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• W1991421191 abstract "Serine proteinase inhibitors, including plasminogen activator inhibitor type 1 (PAI-1) and antithrombin, are key regulators of hemostatic processes such as thrombosis and wound healing. Much evidence suggests that PAI-1 can influence such processes, as well as pathological events like tumor metastasis, through its ability to directly regulate binding of blood platelets and cells to extracellular substrata. One way that PAI-1 influences these processes may be mediated through its binding to the plasma protein vitronectin. Binding to PAI-1 results in the incorporation of vitronectin into a higher order complex with a potential for multivalent interactions (Podor, T. J., Shaughnessy, S. G., Blackburn, M. N., and Peterson, C. B. (2000) J. Biol. Chem. 275, 25402–25410). In this study, evidence is provided to support this concept from studies on the effects of PAI-1-induced multimerization on the interactions of vitronectin with matrix components and cell surface receptors. By monitoring complex formation and stability over time using size-exclusion high performance liquid chromatography, a correlation is made between PAI-1-induced multimerization and enhanced cell/matrix binding properties of vitronectin. This evidence indicates that PAI-1 alters the adhesive functions of vitronectin by converting the protein via the higher order complex to a self-associated, multivalent species that is functionally distinct from the abundant monomeric form found in the circulation. Serine proteinase inhibitors, including plasminogen activator inhibitor type 1 (PAI-1) and antithrombin, are key regulators of hemostatic processes such as thrombosis and wound healing. Much evidence suggests that PAI-1 can influence such processes, as well as pathological events like tumor metastasis, through its ability to directly regulate binding of blood platelets and cells to extracellular substrata. One way that PAI-1 influences these processes may be mediated through its binding to the plasma protein vitronectin. Binding to PAI-1 results in the incorporation of vitronectin into a higher order complex with a potential for multivalent interactions (Podor, T. J., Shaughnessy, S. G., Blackburn, M. N., and Peterson, C. B. (2000) J. Biol. Chem. 275, 25402–25410). In this study, evidence is provided to support this concept from studies on the effects of PAI-1-induced multimerization on the interactions of vitronectin with matrix components and cell surface receptors. By monitoring complex formation and stability over time using size-exclusion high performance liquid chromatography, a correlation is made between PAI-1-induced multimerization and enhanced cell/matrix binding properties of vitronectin. This evidence indicates that PAI-1 alters the adhesive functions of vitronectin by converting the protein via the higher order complex to a self-associated, multivalent species that is functionally distinct from the abundant monomeric form found in the circulation. plasminogen activator inhibitor type-1 N, N′-dimethyl-N-(acetyl)-N′-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) ethylenediamine horseradish peroxidase immunoglobulin G smooth muscle cell serine protease inhibitor urokinase-type and tissue-type plasminogen activators extracellular matrix high pressure liquid chromatography 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) enzyme-linked immunosorbent assay phosphate-buffered saline bovine serum albumin The interactions that occur between cellular receptors and proteins that constitute the extracellular matrix are vital to physiological control of processes like cell adhesion and pericellular proteolysis. Events that alter these interactions can be deleterious, leading to pathological sequelae such as improper wound healing and tumor cell migration or metastasis. For example, components of the humoral response system known as fibrinolysis, which play a role in modulating various cell-binding properties of the extracellular matrix, can be exploited by cancerous cells. By altering the content and activity of proteins related to fibrinolysis, abnormally developing cells can acquire the ability to exit residing tissues and eventually invade and metastasize. An example involves the main regulator of fibrinolysis, PAI-1,1 in which increased levels of the protein in plasma correlate with the presence of malignant ovarian cancer and higher incidence of disease (1Ho C.H. Yuan C.C. Liu S.M. Gynecol. Oncol. 