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At least 19 recordsLinked to original sources
PubMed1988

Conformational states of vitronectin: preferential expression of an antigenic epitope when vitronectin is covalently and noncovalently complexed with thrombin-antithrombin III or treated with urea.

A difference in recognition of the adhesive glycoprotein vitronectin (also called S-protein, serum spreading factor, and epibolin) by monoclonal antibody 8E6 (Hayman EG, et al, Proc Natl Acad Sci USA 80:4003, 1983) was investigated using a competitive enzyme-immunosorbent assay and immunoaffinity chromatography. Recognition of vitronectin in serum was approximately 50-fold greater than recognition of vitronectin in plasma. Recognition of vitronectin incubated with heparin, thrombin-antithrombin III complex, or heparin and thrombin-antithrombin III complex together was 2.5-, 7-, or 32-fold greater, respectively, than recognition of vitronectin alone. Thrombin or antithrombin III by itself did not induce the antigenic change. Factor Xa-antithrombin III was less effective than thrombin-antithrombin III in induction of the change. Dextran sulfate and fucoidan were more potent than heparin in induction of the antigenic change, whereas dermatan sulfate, hyaluronic acid, heparan sulfate, chondroitin sulfate, or keratan sulfate were less effective. Immunoblotting analysis of serum and of vitronectin incubated with thrombin and antithrombin III demonstrated the presence of complexes composed of vitronectin and thrombin-antithrombin III that could only be dissociated with reducing agent. N-ethylmaleimide completely blocked the formation of the presumably disulfide-bonded complexes and partially blocked the antigenic change. Both non-disulfide-bonded and disulfide-bonded vitronectin bound to antibody-Sepharose from a mixture of vitronectin and thrombin-antithrombin III. Treatment of vitronectin with 8 mol/L urea resulted in enhanced recognition by the monoclonal antibody. Thus, the 8E6 antibody reacts with an epitope that is preferentially expressed by noncovalently and covalently linked vitronectin/thrombin-antithrombin III complexes and by urea-treated vitronectin. The change in vitronectin induced by thrombin-antithrombin III, therefore, is a physiological correlate of urea treatment and of adsorption of vitronectin onto tissue culture plastic (as is done in cell adhesion assays). The change may be important for expression of vitronectin activity.

Antithrombin III↗
PubMed1991

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.

Amino Acid Sequence↗
PubMed1992

IL-3-dependent mast cells attach to plate-bound vitronectin. Demonstration of augmented proliferation in response to signals transduced via cell surface vitronectin receptors.

The adhesive interactions of activated mast cells with the extracellular matrix play an important role in anchorage and cellular motility. In this report we demonstrate that IL-3-dependent bone marrow-derived mast cells adhere to plate-bound vitronectin with high affinity in a saturable and dose-dependent manner. This adhesion interaction is unique in that it does not require prior mast cell activation through Fc epsilon RI or after treatment with PMA. It is inhibited by divalent cation chelation and by competitive inhibition with a synthetic Arginine-Glycine-Aspartate-Serine tetrapeptide. Polyclonal antisera for alpha v beta 3, an integrin known to bind vitronectin, inhibits attachment to plate-bound vitronectin in a dose-dependent manner. Comparison of the adhesion interactions for vitronectin, fibronectin, and laminin indicate that adhesion to vitronectin is greater than that seen with either fibronectin or laminin, either in the presence or absence of PMA. FACS analysis using a monoclonal hamster anti-murine vitronectin receptor (alpha v) antibody followed by a fluorescein-conjugated rabbit anti-hamster IgG revealed no change in surface vitronectin receptor expression after Fc epsilon RI-mediated cell activation. Proliferation assays with correction for cell viability revealed a 25% increase in cell number above the maximal IL-3 response over a 24-h period of adhesion to a vitronectin-coated surface and a 41% increase over 96 h of adhesion to vitronectin. Binding to plate-bound vitronectin was not able to sustain cell viability in the absence of IL-3. Thus, IL-3-dependent bone marrow-derived mast cells adhere to vitronectin, an extracellular matrix protein present throughout connective tissues. This interaction generates a signal that results in the augmentation of the maximal IL-3-dependent mast cell proliferative response, thus demonstrating at least one way in which the interaction between mast cells and extracellular matrix alter the biologic responsiveness of the mast cell.

