesion to the substratum. In studies with other types of cells, vitronectin increased migration of several types of cells in a Boyden chamber. Because some agents that enhance spreading and adhesion, such as collagen and fibronectin, also increase motility, we tested whether vitronectin increased motility of keratinocytes. By photographing and quantitating motility of keratinocytes plated on a bed of colloidal gold particles, we determined that vitronectin increased local movement of keratinocytes in a concentration-dependent fashion, resulting in clearing of gold particles in a circular pattern around the cells, but did not cause the production of tracks found in cultures plated on collagen or fibronectin. The small increases in clearing of the gold particles that occurred in the presence of vitronectin were abolished by antibody to vitronectin. Furthermore, the marked increase in motility produced by type I collagen was significantly reduced when the keratinocytes were treated with vitronectin. Antibody to vitronectin also abrogated the vitronectin-induced reduction in collagen-stimulated motility, confirming that this action was specific for vitronectin. Serum, which contains vitronectin, stimulated motility in a fashion identical to purified vitronectin, but serum lacking vitronectin was inactive. These studies show that vitronectin causes a localized increase in movement associated with spreading resulting in a halo around individual cells, that vitronectin does not enhance directional motility of keratinocytes in this assay but in contrast antagonizes such motility produced by collagen, and that vitronectin is the factor in serum responsible for this effect. The findings with vitronectin and collagen show that these agents stimulate different types of motility. The roles in wound healing of agents stimulating different types of motility are unclear and require further study.

Blood Physiological Phenomena↗
PubMed1998

The alphavbeta5 integrin functions as an endocytic receptor for vitronectin.

Endocytosis and degradation of vitronectin by human skin fibroblasts are regulated by the beta5 integrin. To determine whether the beta5 integrin is directly mediating the internalization of vitronectin, both vitronectin and the beta5 integrin were localized by indirect immunofluorescence during the endocytic process. This analysis showed that both vitronectin and beta5 were found in intracellular vesicles within 5 minutes of the addition of exogenous vitronectin to fibroblast cell layers. By 15 minutes, approximately 20% of the vitronectin-containing vesicles stained positively for beta5. In contrast, the beta3 integrin was not found in any intracellular vesicles. Within 30 minutes, more than 50% of vitronectin-containing vesicles also stained for lamp-1, indicating that internalized vitronectin traveled to lysosomes. Inhibition of clathrin assembly by either potassium depletion or hypertonic buffer inhibited vitronectin internalization, suggesting that vitronectin internalization occurred through coated pits. Confocal analysis confirmed the colocalization of vitronectin and alphavbeta5 in intracellular compartments and further demonstrated that the highest colocalization of the two proteins occurred within 1.8 microm from the ventral surface of the cell, suggesting endocytosis occurred at the substrate level. Pretreatment of cells with the PI-3 kinase inhibitor, wortmannin, resulted in a marked increase in the coincidence of vitronectin and beta5 staining within vesicles and prevented the accumulation of vitronectin within lysosomes. This suggests that following internalization, vitronectin and the alphavbeta5 integrin are segregated to different cellular compartments. This study provides the first evidence that the alphavbeta5 vitronectin receptor directly mediates the internalization of vitronectin.

Androstadienes↗
PubMed1993

Receptor-mediated endocytosis of vitronectin is regulated by its conformational state.

Vitronectin is a structurally labile molecule with a native, non-heparin binding form and a conformationally altered, heparin binding form. To understand the physiological significance of the two conformers of vitronectin, we examined the metabolism of both conformers by cultured human skin fibroblasts. Both native and altered vitronectin bound to confluent fibroblast monolayers. Both conformers of vitronectin competed equally well for the binding of altered vitronectin to the cell layer, suggesting that both conformers bound to the same site in the cell layer. In contrast, 125I-altered vitronectin, but not 125I-native vitronectin, was degraded to trichloroacetic acid-soluble radioactivity by the fibroblast monolayer. Degradation of vitronectin was saturable, sensitive to chloroquine, and occurred intracellularly, suggesting that vitronectin was degraded through a lysosomal pathway. Heparin and thrombospondin inhibited the degradation of altered vitronectin. The degradation of native vitronectin was induced by addition of gamma-thrombin which exposes vitronectin's cryptic heparin-binding domain. These studies suggest that the heparin-binding domain in vitronectin is required for the clearance of vitronectin from the matrix. In addition, these data demonstrate that the conformation of vitronectin regulates its half-life in the matrix. These studies provide the first evidence for a distinct function for the conformers of vitronectin.