1999; 75: 397-400Abstract Full Text PDF PubMed Scopus (30) Google Scholar). PAI-1 is a member of the serpin family and represents a key regulatory protein in proteolytic processes responsible for tissue remodeling and tumor metastasis. This property is owed to the fact that PAI-1 is the main inhibitor of both plasminogen activators, uPA and tPA. Interesting features of PAI-1 include its structural lability and the propensity it exhibits to spontaneously adopt a more stable, but inactive conformation. This feature of PAI-1 is unique among the serpin family, with active PAI-1 exhibiting a half-life of ∼90 min (2Hekman C.M. Loskutoff D.J. J. Biol. Chem. 1985; 260: 11581-11587Abstract Full Text PDF PubMed Google Scholar). In the body, however, this transition of PAI-1 into an inactive form is prevented by another important homeostatic protein, vitronectin, which binds to PAI-1 and increases the half-life of the active form approximately 2–4-fold (3De Declerck P.J. Mol M. Alessi M.C. Baudner S. Paques E.P. Preissner K.T. Muller-Berghaus G. Collen D. J. Biol. Chem. 1988; 263: 15454-15461Abstract Full Text PDF PubMed Google Scholar). The fact that nearly all PAI-1 in the human vasculature is complexed with vitronectin underscores the importance of considering vitronectin when investigating PAI-1-related functions such as cell attachment and spreading (3De Declerck P.J. Mol M. Alessi M.C. Baudner S. Paques E.P. Preissner K.T. Muller-Berghaus G. Collen D. J. Biol. Chem. 1988; 263: 15454-15461Abstract Full Text PDF PubMed Google Scholar). Vitronectin is a versatile, multifunctional protein found both in circulation and the ECM. It is involved in several pathways relevant to such physiological events as blood coagulation and fibrinolysis, cellular immunity, and tumor metastasis (4Preissner K.T. Blut. 1989; 59: 419-431Crossref PubMed Scopus (65) Google Scholar, 5Preissner K.T. Jenne D. Thromb. Haemostasis. 1991; 66: 189-194Crossref PubMed Scopus (34) Google Scholar, 6Preissner K.T. Jenne D. Thromb. Haemostasis. 1991; 66: 123-132Crossref PubMed Scopus (87) Google Scholar, 7Tomasini B.R. Mosher D.F. Prog. Hemostasis Thromb. 1991; 10: 269-305PubMed Google Scholar). The diverse functionality of vitronectin arises from the ability of the protein to interact with many ligands in addition to PAI-1, including cellular receptors (such as integrins and uPA receptor), glycosaminoglycans (such as heparin), extracellular components (such as collagen), and complement complexes (such as C5b-9a). Interestingly, binding to some of these ligands, including PAI-1 and complement components, causes vitronectin to experience conformational changes that alter certain functions. Reports from this laboratory (8Podor T.J. Shaughnessy S.G. Blackburn M.N. Peterson C.B. J. Biol. Chem. 2000; 275: 25402-25410Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar) and others (9Seiffert D. Loskutoff D.J. J. Biol. Chem. 1996; 271: 29644-29651Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar) indicate that these conformational changes promote the formation of large oligomeric forms of vitronectin. These multimeric forms of vitronectin exhibit an apparent increase in heparin affinity because of multivalent binding sites for the polyanionic glycosaminoglycan (10Zhuang P. Chen A.I. Peterson C.B. J. Biol. Chem. 1997; 272: 6858-6867Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Are other ligand-binding sites clustered in a similar fashion on the surface of the multimeric form of vitronectin, resulting in enhanced binding? It is intriguing to consider the potential effects of PAI-1 binding and induced multimerization of vitronectin on other functional properties of vitronectin such as cell adhesion and migration. Several lines of evidence indicate that PAI-1 and vitronectin may serve as co-factors in these processes. Vitronectin becomes incorporated into the matrix from a circulatory pool by unknown mechanisms, and its deposition into the ECM of many tissues may be a means of localizing PAI-1 function to specific sites, i.e. regions of tissue injury or tumor invasion. Vitronectin contains an important integrin-binding site (an RGD sequence) near its amino-terminal end that is capable of binding cellular integrins (11Ginsberg M.H. Loftus J.C. Plow E.F. Thromb. Haemostasis. 