Animals↗
PubMed1985

Complete amino acid sequence of human vitronectin deduced from cDNA. Similarity of cell attachment sites in vitronectin and fibronectin.

cDNA clones for vitronectin, a cell adhesion-promoting plasma and tissue protein, were isolated from a lambda gt11 library containing cDNA inserts made from human liver mRNA. The library was screened with anti-vitronectin antibodies and the positive clones were further identified with synthetic oligonucleotide probes deduced from the partial amino acid sequence of vitronectin. Nucleotide sequence analysis showed that the largest insert was 1545 bp long and contained the whole sequence corresponding to plasma vitronectin. It showed that vitronectin contains the entire 44-amino acid somatomedin B peptide at its NH2 terminus and, near its COOH terminus, a 34-amino acid glycosaminoglycan binding site in which half of the amino acids are basic residues. Three potential carbohydrate attachment sites are present in the sequence. An Arg-Gly-Asp sequence, which has previously been shown to be the cell attachment site in fibronectin, was found in vitronectin immediately after the NH2-terminal somatomedin B sequence. No other homologies with fibronectin were found. The Arg-Gly-Asp sequence appears to constitute the cell attachment site of vitronectin, since it is in the region where we have previously localized the cell attachment site, its presence correlate with cell attachment activity among the insert-coded polypeptides, and because previous results have shown that synthetic peptides containing the Arg-Gly-Asp sequence inhibit the cell attachment function of vitronectin. The discovery of an Arg-Gly-Asp cell attachment site in a protein with a known cell attachment function emphasizes the general importance of this sequence in cell recognition.

Amino Acid Sequence↗
PubMed1990

Polymorphism of the human vitronectin gene causes vitronectin blood type.

Human blood plasma/sera are classified into three distinct vitronectin types based on the relative amount of the 75 kDa polypeptide to its cleavage product of 65 kDa. We asked whether the vitronectin blood types correlated with the polymorphism of the vitronectin gene. A portion of the vitronectin gene was amplified by using polymerase chain reaction and digested with a restriction enzyme PmaC I which may distinguish the base sequence causing the polymorphic change at the amino acid position 381. Amplified DNAs of the blood type I (75 kDa-rich), II (75/65 kDa-even), and III (65 kDa-rich) were shown to be resistant, moderately sensitive and completely sensitive to PmaC I, respectively. These results suggest that Thr at position 381 is essential for the cleavage of the vitronectin 75 kDa polypeptide and that three possible combinations of two codominant alleles of vitronectin determine three vitronectin blood types.

Amino Acid Sequence↗
PubMed1990

Specific binding of plasminogen to vitronectin. Evidence for a modulatory role of vitronectin on fibrin(ogen)-induced plasmin formation by tissue plasminogen activator.

Vitronectin immobilized onto polystyrene microtiter wells was demonstrated to specifically bind plasminogen in a concentration-dependent manner, yielding an estimated KD = 0.4 microM. Heparin only moderately interfered with the vitronectin-plasminogen interaction, whereas high concentrations of 6-amino-hexanoic acid inhibited binding. Utilizing a ligand-blotting procedure in which plasminogen was reacted with proteolytic fragments of vitronectin, transblotted onto nitrocellulose, the plasminogen-binding site of vitronectin was localized to the heparin-binding domain of the adhesive protein. Moreover, vitronectin was found to inhibit in a dose-dependent fashion the fibrin(ogen)-induced activation of plasminogen by tissue plasminogen activator. These results provide the first evidence for a novel vitronectin-mediated control of plasminogen activation potentially relevant for directional clot-lysis and plasmin-dependent proteolysis in extracellular matrices.

Electrophoresis, Polyacrylamide Gel↗
PubMed1992

Platelets and vitronectin: immunocytochemical localization and platelet interaction with exogenously added vitronectin.

Vitronectin stores were localized in human platelets by immunofluorescence, confocal laser microscopy, and immunoelectron microscopy. In ultrathin cryosections, the immunogold labelling was mainly observed in the alpha-granules, although occasional immunolabelling of the platelet surface was present. The interaction between thrombin-stimulated platelets and exogenously added vitronectin was also explored. This vitronectin had been purified by heparin-affinity chromatography. Binding of vitronectin to thrombin-stimulated platelets was demonstrated by flow cytometry. The immunoelectron microscopical studies revealed that this binding was mainly restricted to clumps of released alpha-granular material on the platelet surface. The possible significance of the interaction between exogenous vitronectin and released platelet proteins is discussed.

