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.     

Vitronectin-induced haptotaxis of vascular smooth muscle cells in vitro.

Vitronectin, a multifunctional glycoprotein present in the plasma and interstitial tissues, has recently been found to be localized in atherosclerotic lesions. In this study we examined the effects of vitronectin on the migration of cultured bovine aortic smooth muscle cells using a modified Boyden chamber assay. The cells migrated to fluid-phase vitronectin in a concentration-dependent fashion. The cells also migrated to membrane filter surfaces precoated with vitronectin for a few minutes in the absence of additional vitronectin in the fluid phase, suggesting that this substance binds easily to the filters and stimulates cell migration by haptotaxis under the conditions described. These observations suggest that vitronectin deposited in the intima may be involved in the pathogenesis of atherosclerosis by recruiting smooth muscle cells from the media into the intima.     

Immunological characterization of human vitronectin and its binding to glycosaminoglycans

The cell-adhesive glycoprotein vitronectin in human plasma was characterized with a monospecific anti-vitronectin antibody. Vitronectin, a mixture of monomeric 75 and 65 kDa polypeptides, was found to have different ratios of amounts of 75 and 65 kDa polypeptides in immunoblots of sera from various healthy human donors. Two states of vitronectin were previously reported; the open state binds to heparin, but the cryptic state does not (Hayashi et al. (1985) J. Biochem. 98, 1135-1138). The anti-vitronectin antibody was suggested to react more strongly with the open state of vitronectin than with the cryptic state. To quantitate all vitronectin regardless of its state, an enzyme-linked immunosorbent assay of vitronectin was developed based on prior boiling of vitronectin-containing samples in 2% (w/v) sodium dodecyl sulfate and 40 mM dithiothreitol to destroy conformational differences. About 12-20% of the vitronectin molecules in plasma were found to bind to heparin-Sepharose under physiological conditions. Vitronectin in plasma bound 30-fold more efficiently to heparin immobilized by amino groups than by carboxyl groups. Its affinity for heparin was higher than for chondroitin sulfate A or C, or dermatan sulfate. Vitronectin was also found to contain covalently-linked small polypeptides of 15 and 13 kDa. These light chains seemed to be disulfide-bonded to the 65 kDa polypeptide, and might be endogenously derived from nicks in the carboxy-terminal portion of the 75 kDa polypeptide in plasma.     

Characterization of Vitronectin-Binding Proteins of Staphylococcus epidermidis

Staphylococcus epidermidis is the most common microorganism that is isolated from the cerebrospinal fluid (CSF) shunt infection patients. Vitronectin adsorbed on the surface of implants may mediate bacterial adhesion and colonization. To characterize vitronectin-binding properties, we analyzed S. epidermidis BD5703 isolated from a CSF shunt infection. Expression of vitronectin-binding protein(s) depended on culture media. Two proteins (60 and 52 kDa) were purified from vitronectin affinity chromatography. Two other vitronectin-binding proteins (21 and 16 kDa) were purified from an ion-exchange column. All purified proteins blocked bacterial binding of immobilized vitronectin significantly except the 16-kDa protein. The N-terminal sequences of the 21- and 16-kDa proteins did not show any appreciable amino acid sequence homology. The 52-kDa protein was sequenced by mass spectrometry and identified as an autolysin. This report demonstrates that interaction of vitronectin with multiple recognition sites on BD5703 surface may contribute to bacterial colonization. Received: 6 September 2000 / Accepted: 6 November 2000     