1988; 59: 1-6Crossref PubMed Scopus (231) Google Scholar, 12Cheresh D.A. Spiro R.C. J. Biol. Chem. 1987; 262: 17703-17711Abstract Full Text PDF PubMed Google Scholar). Current understanding dictates that PAI-1 binding to vitronectin antagonizes the cell-binding functions of vitronectin. This effect is proposed to result from steric hindrance because of the proximity of the PAI-1-binding site near the N terminus and an adjacent RGD site comprising residues 45–47 (13Stefansson S. Lawrence D.A. Nature. 1996; 383: 441-443Crossref PubMed Scopus (607) Google Scholar, 14Germer M. Kanse S.M. Kirkegaard T. Kjoller L. Felding-Habermann B. Goodman S. Preissner K.T. Eur. J. Biochem. 1998; 253: 669-674Crossref PubMed Scopus (40) Google Scholar, 15Gladson C.L. Stewart J.E. Olman M.A. Chang P.L. Schnapp L.M. Grammer J.R. Benveniste E.N. Neurosci. Lett. 2000; 283: 157-161Crossref PubMed Scopus (17) Google Scholar). On the other hand, the effects on cell binding activity that result from PAI-1-induced changes in the oligomeric state of vitronectin have not been carefully studied. Does self-association of vitronectin into multimers, as a consequence of PAI-1 binding, result in an enhanced binding to integrins and components of the ECM because of its multivalent nature? The main objective of this study was to investigate the effect that PAI-1-induced multimerization has on the adhesive functions of vitronectin. Native vitronectin was purified from human blood plasma using a modified protocol of the method developed by Dahlback and Podack (16Bittorf S.V. Williams E.C. Mosher D.F. J. Biol. Chem. 1993; 268: 24838-24846Abstract Full Text PDF PubMed Google Scholar, 17Dahlback B. Podack E.R. Biochemistry. 1985; 24: 2368-2374Crossref PubMed Scopus (148) Google Scholar). Human GPIIbIIIa was obtained from Enzyme Research Laboratories. Human αvβ3 came from Calbiochem. Recombinant human PAI-1 (wild-type) was obtained from Molecular Innovations. Latent PAI-1 was either prepared by 24-hour incubation of wild-type PAI-1 at 37 °C, or it was purchased from Molecular Innovations. The S119C mutant of PAI-1 was labeled with NBD as previously described (18Gibson A. Baburaj K. Day D.E. Verhamme I. Shore J.D. Peterson C.B. J. Biol. Chem. 1997; 272: 5112-5121Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). The labeled PAI-1 was a generous gift from Duane Day and Joseph Shore (Henry Ford Hospital, Detroit, MI). Human ECM and Matrigel were purchased from Becton Dickinson. Polyclonal antibodies against human vitronectin were generated in rabbits by contract with Rockland Laboratories. A monoclonal antibody specific for PAI-1 was the kind gift of Dr. Tom Podor (Hamilton Civic Hospitals Research Center, Hamilton, Ontario, Canada). Peroxidase-labeled secondary antibodies (anti-rabbit and anti-mouse) were obtained from Vector Laboratories. Protein standards used for HPLC came from Amersham Biosciences, Inc. Native vitronectin was phosphorylated using [γ-32P]ATP (Amersham Biosciences, Inc.) and protein kinase A (catalytic subunit, product of Sigma). Unincorporated radiolabel was removed by a desalting centrifugation step using Bio-Spin 30 columns. Specific radioactivity was determined directly by quantifying vitronectin in a BCA assay (Pierce) and measuring radioactivity in a Beckman LS3801 Scintillation Counter. Specific radioactivity was 100,000 cpm/pmol of vitronectin. Chromatography of vitronectin-PAI-1 mixtures was carried out using a Phenomenex Biosep SEC-S3000 (300 × 7.8 mm) column attached to a Hewlett-Packard 1100 series HPLC system. PBS containing 0.005% sodium azide was used for the reaction and isocratic mobile phase. Equimolar concentrations of vitronectin and PAI-1 were mixed and incubated for various times at 37 °C. In some experiments, an equimolar amount of tPA was added at the end of the incubation period to inactivate PAI-1 and dissociate it from vitronectin. On occasion, NBD-S119C PAI-1 was used in the experiment to explicitly monitor the absorbance of NBD at 494 nm as a probe for PAI-1. Samples (50 μl) were injected onto the pre-equilibrated size-exclusion column and chromatographed at room