Amino Acid Sequence↗
PubMed1990

The integrin beta 1 subunit associates with the vitronectin receptor alpha v subunit to form a novel vitronectin receptor in a human embryonic kidney cell line.

We describe a novel integrin heterodimer on the surface of the human embryonic kidney cell line 293. This receptor is comprised of alpha v and beta 1 subunits, each of which has been previously found in association with other integrin subunits. This alpha v.beta 1 complex was identified as the predominant vitronectin receptor (VnR) on the surface of 293 cells by immunoprecipitation with antibodies raised against the alpha v subunit. Polymerase chain reaction analysis detected mRNAs for alpha v and beta 1 subunits while no evidence was obtained for beta 2, beta 3, or alpha IIb integrin subunit mRNA. Immunoprecipitation of surface-iodinated proteins with antibodies to alpha v gave bands of 150 and 120 kDa. The 120-kDa band reacted with antibodies to beta 1 in immunoblotting experiments. 293 cells adhere to vitronectin, fibronectin, laminin, and collagen IV, while von Willebrand factor and fibrinogen, known ligands of the VnR (alpha v.beta 3), did not support adhesion. A polyclonal antibody directed against both subunits of the VnR (alpha v, beta 3) inhibits attachment of 293 cells to vitronectin but not to other adhesive proteins. A beta 1-specific monoclonal inhibited attachment to fibronectin, laminin, and collagen IV, known ligands of beta 1 integrins, as well as vitronectin. This novel (alpha v. beta 1) VnR thus appears to mediate cell adhesion exclusively to vitronectin, in contrast to previously described VnRs which have multiple ligands.

Antibodies↗
PubMed1991

Vitronectin and proliferative intraocular disorders. I. A colocalisation study of the serum spreading factor, vitronectin, and fibronectin in traction membranes from patients with proliferative vitreoretinopathy.

The presence of a scaffold for cellular spreading and proliferation is a precondition for the development of traction membranes in proliferative vitreoretinopathy (PVR). This study shows the presence of the serum spreading factor, vitronectin, in the extracellular matrix of periretinal membranes removed during vitreoretinal surgery. By means of a double label immunofluorescence protocol, a partial colocalisation of vitronectin with fibronectin at the magnification of light microscopy can be detected. Fibronectin is a high-molecular glycoprotein with multiple biological functions including the mediation of cell attachment and migration. Both proteins share a special cell recognition site which could be a target for experimental pharmacological approaches to PVR. Preliminary studies of vitreous aspirates using electrophoresis and Western blotting indicate that vitronectin may play a more important role in post-traumatic PVR as compared to PVR following rhegmatogenous retinal detachment.

Blotting, Western↗
PubMed1991

Vitronectin and proliferative intraocular disorders. II. Expression of cell surface receptors for fibronectin and vitronectin in periretinal membranes.

Several cell types participate in the formation of vitreoretinal traction membranes in proliferative intraocular disorders. The communication between these cells involves hormones, growth factors, and the interaction with extracellular matrix molecules. We have previously demonstrated a partial colocalisation of two potent mediators of cell attachment, fibronectin and vitronectin, in periretinal membranes from patients with proliferative vitreoretinopathy (PVR). We found a similar pattern of vitronectin and fibronectin deposition in proliferative diabetic retinopathy (PDR) (n = 6). Now we show the expression of the corresponding cell surface receptors, integrins, for fibronectin and vitronectin by proliferating cells in 22 periretinal membranes, including traumatic (n = 8) and idiopathic (n = 8) PVR as well as PDR membranes (n = 6). Integrins are membrane receptors for extracellular matrix macromolecules which are involved in such basic biological phenomena as embryogenesis and metastasis. Future studies on the pathogenesis of vitreoretinal proliferation will have to focus on the initiation, maintenance, and regulation of this intercellular communication network involving attachment proteins and integrins.

Diabetic Retinopathy↗
PubMed1992

Binding of vitronectin-thrombin-antithrombin III complex to human endothelial cells is mediated by the heparin binding site of vitronectin.