Involvement of vitronectin in lipopolysaccaride-induced acute lung injury

Vitronectin is present in large concentrations in serum and participates in regulation of humoral responses, including coagulation, fibrinolysis, and complement activation. Because alterations in coagulation and fibrinolysis are common in acute lung injury, we examined the role of vitronectin in LPS-induced pulmonary inflammation. Vitronectin concentrations were significantly increased in the lungs after LPS administration. Neutrophil numbers and proinflammatory cytokine levels, including IL-1beta, MIP-2, KC, and IL-6, were significantly reduced in bronchoalveolar lavage fluid from vitronectin-deficient (vitronectin(-/-)) mice, as compared with vitronectin(+/+) mice, after LPS exposure. Similarly, LPS induced increases in lung edema, myeloperoxidase-concentrations, and pulmonary proinflammatory cytokine concentrations were significantly lower in vitronectin(-/-) mice. Vitronectin(-/-) neutrophils demonstrated decreased KC-induced chemotaxis as compared with neutrophils from vitronectin(+/+) mice, and incubation of vitronectin(+/+) neutrophils with vitronectin was associated with increased chemotaxis. Vitronectin(-/-) neutrophils consistently produced more TNF-alpha, MIP-2, and IL-1beta after LPS exposure than did vitronectin(+/+) neutrophils and also showed greater degradation of IkappaB-alpha and increased LPS-induced nuclear accumulation of NF-kappaB compared with vitronectin(+/+) neutrophils. These findings provide a novel vitronectin-dependent mechanism contributing to the development of acute lung injury.     

Activation of vitronectin (serum spreading factor) binding of heparin by denaturing agents

Vitronectin (serum spreading factor), a cell-adhesive glycoprotein present in mammalian serum, has previously been the subject of conflicting reports concerning its binding to heparin. Vitronectin purified from human plasma does not bind to heparin under physiological conditions, but it does so after treatment with denaturing agents including 8 M urea or 6 M guanidine-HC1, or heating at 100 degrees C for 5 min. These treatments seem to expose a heparin-binding site in vitronectin; this finding thus resolves the conflicts concerning this function.     

Kinetic analysis of the interaction between type 1 plasminogen activator inhibitor and vitronectin and evidence that the bovine inhibitor binds to a thrombin-derived amino-terminal fragment of bovine vitronectin.

The interaction between guanidine-activated bovine type 1 plasminogen activator inhibitor (PAI-1) and bovine vitronectin was investigated. Activated PAI-1 bound to vitronectin in a dose- and time-dependent manner, and binding was saturable. The dissociation constant (Kd) for this interaction was estimated to be 3.10(-10) mol/l by Scatchard analysis. Complexes of activated PAI-1 and vitronectin were relatively stable at 4 degrees C (T1/2 greater than 24 h), but dissociated with a T1/2 of 4 h at 37 degrees C. The half-life of PAI-1 activity was increased from 2.5 to 4.5 h upon binding to immobilized vitronectin. In order to identify the binding domain(s) in vitronectin for activated PAI-1, the ability of PAI-1 to bind to vitronectin fragments was assessed. Vitronectin was cleaved by thrombin in a dose- and time-dependent manner, generating fragments of Mr 60,000, 54,000 and 38,000. The PAI-1 binding domain(s) were not destroyed by this treatment, since the digested vitronectin competed with immobilized vitronectin for PAI-1 binding to the same extent as uncleaved vitronectin. The thrombin digested vitronectin fragments were fractionated by SDS-PAGE and analyzed by PAI-1 ligand binding. The smallest fragment (Mr 38,000) retained PAI-1 binding function, and sequence analysis demonstrated that this fragment contained the NH2-terminus of bovine vitronectin. These results suggest that the high-affinity binding site for activated PAI-1 is located in the NH2-terminal region of the bovine vitronectin molecule.     