temperature at a flow rate of 1 or 0.5 ml/min. Absorbance was detected directly in the flow cell at 280 or 494 nm. Samples from HPLC (100 μl) were analyzed by PAGE on 10% polyacrylamide SDS gels (19Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207529) Google Scholar). Proteins were transferred to nitrocellulose filters using a Semi-Dry Blotter (Bio-Rad) in transfer buffer (0.35 m glycine, 25 mm Tris, containing 20.0% methanol). The filters were blocked as with 10% nonfat dry milk in PBS. The nitrocellulose membrane was washed three times after this and subsequent incubation steps with PBS containing 0.05% Tween 20. Immunoblotting was performed to detect vitronectin and PAI-1 simultaneously by incubating the filter with a 1:2500 dilution of polyclonal anti-vitronectin rabbit serum and a 1:2500 dilution of a monoclonal antibody to PAI-1 in a 2.0% solution of nonfat dry milk in PBS. The blot was then incubated with a 1:1000 dilution of both HRP-linked goat anti-rabbit and HRP-linked rabbit anti-mouse antibodies in a 20-ml solution of 2.0% milk in PBS. Immunostained protein bands were visualized by developing in a freshly prepared solution of 30 ml of PBS containing 50 mg/ml 4-chloronapthol and a 1:3,000 dilution of 30% H2O2. Changes in the fluorescence emission of NBD-S119C-PAI-1 were measured using the PerkinElmer LS50B luminescence spectrometer with an excitation wavelength of 480 nm and emission wavelength of 525 nm. Excitation and emission slits were set at 4 and 7 mm, respectively. Solutions of NBD-PAI-1 (200 nm) in HEPES buffer (100 mm HEPES, 150 mm NaCl, 1 mm EDTA, 0.1% (w/v) polyethylene glycol 8000, pH 7.4) were titrated with small aliquots of monomeric human vitronectin to a final concentration of 550 nm. Samples were mixed in a total volume of 2.0 ml in acrylic cuvettes (Sarstedt) that were pre-coated with a 1.0% (w/v) solution of polyethylene glycol 20,000, according to the procedure of Latallo and Hall (20Latallo Z.S. Hall J.A. Thromb. Res. 1986; 43: 507-521Abstract Full Text PDF PubMed Scopus (26) Google Scholar). Titrations were performed in triplicate. All data were mathematically corrected for dilution. Change in emission was calculated using the equation (F −F 0)/F 0, whereF is the emission intensity and F 0 is the starting emission intensity of NBD-PAI-1 in the absence of vitronectin. Emission scans were performed over a wavelength range of 500–600 nm using an excitation wavelength of 480 nm. Measurements were plotted as a function of titrant concentration using the graphing program KaleidaGraph (Synergy Software). Data were fit to the Hill equation. ΔF/F0=ΔFmax/F0·[S] n/((K0.5) n+[S] n)Equation 1 S is the total titrant concentration,K 0.5 corresponds to the titrant concentration at half-maximal saturation, and n is the Hill coefficient. Rabbit SMCs (American Red Cross) were grown to near confluence in tissue culture dishes in Dulbecco's modified Eagle's medium. Cells harvested by trypsinization were subsequently incubated in Dulbecco's modified Eagle's growth media containing 32P-labeled vitronectin with or without PAI-1. Control cell binding experiments also were conducted using latent PAI-1. These mixtures were then incubated at 37 °C for 1 h. Following incubation, cells were pelleted by centrifugation at 12,000 × g and washed three times with PBS followed by re-centrifugation. Washed cell pellets were then placed in a liquid scintillation mixture, and their radioactivity was measured in a Beckman LS3801 scintillation counter. 96-well ELISA plates (polypropylene or polystyrene, CoStar) were coated with 500 ng of human ECM or 2.5 μg of Matrigel in PBS. Wells were washed three times with PBS and then blocked with 3.5% BSA for 1 h at 37 °C. Vitronectin and PAI-1 were mixed in variable ratios in PBS containing 0.2% BSA and 0.1% Tween 20 (to prevent nonspecific binding), and the samples were incubated for various times at 37 °C. Following washing with buffer containing BSA and Tween, blocked wells were layered with vitronectin-PAI-1 mixtures and incubated at 37 °C for 1 h. Following incubation, mixtures were removed, wells washed, and bound vitronectin was detected by two different methods. For the human ECM binding assays, detection was immunochemical, using a polyclonal anti-vitronectin IgG and a peroxidase-conjugated anti-rabbit IgG. The