Blood Proteins↗
PubMed1996

Heparan sulfate proteoglycans function in the binding and degradation of vitronectin by fibroblast monolayers.

Vitronectin, a 75-kDa plasma protein is also found in the extracellular matrix, where it is believed to promote cell adhesion and migration. In addition to its role in adhesion, matrix vitronectin is also believed to function as an opsonin promoting the clearance of thrombin-serpin complexes from the matrix. Vitronectin is cleared from the matrix by receptor-mediated endocytosis followed by lysosomal degradation, suggesting that cells can regulate the levels of vitronectin present in the matrix. However, the mechanism by which plasma vitronectin associates with the extracellular matrix remains unclear. Studies were conducted to define the binding site(s) for vitronectin in fibroblast cell layers. Sodium chlorate, a competitive inhibitor of proteoglycan sulfation, produced a dose-dependent decrease in both binding and degradation of vitronectin. This inhibition was reversible in that removal of chlorate returned both binding and degradation of vitronectin to near control levels within 24 h. The binding of vitronectin to cell layers was not dependent on cells because vitronectin bound directly to isolated matrix. Isolated matrices prepared from cell layers treated with sodium chlorate also exhibited a dose-dependent decrease in vitronectin binding, consistent with the binding site for vitronectin in the matrix being sulfated proteoglycans. Binding and degradation of vitronectin were also sensitive to the addition of exogenous heparin, suggesting that the heparin binding domain of vitronectin was mediating binding to the matrix. Incubating fibroblast monolayers with heparinase III resulted in a 40% decrease in binding and degradation of vitronectin. Taken together, the above findings suggest that vitronectin's binding to the matrix and its subsequent degradation are dependent on heparan sulfate proteoglycans.

Binding Sites↗
PubMed1995

Specific binding of urinary-type plasminogen activator (u-PA) to vitronectin and its role in mediating u-PA-dependent adhesion of U937 cells.

The present paper described interactions of urinary-type plasminogen activator (u-PA) with isolated protein components of the extracellular matrix (ECM) using kinetic and ligand-blotting analyses, as well as adhesion studies with u-PA-saturated U937 monocytic cells. Kinetic analyses showed that fibronectin and laminin were moderately effective at decreasing activation of plasminogen by u-PA (3-4-fold decrease in kcat/Km), while activation was stimulated slightly by collagen types I and IV (2-4-fold increase in kcat/Km). Ligand-blotting experiments using intact immobilized ECM proteins demonstrated that u-PA binds predominantly to vitronectin. This was supported by ELISA studies, which showed concentration dependent, saturable, reversible binding of u-PA to vitronectin (Kd,app. of 97 nM). Limited proteolysis of vitronectin followed by ligand-blotting analysis demonstrated u-PA binding to a specific vitronectin fragment (M(r) 49,000), and binding was shown to occur through the N-terminal fragment of u-PA. N-terminal sequence analysis indicated that this binding fragment of vitronectin originates with Thr-122 and comprises the hemopexin domain, including the heparin-binding region of the vitronectin molecule. Plasminogen activator inhibitor type I did not compete with u-PA for binding to vitronectin, suggesting both molecules may co-localize on vitronectin. In contrast, binding of u-PA to vitronectin was significantly inhibited by plasminogen, suggesting these molecules share a common binding site on vitronectin. In addition to in vitro studies, experiments were performed to assess the contribution of direct binding of u-PA to vitronectin on the adhesive behaviour of U937 cells. Binding of u-PA-saturated U937 cells to vitronectin was inhibited 66% by excess vitronectin, suggesting that direct binding of u-PA to vitronectin is the mechanism by which u-PA-dependent adhesion of U937 cells to vitronectin is mediated.

Amino Acid Sequence↗
PubMed1998

Degradation of distinct forms of multimeric vitronectin by human fibroblasts.