Purification of a protein from bovine plasma that binds to type 1 plasminogen activator inhibitor and prevents its interaction with extracellular matrix. Evidence that the protein is vitronectin

Type 1 plasminogen activator inhibitor (PAI-1) binds to the extracellular matrix of cultured bovine aortic endothelial cells. Bovine plasma and bovine lung extract contain protein(s) that bind to PAI-1 and prevent this interaction. One of these proteins was purified approximately 425-fold from ammonium sulfate-fractionated plasma using standard chromatographic procedures together with affinity chromatography on PAI-1-Sepharose. The final product consisted of a major polypeptide of Mr 65,000 and two minor polypeptides of Mr 80,000 and 57,000. NH2-terminal amino acid sequence analysis of the Mr 65,000 polypeptide revealed that it was homologous with vitronectin, and antiserum against this purified binding protein recognized vitronectin and vice versa. Immunological analysis using these antisera demonstrated that the three peptides were immunologically related, and that vitronectin was present in the extracellular matrix of bovine endothelial cells and also in bovine lung.     

Incorporation of vitronectin into fibrin clots. Evidence for a binding interaction between vitronectin and gamma A/gamma' fibrinogen.

Vitronectin is an abundant plasma protein that regulates coagulation, fibrinolysis, complement activation, and cell adhesion. Recently, we demonstrated that plasma vitronectin inhibits fibrinolysis by mediating the interaction of type 1 plasminogen activator inhibitor with fibrin (Podor, T. J., Peterson, C. B., Lawrence, D. A., Stefansson, S., Shaughnessy, S. G., Foulon, D. M., Butcher, M., and Weitz, J. I. (2000) J. Biol. Chem. 275, 19788-19794). The current studies were undertaken to further examine the interactions between vitronectin and fibrin(ogen). Comparison of vitronectin levels in plasma with those in serum indicates that approximately 20% of plasma vitronectin is incorporated into the clot. When the time course of biotinylated-vitronectin incorporation into clots formed from (125)I-fibrinogen is monitored, vitronectin incorporation into the clot parallels that of fibrinogen in the absence or presence of activated factor XIII. Vitronectin binds specifically to fibrin matrices with an estimated K(d) of approximately 0.6 microm. Additional vitronectin subunits are assembled on fibrin-bound vitronectin multimers through self-association. Confocal microscopy of fibrin clots reveals the globular vitronectin aggregates anchored at intervals along the fibrin fibrils. This periodicity raised the possibility that vitronectin interacts with the gamma A/gamma' variant of fibrin(ogen) that represents about 10% of total fibrinogen. In support of this concept, the vitronectin which contaminates fibrinogen preparations co-purifies with the gamma A/gamma' fibrinogen fraction, and clots formed from gamma A/gamma' fibrinogen preferentially bind vitronectin. These studies reveal that vitronectin associates with fibrin during coagulation, and may thereby modulate hemostasis and inflammation.     

Phosphorylation of vitronectin by a protein kinase in human plasma. Identification of a unique phosphorylation site in the heparin-binding domain

Incubation of human plasma with 27 nM [gamma-32P]ATP in the presence of 20 mM MnCl2 results in the phosphorylation of several proteins detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. About 60% of the incorporated radioactivity is found in a 75-kDa protein containing [32P] phosphoserine. The amino-terminal amino acid sequence of the purified 75-kDa [32P]phosphoprotein is identical to that of vitronectin (also termed serum spreading factor or complement S protein). Rabbit antiserum against vitronectin precipitates greater than 90% of the 75-kDa [32P]phosphoprotein from plasma. Reverse phase chromatography of [32P]vitronectin degraded sequentially with CNBr and chymotrypsin yields one major labeled peptide. The sequence of the peptide, Ser-Arg-Arg-Pro-[32PO4]Ser-Arg-Ala-Thr, corresponds to residues 374-381 which are located in the heparin-binding fragment of vitronectin identified by Suzuki et al. [1984) J. Biol. Chem. 259, 15307-15314). Vitronectin could potentially be phosphorylated in vivo with ATP released from injured cells or secreted by platelets activated during hemostasis.     