The interaction of vitronectin-thrombin-antithrombin III (VN.TAT) complex with endothelial cells (EC) was investigated. Binding was specific and time- and concentration-dependent. Kinetics revealed an apparent dissociation constant of 16 nM and 1.7 x 10(5) binding sites/endothelial cell. The binding determinant of the ternary complex was located on the VN moiety. Since the association of VN to TAT adds its specific properties to the VN.TAT complex, the involvement of the heparin binding domain and the cell attachment site of VN was investigated. Neither addition of RGD peptide nor blocking of the vitronectin receptor with a monoclonal antibody interfered with VN.TAT binding to EC. Addition of heparin, a VN-derived peptide comprising two heparin binding consensus sequences or a monoclonal antibody directed against the heparin binding domain on VN, completely inhibited VN.TAT binding to EC. These results indicate that the interaction is mediated through the heparin binding domain of VN. Digestion of heparan sulfate proteoglycans resulted in a decrease of VN.TAT binding to EC, indicating the involvement of heparin-like structures on the EC surface. Our findings point to an unrecognized mechanism by which VN may act as scavenger in order to enhance the clearance of end products of the clotting system via binding of the ternary VN.TAT complex to the luminal surface of EC.

Amino Acid Sequence↗
PubMed1989

Biogenesis of the vitronectin receptor in human endothelial cell: evidence that the vitronectin receptor and GPIIb-IIIa are synthesized by a common mechanism.

Human endothelial cells express a membrane glycoprotein alpha beta heterodimer similar to the human platelet glycoprotein IIb-IIIa complex (GPIIb-IIIa). This noncovalently associated complex is the vitronectin receptor (VNR). These two receptors belong to the cytoadhesin family and share the same beta subunit. They express different recognition specificities: platelet GPIIb-IIIa is a receptor for fibrinogen, fibronectin, and von Willebrand factor (vWF), whereas VNR is a receptor for vitronectin, and is possibly a receptor for fibrinogen and vWF. We analyzed the biosynthesis of the endothelial cell VNR. Our data show that VNR alpha is a two-chain protein which is biosynthesized as a single-chain precursor: the pro-VNR alpha. Pro-VNR alpha forms a complex with VNR beta, and this association occurs prior to the Golgi-mediated processing of the oligosaccharide side chains. Mature VNR beta is glycosylated by not fully processed oligosaccharide side chains because it remains endoglycosidase H (endo H) sensitive, even when the complex is expressed on the cell surface. This characteristic appears as a common feature for the members of the cytoadhesin family. These results indicate that although VNR and GPIIb-IIIa are biosynthesized in different cells, their expression is controlled by similar mechanisms, providing further support for the concept that the cytoadhesin family constitutes a distinct group of adhesion receptors.

Cells, Cultured↗
PubMed1992

Mapping of binding sites for heparin, plasminogen activator inhibitor-1, and plasminogen to vitronectin's heparin-binding region reveals a novel vitronectin-dependent feedback mechanism for the control of plasmin formation.

Vitronectin (VN) has been implicated as a major matrix-associated regulator component of plasminogen activation by serving as a potent stabilizing cofactor of plasminogen activator inhibitor-1 (PAI-1). The direct binding of heparin, plasminogen as well as PAI-1 in its latent and active form to immobilized VN was studied in the absence or presence of competitors. Monoclonal antibodies against the carboxyl-terminal portion of VN inhibited both PAI-1 and plasminogen binding, whereas heparin, heparan sulfate with a high degree of sulfation, or dextran sulfate interfered with PAI-1 binding (KD = 20 nM) only. Utilizing synthetic peptides encompassing overlapping sequences of the heparin-binding domain of VN, adjacent heparin and PAI-1-binding sites were localized within the sequence 348-370 of VN. Although a number of other serine protease inhibitors which do not form binary complexes with VN contain a reactive-site Ser at their P1'-position, a reactive-site P1' mutant of PAI-1 (Met----Ser) showed comparable if not increased binding to VN. Binding of Lys-plasminogen and active-site-blocked plasmin was at least 10-fold higher in affinity (KD = 85-100 nM) compared to Glu-plasminogen (KD approximately 1 microM) and could be inhibited by lysine analogs but not by glycosaminoglycans or PAI-1, indicating that heteropolar plasmin(ogen) binding of VN occurs to an adjacent segment upstream to the heparin and PAI-1-binding sites. This contention was further supported in binding studies with plasmin-modified VN which lost both heparin and PAI-1 binding but exhibited 2-3-fold higher capacity to bind plasminogen. The essential plasmin(ogen)-binding site was mapped by ligand blot analysis to the carboxyl-terminal portion of proteolytically trimmed VN (M(r) = 61,000). Moreover, treatment of the extracellular matrix of human umbilical vein endothelial cells with plasmin resulted in partial degradation of matrix-associated VN and concomitant release of PAI-1, but increased the ability of the matrix by about 2-fold to bind plasminogen. These results are indicative of differential interactions of VN with components of the plasminogen activation system, whereby plasmin itself may provoke the switch of VN from an anti-fibrinolytic into a pro-fibrinolytic cofactor. This process reflects a novel role for the adhesive protein and its degradation product(s) in the possible feedback regulation of localized plasmin formation at extracellular sites.