The plasma protein vitronectin is thought to be an important regulator of extravascular plasminogen activation. In previous studies we have shown that a disulfide stabilized multimeric form of vitronectin is endocytosed and degraded by fibroblast cells (T.S. Panetti, P.J. McKeown-Longo, J. Biol. Chem. 268 (1993) 11988-11993; P.J. McKeown-Longo, T.S. Panetti, in: K.T. Preissner, S. Rosenblatt, C. Kost, J. Wegerhoff, D.F. Mosher (Eds.), Biology of Vitronectins and their Receptors, Elsevier Science Publishers, Amsterdam, 1993, pp. 111-118). The preparation of multimeric vitronectin used in these earlier studies was in the form of high molecular weight disulfide-bonded aggregates which were stable in sodium dodecyl sulfate (SDS). To address the question of whether vitronectin needed to be in the form of disulfide stabilized multimers in order to be endocytosed, a multimeric vitronectin, which was not disulfide stabilized, was prepared from vitronectin that had been treated with reducing agent and alkylated with iodoacetamide. The resulting protein migrated as a 65/75 kDa protein on SDS gels in the absence of reducing agent, confirming that this form of vitronectin was no longer stabilized into disulfide-bonded aggregates. However, the protein was still multimeric when analyzed by native gels and could be converted to SDS stable multimers by cross-linking agents. This result demonstrated that reduced and alkylated vitronectin aggregates into multimeric forms which are not stable in SDS. Similar to disulfide stabilized multimers, alkylated multimers of vitronectin bound to sulfated proteoglycans in the extracellular matrix and were endocytosed and degraded. Degradation of both forms of vitronectin was inhibited with arginine-glycine-aspartic acid peptides, an anti-alphavbeta5 antibody and heparin. Chloroquine and wortmannin were also able to inhibit degradation of both forms of vitronectin, indicating that both multimeric forms were following the same endocytic and degradative pathway. These results suggest that the organization of vitronectin into a multimeric form which will be recognized for endocytosis does not require disulfide bond stabilization. This study further suggests that recognition of vitronectin for endocytosis is dependent upon its conversion from a monomeric to a multivalent form (C.E. Wilkins-Port, P.J. McKeown-Longo, Mol. Biol. Cell 8:S:64A (1997).

Alkylation↗
PubMed1992

Role of vitronectin in embryonic rat endocardial cell migration in vitro.

Vitronectin is one of the extracellular matrices that mediate cell spreading and attachment in vitro. In the present paper, we demonstrate the involvement of vitronectin in the migration of cushion mesenchymal cells of the embryonic rat heart. Immunohistochemistry established the localization of vitronectin in the myocardial cells and in some of the cushion mesenchymal cells of the truncus arteriosus and atrioventricular canal. In vitro, vitronectin, fibronectin, and collagen type-I revealed significant stimulating activity for cushion mesenchymal cell migration. The distance migrated by cushion mesenchymal cells cultured on vitronectin, collagen type-I, or both vitronectin and fibronectin was similar, but that on fibronectin was significantly shorter. Following the addition of anti-vitronectin IgG to the medium, the migration distance of cushion mesenchymal cells on fibronectin was remarkably increased. Most explants on vitronectin or on both vitronectin and fibronectin became detached from dishes after the addition of the antivitronectin antibody. Immunostaining revealed that cushion mesenchymal cells cultured on substrata other than vitronectin synthesized vitronectin. From these results, it is suggested that vitronectin is synthesized by myocardial cells and some cushion mesenchymal cells, and that vitronectin inhibits cell movement on fibronectin. This feature of vitronectin may be important in the regulation of the migration of cushion mesenchymal cell in vivo.

Animals↗
PubMed1998

Vitronectin binds to the gonococcal adhesin OpaA through a glycosaminoglycan molecular bridge.