Inhibition of cell adhesion by high molecular weight kininogen

An anti-cell adhesion globulin was purified from human plasma by heparin-affinity chromatography. The purified globulin inhibited spreading of osteosarcoma and melanoma cells on vitronectin, and of endothelial cells, platelets, and mononuclear blood cells on vitronectin or fibrinogen. It did not inhibit cell spreading on fibronectin. The protein had the strongest antiadhesive effect when preadsorbed onto the otherwise adhesive surfaces. Amino acid sequence analysis revealed that the globulin is cleaved (kinin-free) high molecular weight kininogen (HKa). Globulin fractions from normal plasma immunodepleted of high molecular weight kininogen (HK) or from an individual deficient of HK lacked adhesive activity. Uncleaved single-chain HK preadsorbed at neutral pH, HKa preadsorbed at pH greater than 8.0, and HKa degraded further to release its histidine-rich domain had little anti-adhesive activity. These results indicate that the cationic histidine-rich domain is critical for anti-adhesive activity and is somehow mobilized upon cleavage. Vitronectin was not displaced from the surface by HKa. Thus, cleavage of HK by kallikrein results in both release of bradykinin, a potent vasoactive and growth-promoting peptide, and formation of a potent anti-adhesive protein.     

Vitronectin exists in two structurally and functionally distinct forms in human plasma

Vitronectin (serum spreading factor, S-protein or epibolin) is a plasma glycoprotein implicated in cell adhesion, as well as in the regulation of complement-mediated cytolysis and antithrombin III function. Vitronectin was found to exist in fresh human plasma as a heterogeneous mixture consisting of 2% heparin-binding form and the remainder as a non-binding species. Heparin-binding vitronectin consisted of 6.5 S aggregates with a Stokes radius of 5.6 nm, which was enriched in the 65 kDa polypeptide, with a high content of molecules and a putative unfolded conformation. In contrast, non-heparin-binding vitronectin was a 4.2 S monomer with a Stokes radius of 3.9 nm, which appeared to be in a folded conformation with an immunologically cryptic site. Both vitronectins displayed similar activities in mediating the spreading of BHK fibroblastic cells on substrates. During blood coagulation, 5% more of the non-heparin-binding vitronectin was converted into the heparin-binding form, producing a greater than 3.5-fold increase in this species. Our results indicate that vitronectin normally exists in circulating blood in at least two structurally and functionally distinct forms which may serve different functions.     

Vitronectin Expression in the Airways of Subjects with Asthma and Chronic Obstructive Pulmonary Disease

Vitronectin, a multifunctional glycoprotein, is involved in coagulation, inhibition of the formation of the membrane attack complex (MAC), cell adhesion and migration, wound healing, and tissue remodeling. The primary cellular source of vitronectin is hepatocytes; it is not known whether resident cells of airways produce vitronectin, even though the glycoprotein has been found in exhaled breath condensate and bronchoalveolar lavage from healthy subjects and patients with interstitial lung disease. It is also not known whether vitronectin expression is altered in subjects with asthma and COPD. In this study, bronchial tissue from 7 asthmatic, 10 COPD and 14 control subjects was obtained at autopsy and analyzed by immunohistochemistry to determine the percent area of submucosal glands occupied by vitronectin. In a separate set of experiments, quantitative colocalization analysis was performed on tracheobronchial tissue sections obtained from donor lungs (6 asthmatics, 4 COPD and 7 controls). Vitronectin RNA and protein expressions in bronchial surface epithelium were examined in 12 subjects who undertook diagnostic bronchoscopy. Vitronectin was found in the tracheobronchial epithelium from asthmatic, COPD, and control subjects, although its expression was significantly lower in the asthmatic group. Colocalization analysis of 3D confocal images indicates that vitronectin is expressed in the glandular serous epithelial cells and in respiratory surface epithelial cells other than goblet cells. Expression of the 65-kDa vitronectin isoform was lower in bronchial surface epithelium from the diseased subjects. The cause for the decreased vitronectin expression in asthma is not clear, however, the reduced concentration of vitronectin in the epithelial/submucosal layer of airways may be linked to airway remodeling.     