Amino Acid Sequence↗
PubMed1990

Highly sulfated glycosaminoglycans augment the cross-linking of vitronectin by guinea pig liver transglutaminase. Functional studies of the cross-linked vitronectin multimers.

Vitronectin (VN) is an adhesive glycoprotein with roles in the complement, coagulation, and immune systems. Many of the functions of VN are mediated by a glycosaminoglycan binding site, near its carboxyl-terminal end. In this paper, we show that the highly sulfated glycosaminoglycans (GAGs), dextran sulfate, pentosan polysulfate, and fucoidan effectively augment [14C]putrescine incorporation into VN and cross-linking of VN into high molecular multimers by guinea pig liver transglutaminase (TG). Other GAGs including heparin, low molecular weight heparin, dermatan sulfate, keratan sulfate, and the nonsulfated dextrans were ineffective in accelerating these reactions. Dextran sulfate of average molecular mass 500 kDa was more effective than dextran sulfate of average molecular mass 5 kDa, supporting a template mechanism of action of the GAGs, in which VN molecules align on the GAG in a conformation suitable for cross-linking. The VN multimers catalyzed by TG retained functional activity in binding [3H]heparin, platelets, and plasminogen activator inhibitor type-1 (PAI-1). [3H]Heparin bound selectively to the 65-kDa monomeric band of VN and to the multimers derived from this band. PAI-1, however, bound equally to both the 75- and 65-kDa monomeric forms of VN, suggesting that the PAI-1 binding site on VN is distinct from the GAG binding site. The interaction of GAGs with the TG-catalyzed cross-linking of VN may facilitate studies of VN structure-function relationships.

Animals↗
PubMed1991

[Changes in plasma vitronectin, fibronectin, and serum laminin P1 levels and immunohistochemical study of vitronectin in the liver of patients with chronic liver diseases].

Vitronectin (VN), fibronectin (FN) and laminin (LM), which are known to be important glycoproteins in cell attachment, are produced by such liver cells as hepatocytes, Kupffer cells endothelial cells and Ito cells. In this study, the levels of plasma VN, FN and serum LM P1 in patients with chronic hepatitis, liver cirrhosis and hepatocellular carcinoma accompanied with cirrhosis were examined and compared with those in normal subjects. Plasma VN levels in patients with chronic hepatitis, compensated cirrhosis and decompensated cirrhosis were less than that in normal subjects. As hepatic dysfunction deteriorated, plasma VN level decreased in chronic liver diseases. Plasma FN levels in patients with compensated and decompensated cirrhosis were also less than that of patients with chronic hepatitis, which was not significantly different from that of normal subjects. Plasma VN and FN levels in patients with hepatocellular carcinoma were similar to those in patients with compensated cirrhosis. Plasma VN and FN levels in patients with chronic liver diseases including hepatocellular carcinoma showed positive correlations with serum albumin content, cholinesterase activity, and normalized normo test value. On the other hand, serum LM P1 levels in patients with chronic hepatitis, compensated cirrhosis and decompensated cirrhosis were higher than that of normal subjects. As hepatic dysfunction deteriorated, serum LM P1 level increased in chronic liver diseases. Level of serum type IV collagen 7S, which is related to hepatic fibrosis, was similar to that of serum LM P1; serum LM P1 concentration in patients with chronic liver diseases showed a significant positive correlation with that of serum type IV collagen 7S. Immunolocalization of VN in liver tissue from patients with chronic hepatitis and cirrhosis was examined by the method of avidin-biotin-complex staining, and positive reaction was observed in enlarged portal tracts, central veins and fibrous septa. These results suggest that decreased levels of plasma VN and FN and increased level of serum LM P1 in patients with chronic liver diseases are related to hepatic dysfunction, and that changes in the levels of these glycoproteins involved in cell attachment are important in the development of hepatic fibrosis in patients with chronic liver diseases.

Adult↗
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↗
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↗
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↗