Several bacterial pathogens including Neisseria gonorrhoeae bind the human serum glycoprotein vitronectin. We aimed at defining the gonococcal receptor for vitronectin. Ligand blots demonstrated that vitronectin bound specifically to the heparin-binding outer-membrane protein OpaA, but that coating OpaA with the sulphated polysaccharide heparin was required for the interaction to occur. Bound vitronectin could be dissociated from OpaA-heparin-vitronectin complexes by the addition of excess heparin, indicating that sulphated polysaccharides provided the main linkage between the two proteins. Binding assays with intact micro-organisms substantiated the requirement of sulphated polysaccharides such as heparin and dextran sulphate for the efficient binding of vitronectin to OpaA+ gonococci. This was underscored by the increased binding of vitronectin to gonococci that had been preincubated with saturating concentrations of dextran sulphate, as opposed to the inhibition of vitronectin binding observed when bacteria were incubated simultaneously with vitronectin and saturating concentrations of dextran sulphate. Binding assays with dextran sulphates of various sizes indicated that vitronectin binding correlated with the size of the polysaccharide rather than with the amount of OpaA produced by the bacteria. The inability of zero-length cross-linking agents to couple vitronectin to OpaA provided further evidence that sulphated polysaccharides formed the linkage between vitronectin and OpaA. Infection experiments demonstrated that proteoglycan-deficient Chinese hamster ovary cells efficiently internalized dextran sulphate/vitronectin-coated gonococci, suggesting that soluble sulphated polysaccharides could substitute for cell surface glycosaminoglycans in the internalization process. On the basis of our results, we propose a novel mechanism of vitronectin binding in which sulphated polysaccharides act as molecular bridges, linking the glycosaminoglycan-binding sites of vitronectin and gonococcal OpaA.

Animals↗
PubMed1990

Specific interaction of vitronectin with the cell-secreted protease inhibitor glia-derived nexin and its thrombin complex.

Interaction of vitronectin with glia-derived nexin (GDN), thrombin, and the complex GDN-thrombin was demonstrated in direct binding assays that indicated the formation of binary and ternary complexes. The concentration of vitronectin necessary to obtain 50% saturation of the immobilized GDN-thrombin complex binding sites (EC50) was about 1 nM. Under similar experimental conditions, the EC50 of vitronectin for the immobilized antithrombin-III-thrombin complex was about fivefold higher. A tight complex was also formed between vitronectin and immobilized GDN (EC50 approximately 1.5 nM) but when vitronectin was immobilized, GDN displayed a reduced affinity for vitronectin (EC50 approximately 10 nM). These results suggest differences between the immobilized and free conformations of GDN and/or vitronectin. In contrast, vitronectin displayed negligible affinity for antithrombin III. Biotinylated GDN was used to characterize further the binding of GDN or the GDN-thrombin complex to vitronectin. The interaction of the biotinylated GDN-thrombin complex with immobilized vitronectin (EC50 approximately 2 nM) was completely blocked by nonbiotinylated complexes of thrombin with either GDN or antithrombin III, whereas free GDN, free thrombin and the GDN-trypsin complex were only weak competitors. Active-site-blocked urokinase and the complex GDN-urokinase also strongly competed for binding of the biotinylated GDN-thrombin complex to vitronectin. Binding of biotinylated GDN to immobilized vitronectin was specific, saturable and was competed with decreasing efficiency by the GDN-thrombin complex, free GDN and free antithrombin III. These interactions between the adhesive component vitronectin and the serine protease inhibitor GDN may relate to localized control of thrombin and/or urokinase action at certain extravascular sites. These results are discussed in terms of binding sites for vitronectin on GDN, thrombin, and the GDN-thrombin complex.

Amino Acid Sequence↗
PubMed1991

Enhancement of vitronectin expression in human HepG2 hepatoma cells by transforming growth factor-beta 1.