plates were then developed with a 0.2 mg/ml solution of ABTS in 50 mm sodium citrate, pH 5.5, containing a 1:2000 dilution of 30% hydrogen peroxide. ELISA plates were read (λ = 405) on a Wallac Victor2 ELISA plate reader. For the Matrigel-binding experiments, 32P-labeled vitronectin was used and detected by scintillation counting. Nonspecific binding was monitored in all experiments and found to be minimal. Effects of nonspecific binding were subtracted from the reported results. Assays were also performed to test the effects of ionic strength and tPA-mediated release of PAI-1, using equimolar concentrations of tPA and PAI-1. ELISA plates (polystyrene, CoStar) were coated with 250 ng of GPIIbIIIa or αvβ3 in integrin-binding buffer (50 mm Tris, 150 mm NaCl, 1 mmMgCl2, 1 mm CaCl2). Wells were washed three times with integrin-binding buffer and then blocked with 3.5% BSA for 1 h at 37 °C. Equimolar or 1:2 mixtures of vitronectin and PAI-1 were incubated in integrin-binding buffer containing 0.2% BSA and 0.1% Tween 20 (to prevent nonspecific binding) for various times at 37 °C. Following washing with integrin-binding buffer containing BSA and Tween, blocked wells were layered with vitronectin-PAI-1 mixtures and incubated at 37 °C for 1 h. Incubation with secondary antibody-enzyme conjugates and development with substrates were performed as described above for the matrix-binding assays. Assays were also performed to test the effects of metals, ionic strength, and tPA-mediated release of PAI-1, using equimolar concentrations of tPA and PAI-1. An underlying hypothesis of this work is that the interaction of vitronectin with circulating target ligands, such as PAI-1, results in the formation of large complexes that may have enhanced binding to other potential targets, including heparin and cell-surface receptors. The increased binding is because of the multivalent nature of the binding sites for these ligands on the self-associated form of vitronectin (10Zhuang P. Chen A.I. Peterson C.B. J. Biol. Chem. 1997; 272: 6858-6867Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). For the purposes of this study, namely to investigate the effects of PAI-1 induced multimerization of vitronectin, an HPLC method of size-exclusion chromatography was employed to monitor the formation of large multivalent complexes. Fig. 1 shows chromatograms of equimolar mixtures of vitronectin and PAI-1 that were incubated for various times. Separation immediately following mixing of vitronectin and PAI-1 demonstrated the fast formation of very large complexes (elution time = 6 min). From 1 to 4 h, significant amounts of the high molecular weight complexes accumulated. Analysis at long times (up to 36 h) demonstrated that high molecular weight forms of vitronectin persisted even after the disassociation of PAI-1 from the complex upon its conversion to a latent form. In the HPLC experiments, NBD-S119C PAI-1 provided a chromophore to directly determine whether PAI-1 was an integral part of the high molecular complexes that form. As shown in Fig.2 A, the elution profile at 494 nm (the extinction maximum for NBD) confirms that PAI-1 is present in the high molecular weight complexes formed after 1 h of incubation. Furthermore, SDS-PAGE and Western blotting (Fig.2 B) demonstrated that these large complexes contained both vitronectin and PAI-1. The same approach indicated that the high molecular weight species observed at long times (e.g.36 h) comprised oligomeric forms of vitronectin without associated PAI-1. As a control, latent PAI-1 did not cause the formation of large complexes when mixed with vitronectin. These results are in agreement with findings from Seiffert and Loskutoff (9Seiffert D. Loskutoff D.J. J. Biol. Chem. 1996; 271: 29644-29651Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), as well as with a recent study from this laboratory that demonstrates self-association of vitronectin when complexed with PAI-1 (8Podor T.J. Shaughnessy S.G. Blackburn M.N. Peterson C.B. J. Biol. Chem. 2000; 275: 25402-25410Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). The information obtained from this HPLC gel filtration approach was instructive for the remainder of this study. By monitoring the formation of complexes upon vitronectin binding to PAI-1 over time, we could correlate