Influence of dose and route of administration of bovine follicular fluid and the suppressive effect of purified bovine inhibin (Mr 31,000) on plasma FSH concentrations in ovariectomized ewes

Ovariectomized Merino ewes were used to develop an in-vivo bioassay for purified bovine inhibin of Mr 31,000. Various doses (0.25, 0.5, 1 or 2 ml) of bovine follicular fluid, given either by the intravenous (i.v.) or intracarotid route (i.c.) resulted in significant linear dose-related suppression of plasma FSH and interval to maximum suppression. Control ewes (1.0 ml steer plasma) showed no significant change in FSH over the same period. Doses of 470 and 2590 U of pure inhibin given i.v. caused a significant suppression of FSH in plasma in all ewes. The in-vivo potency estimate of the high dose (2760 U, 1420-4690 fiducial limits) agreed well with the in-vitro assay of potency. There were no significant changes observed in mean plasma LH after treatment with the higher dose of pure inhibin. There were no rebound effects of treatment with bovine follicular fluid or pure inhibin on FSH concentrations above that of controls. It is concluded that the form of bovine inhibin of Mr 31,000, which is believed to be the predominant circulating form, is biologically active when administered in vivo.     

Activation of the collagen-binding of endogenous serum vitronectin by heating, urea and glycosaminoglycans.

The present study describes that the collagen-binding activity of vitronectin in human serum increases by treatment with heparin, heating and urea. Vitronectin purified from human serum was bound to native collagen, whereas endogenous vitronectin in the serum was not. We have examined the conditions to change the collagen-binding activity of endogenous vitronectin. Endogenous vitronectin in human serum became considerably bound to collagen when the serum was boiled in 4-8 M urea for 5 min and mixed with heparin (0.5-5 micrograms/ml). Each treatment of heating, urea or heparin alone, and any combination of the two factors, inefficiently activated the binding. Dextran sulfate could substitute for heparin, but dermatan sulfate, keratan sulfate, chondroitin sulfate A and C, heparan sulfate and hyaluronan could not. Possible explanations for the activation of endogenous vitronectin are discussed.     

Sequence location of a putative transglutaminase cross-linking site in human vitronectin.

We attempted to locate the glutamine residue in human vitronectin, susceptible to cross-linking by transglutaminases. Vitronectin was incubated with 14C-labelled putrescine and plasma factor XIIIa and, after reduction and alkylation, the vitronectin was digested with trypsin. HPLC of the digest followed by scintillation counting revealed one major and two minor radioactivity labelled peaks. Sub-digestion with Staphylococcus aureus protease, sequence analysis and mass-spectrometry of the resulting peptides demonstrated that Gln-93 of vitronectin had incorporated putrescine. Additionally, Gln-73, Gln-84 and Gln-86 were found to be minor sites for incorporation.     

Adsorption of vitronectin in human serum onto plastics is augmented by sodium dodecyl sulfate

We have investigated the adsorption of cell-spreading activity in human serum onto polystyrene plates after treatment of the serum with sodium dodecyl sulfate (SDS). Vitronectin in human serum was remarkably adsorbed onto the plate after boiling the serum with 0.1% SDS for 5 min. SDS was effective over the concentration range from 0.05 to 0.25%. Increase of the vitronectin adsorption was accompanied by an increase of cell spreading on the plates. The cell-spreading activity in SDS-treated serum was impeded by anti-vitronectin antibody but not by anti-fibronectin antibody. After treatment with SDS, fibronectin-depleted serum could induce cell spreading but vitronectin-depleted serum could not. These results indicate that vitronectin alone was the cell-spreading factor in SDS-treated human serum. However, SDS-treated pure vitronectin itself did not retain the cell-spreading activity. The activity was recovered when bovine serum albumin was added to pure vitronectin before or after boiling with 0.1% SDS. Therefore, vitronectin adsorbed from SDS-treated serum might retain the cell-spreading activity with the aid of serum protein. Treatment of serum with SDS provides an easy, specific, and efficient method of coating polystyrene plates with vitronectin.     