Liver cells are considered the principal source of plasma vitronectin. The human hepatoma cell line HepG2 produces vitronectin into its culture medium. In the current work we have analyzed the regulation of vitronectin by transforming growth factor-beta 1 (TGF beta 1) in this hepatoma cell line by Northern hybridization, polypeptide and immunoprecipitation analyses and compared the response to another TGF beta-regulated gene, plasminogen activator inhibitor (PAI-1). Rabbit antibodies raised against human plasma-derived vitronectin were used in immunodetection. Polypeptide and immunoprecipitation analyses of the medium and cells, as well as immunoblotting analysis of the cells and their extracellular matrices, indicated enhanced TGF beta 1-induced production and extracellular deposition of vitronectin. Accordingly, TGF beta 1 enhanced the expression of vitronectin mRNA at picomolar concentrations (2-20 ng/ml) as shown by Northern hybridization analysis. Comparison of the temporal TGF beta induction profiles of vitronectin and PAI-1 mRNAs showed that vitronectin was induced more slowly but the vitronectin mRNAs persisted longer. In addition, platelet-derived and epidermal growth factors had an effect on vitronectin expression, but it was of lower magnitude. TGF beta 1 enhanced the expression of PAI-1 but, unlike previous reports, epidermal growth factor did not have any notable effect on PAI-1 in these cells. The results indicate that TGF beta 1 is an efficient regulator of the production of vitronectin by HepG2 cells and that PAI-1 and vitronectin are not coordinately regulated. In addition, with affinity purified antibodies to vitronectin receptor, we observed strong enhancement of the alpha subunit of the receptor in response to TGF beta 1. These effects of TGF beta are probably involved in various processes of the liver where matrix induction and controlled pericellular proteolysis is needed, as in tissue repair.

Blood Proteins↗
PubMed1994

Identification of a PAI-1 binding site in vitronectin.

Active PAI-1 (plasminogen activator inhibitor 1) is bound to vitronectin in plasma and in the extracellular matrix. In this study we aimed at identifying the PAI-1 binding site in vitronectin, which at present is a matter of dispute. Vitronectin was cleaved with trypsin and the fragments were tested for inhibitory effect on the PAI-1/vitronectin interaction using vitronectin-coated microtiter plates. Intact vitronectin and the tryptic digest of vitronectin both caused a 50% reduction in PAI-1 binding at a concentration of about 2 nmol/I. Gel-filtration on Sephadex G-50 superfine of the tryptic peptides resulted in one main peak of inhibitory activity. The elution volume, Kav, was 0.55 indicating (a) medium-size peptide(s). The peptide was further purified by reverse-phase HPLC. Structural analysis revealed that it constituted the 45 NH2-terminal amino-acid residues in vitronectin. The NH2-terminal vitronectin peptide caused a 50% decrease in PAI-1 binding to the vitronectin-coated microtiter plates at a concentration of about 13 nmol/l. Thus, the peptide is a little less effective in this respect than intact vitronectin. Reduced and S-carboxymethylated peptide had no effect on the interaction. The NH2-terminal vitronectin fragment increased the stability of active PAI-1 by about 60%, which is a little less than with intact vitronectin. The peptide also prevented PAI-1 from oxidation with chloramine T. The half-life was prolonged about 4-fold as compared to about 30-fold with intact vitronectin.

Amino Acid Sequence↗
PubMed1991

Adhesive properties of the beta 3 integrins: comparison of GP IIb-IIIa and the vitronectin receptor individually expressed in human melanoma cells.

Glycoprotein IIb-IIIa (alpha IIb beta 3) and the vitronectin receptor (alpha v beta 3), two integrins that share the common beta 3 subunit, have been reported to function as promiscuous receptors for the RGD-containing adhesive proteins fibrinogen, vitronectin, fibronectin, von Willebrand factor, and thrombospondin. The present study was designed to establish a cell system for the expression of either GP IIb-IIIa or the vitronectin receptor in an otherwise identical cellular environment and to compare the adhesive properties of these two integrins with those of native GP IIb-IIIa and the vitronectin receptor constitutively expressed in HEL cells or platelets. M21 human melanoma cells lack GP IIb-IIIa and use the vitronectin receptor to attach to vitronectin, fibrinogen, fibronectin, and von Willebrand factor. To study the functional properties of GP IIb-IIIa in these cells, we transfected GP IIb into M21-L cells, a variant of M21 cells (Cheresh, D.A., and R.C. Spiro. 1987. J. Biol. Chem. 262:17703-17711), which lack the expression of functional alpha v and are therefore unable to attach to vitronectin, fibrinogen, and von Willebrand factor. Transfectants expressing GP IIb were isolated by immunomagnetic beads and surface expression of the GP IIb-IIIa complex was documented by FACS analysis and immunoprecipitation experiments performed with 125I-labeled M21-L/GP IIb cells. Comparative functional studies demonstrated that GP IIb-IIIa expressed in M21-L/GPIIb cells as well as native GP IIb-IIIa constitutively expressed in HEL-5J20 cells (an HEL variant lacking alpha v beta 3) mediated cell attachment to immobilized fibrinogen, but not to vitronectin or von Willebrand factor, whereas the vitronectin receptor expressed in M21 cells and HEL-AD1 cells (an HEL variant expressing alpha v beta 3) mediated cell attachment to fibrinogen, vitronectin, and von Willebrand factor. Similarly, PGl2-treated resting platelets attached to immobilized fibrinogen but not to vitronectin or von Willebrand factor, and this attachment could be inhibited by mAb A2A9 (directed against a functional site on the GP IIb-IIIa complex). However, in contrast to platelets, which adhered to vitronectin and von Willebrand factor after stimulation by thrombin or PMA, activation of the protein kinase C pathway in M21-L/GP IIb or HEL cells did not induce cell adhesion to vitronectin or von Willebrand factor.(ABSTRACT TRUNCATED AT 400 WORDS)

Blood Platelets↗
PubMed2004

Regulation of the hyperpolarization-activated cationic current Ih in mouse hippocampal pyramidal neurones by vitronectin, a component of extracellular matrix.

Because the hyperpolarization-activated cation-selective current I(h) makes important contributions to neural excitability, we examined its long-term regulation by vitronectin, an extracellular matrix component commonly elevated at injury sites and detected immunochemically in activated microglia. Focusing on mouse hippocampal pyramidal neurones in organotypic slice cultures established at postnatal day 0 or 1 and examined after 3-4 days in vitro, we observed differences in the amplitude and activation rate of I(h) between neurones in naive and vitronectin-exposed slices (10 microg ml(-1) added to serum-free medium), and between neurones in slices derived from wild-type and vitronectin-deficient mice. The potassium inward rectifier I(K(ir)), activated at similar voltages to I(h), was not affected by vitronectin. In CA1, differences in I(h) amplitude primarily reflected changes in maximum conductance (G(max)): a 23.3% increase to 3.18 +/- 0.64 nS from 2.58 +/- 0.96 nS (P < 0.05) in vitronectin-exposed neurones, and a 17.9% decrease to 2.24 +/- 0.26 nS from 2.73 +/- 0.64 nS (P < 0.05) in neurones from vitronectin-deficient slices. The voltage of one-half maximum activation (V(1/2)) was not significantly affected by vitronectin exposure (-78.1 +/- 2.3 mV versus -80.0 +/- 4.9 mV in naive neurones; P > 0.05) or vitronectin deficiency (-83.8 +/- 3.1 mV versus -82.0 +/- 2.9 mV in wild-type neurones; P > 0.05). In CA3 neurones, changes in I(h) reflected differences in both G(max) and V(1/2): in vitronectin-exposed neurones there was a 35.4% increase in G(max) to 1.30 +/- 0.49 nS from 0.96 +/- 0.26 nS (P < 0.01), and a +3.0 mV shift in V(1/2) to -89.8 mV from -92.8 mV (P < 0.05). The time course of I(h) activation could be fitted by the sum of two exponential functions, fast and slow. In both CA1 and CA3 neurones the fast component amplitude was preferentially sensitive to vitronectin, with its relatively larger contribution to total current in vitronectin-exposed cells contributing to the acceleration of I(h) activation. Further, HCN1 immunoreactivity appeared elevated in vitronectin-exposed slices, while HCN2 levels appeared unaltered. We suggest that vitronectin-stimulated increases in I(h) may potentially affect excitability under pathological conditions.

Animals↗
PubMed1991

Vitronectin in bronchoalveolar lavage fluid is increased in patients with interstitial lung disease.