Vitronectin—A major cell attachment-promoting protein in fetal bovine serum

Bovine serum is a constituent of most media used for the culture of animal cells. The adhesion-promoting properties of serum are generally attributed to fibronectin, yet there have been frequent reports of other adhesion-promoting molecules in bovine serum. Using a technique in which adhesive proteins are visualized after separation by SDS-PAGE, we graphically confirm the presence of a second cell attachment protein in bovine serum and present the evidence that this molecule is the bovine equivalent of vitronectin. The molecular size of this protein is in the same range as the size of the adhesive human plasma protein, vitronectin. The bovine protein also shared with human vitronectin an affinity for glass, and it could be purified by a combination of glass bead and ion exchange chromatography. The isolated bovine protein had varying proportions of an 80 and a 65 kD polypeptide. It showed immunological cross-reactivity with anti-human vitronectin and with anti-human somatomedin B. Somatomedin B is a serum peptide which has a NH2-terminal sequence identical to that of human vitronectin. The identity of the bovine protein as vitronectin was established by showing that its NH2-terminal amino acid sequence is strongly homologous with those of human vitronectin and somatomedin B. Quantitation of the adhesive activities of fibronectin and vitronectin in bovine plasma and fresh serum showed that more activity is associated with vitronectin than with fibronectin. The preponderance of vitronectin was particularly clear in fetal bovine serum intended for cell culture. In various batches, cell attachment activity attributable to vitronectin was 8-16-fold greater than that of fibronectin, making vitronectin the main adhesive protein in routine cell culture media.     

Vascular smooth muscle cell growth-promoting factor/F-spondin inhibits angiogenesis via the blockade of integrin alphavbeta3 on vascular endothelial cells

Vascular smooth muscle cell growth-promoting factor (VSGP) was originally isolated from bovine ovarian follicular fluid as a stimulator of vascular smooth muscle cell proliferation. Homology searches indicate that bovine and human VSGPs are orthologs of rat F-spondin. Here, we examined whether recombinant human VSGP/F-spondin affected the biological activities of endothelial cells. VSGP/F-spondin did not affect the proliferation of human umbilical vein endothelial cells (HUVECs); however, it did inhibit VEGF- or bFGF-stimulated HUVEC migration. To clarify the mechanism of this inhibitory effect, we examined the adhesion of HUVECs to extracellular matrix proteins. VSGP/F-spondin specifically inhibited the spreading of HUVECs on vitronectin via the functional blockade of integrin alphavbeta3. As a result, VSGP/F-spondin inhibited the tyrosine phosphorylation of focal adhesion kinase (FAK) when HUVECs were plated on vitronectin. Moreover, VSGP/F-spondin inhibited the activation of Akt when HUVECs on vitronectin were stimulated with VEGF. VSGP/F-spondin inhibited tube formation by HUVECs in vitro and neovascularization in the rat cornea in vivo. These results indicate that VSGP/F-spondin inhibits angiogenesis at least in part by the blockade of endothelial integrin alphavbeta3.     

蛋白凝胶基质的制备与基质内的血管生成反应

利用肝素亲和层析从血清中提取玻璃粘连蛋白（vitronectin）,以硫酸铵沉淀法从血浆中粗提含纤维蛋白原的复合蛋白质组分,向血浆蛋白、胎牛血清和DMEM组成的复合成分中加入凝血酶,制成蛋白质凝胶.观察血管内皮细胞在此基质上或在基质中的生长及在碱性成纤维细胞生长因子(basic fibroblast growth factor, bFGF)的诱导下形成的血管样结构.结果表明血管内皮细胞可粘附在此凝胶基质表面正常生长,在bFGF的诱导下,内皮细胞向胶内迁移、生长并形成管状结构,多个管状结构连接、融合形成毛细血管网状结构.