Vitronectin, also known as S-protein, is a 75,000-dalton serum glycoprotein that has a variety of functions, including the capacity to interact with the terminal components of the complement cascade, the coagulation system, and cell surfaces. By virtue of its ability to interact with cells, vitronectin is capable of mediating cell-spreading and adhesion and may also influence cell differentiation and cell growth. To investigate the possibility that vitronectin might contribute to the pathogenesis of interstitial lung disease, vitronectin was measured in bronchoalveolar lavage fluid from patients with sarcoidosis, idiopathic pulmonary fibrosis, and, for comparison, normal volunteers. Vitronectin was detected in lavage fluid and serum of all study subjects. Increased lavage fluid concentrations were found in patients with interstitial lung disease when compared with normal subjects (p less than 0.005), and glucocorticoid-treated patients with interstitial lung disease had lower vitronectin levels than did untreated patients. Furthermore, on SDS-PAGE and Western blot analysis lavage fluid vitronectin comigrated with serum vitronectin, suggesting similar molecular size. Thus, vitronectin is a normal constituent of the epithelial lining fluid, and lavage fluid vitronectin is similar to serum vitronectin. The increase of vitronectin concentrations in the epithelial lining fluid of patients with interstitial lung disease suggests that vitronectin may contribute to the pathogenesis of interstitial lung disease.

Blood Proteins↗
PubMed1986

Interaction of vitronectin with collagen.

Purified human plasma vitronectin was demonstrated to bind to type I collagen immobilized on plastic as measured by enzyme-linked immunosorbent assay and by binding of 125I-radiolabeled vitronectin to a collagen-coated plastic surface. Vitronectin did not bind to immobilized laminin, fibronectin, or albumin in these assays. Vitronectin showed similar interaction with all types of collagen (I, II, III, IV, V, and VI) tested. Collagen unfolded by heat treatment bound vitronectin less efficiently than native collagen. Vitronectin-coated colloidal gold particles bound to type I collagen fibrils as shown by electron microscopy. Salt concentrations higher than physiological interfered with the binding of vitronectin to collagen, suggesting an ionic interaction between the two proteins. Binding studies conducted in the presence of plasma showed that purified vitronectin added to plasma bound to immobilized collagen, whereas the endogenous plasma vitronectin bound to collagen less well. Although fibronectin did not interfere with the binding of vitronectin to native collagen, vitronectin inhibited the binding of fibronectin to collagen. These results show that vitronectin has a collagen-binding site(s) which, unlike that of fibronectin, preferentially recognizes triple-helical collagen and that the binding between vitronectin and collagen has characteristics compatible with the occurrence of such an interaction in vivo.

Blood Proteins↗
PubMed1988

Plasma vitronectin polymorphism in normal subjects and patients with disseminated intravascular coagulation.

Vitronectin, also known as serum-spreading factor or S-protein, mediates cell adhesion and inhibits formation of the membrane-lytic complex of complement and the rapid inactivation of thrombin by antithrombin III in the presence of heparin. Vitronectin is normally present in plasma at a concentration of approximately 300 micrograms/mL. The investigators quantified plasma vitronectin with an enzyme-linked immunosorbent assay and visualized reduced and nonreduced vitronectin by immunoblotting after separation of plasma or serum by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The concentration of plasma vitronectin was markedly reduced in some patients with disseminated intravascular coagulation, especially in those with liver failure; it was near normal in patients with metastatic cancer and acute leukemia. Patients with vitronectin levels less than 40% normal invariably had low fibrinogen and antithrombin III and a prolonged prothrombin time. In both normal and patient plasmas there was heterogeneity in the ratio of the 75,000- and 65,000-mol wt polypeptides of reduced vitronectin: 18% had mostly the 75,000-mol wt polypeptide, 59% had roughly equal amounts of the two polypeptides, and 22% had mostly the 65,000-mol wt polypeptide. This polymorphism is inherited and appears to be due to two alleles that are present with approximately equal frequency. The blotting patterns of vitronectin in reduced and nonreduced plasmas were largely unaltered in plasma of patients with defibrination syndrome, fibrinolysis, liver failure, sepsis, metastatic cancer, and acute leukemia. There was no evidence of fragmentation of vitronectin or formation of the disulfide-bonded complex of vitronectin and thrombin-antithrombin III that is found when blood is clotted. Thus these results corroborate in vitro observations that the liver is the major source of plasma vitronectin, suggest that vitronectin may become depleted during disseminated intravascular coagulation, and define a genetic polymorphism of vitronectin.

Animals↗