this information with effects that PAI-1-induced multimerization has on the adhesive properties of vitronectin. We were especially interested in functions relative to matrix and/or cell interactions. Research from this laboratory (10Zhuang P. Chen A.I. Peterson C.B. J. Biol. Chem. 1997; 272: 6858-6867Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar), which demonstrates the positive effect of increased valency (upon vitronectin multimerization) on the binding of vitronectin to heparin, raises the possibility that PAI-1-induced oligomerization of vitronectin may also enhance the binding of the protein to other ligands. Of particular interest is the association of vitronectin and its complexes with the ECM, which will be enhanced by interaction with the multivalent substrates in the matrix and which may be mediated by heparin-like glycosaminoglycans. The ability of vitronectin to interact with both integrins and matrix proteins places vitronectin at the boundary of cell-matrix interactions and activities. Results presented in Fig.3 address this idea by demonstrating that vitronectin, when incubated with PAI-1, shows increased binding to matrix-coated ELISA plates. The enhanced binding of vitronectin upon incubation with PAI-1 was demonstrated using two commercially available matrices, human ECM and Matrigel. Binding of complexes is observed with both matrices and is detected using either immunochemical (panel A) or radiological detection (panel B), indicating a specific effect of PAI-1 on matrix incorporation of vitronectin. These findings support a model in which PAI-1 promotes formation of vitronectin multimers that have enhanced affinity for components that constitute the ECM. Although equimolar mixtures of vitronectin and PAI-1 exhibited enhanced association with the ECM, as shown in Fig. 3, we considered whether nonstoichiometric mixtures of the two proteins would vary in matrix-binding behavior. This experiment was initiated in light of our recently published results that demonstrate the association of vitronectin and PAI-1 into complexes with a 2:4 stoichiometry (8Podor T.J. Shaughnessy S.G. Blackburn M.N. Peterson C.B. J. Biol. Chem. 2000; 275: 25402-25410Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Thus, the concentration of vitronectin was fixed in the ECM-binding experiments, whereas the concentration of PAI-1 was varied from less than stoichiometric to a 4-fold excess relative to vitronectin. As shown in Fig. 4, matrix binding is dependent on the amount of PAI-1 present, saturating at a higher value for relative binding than observed for a simple 1:1 ratio of the two proteins in Fig. 3. For this reason, subsequent ECM-binding experiments were performed using a 2:1 ratio of PAI-1:vitronectin, and these results show a greater enhancement in ECM binding than the titrations shown in Fig. 3. Latent PAI-1, tested over a wide range in concentrations, had no effect on matrix binding by vitronectin. To correlate the observed effects of PAI-1 binding on vitronectin adhesive functions and the formation of large multimeric complexes as a result of this interaction, these properties were carefully monitored over time. Mixtures of vitronectin and PAI-1 were incubated for various times and then added to ECM-coated ELISA plates. Fig.5 A demonstrates the significant increase in the amount of vitronectin bound to matrix when PAI-1 is added versus native vitronectin alone. Strikingly, this increase in matrix deposition persists at long times after which PAI-1 is no longer present in the large complexes. These results indicate that PAI-1 binding causes a consistent enhancement of the adhesive form of vitronectin in a temporal pattern similar to that of complex formation and vitronectin multimerization observed in the HPLC time course described above. For an explicit test of the requirement for PAI-1 in the complexes for the enhancement in matrix binding, tPA was added to the incubation mixtures at timed intervals. This protease is known to associate with PAI-1 to form a stable acyl-complex, thus disrupting the vitronectin-PAI-1 interaction (21Lawrence D.A. Palaniappan S. Stefansson S. Olson S.T. Francis-Chmura A.M. Shore J.D. Ginsburg D. J. Biol. Chem. 1997; 272: 7676-7680Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Dissociation of the vitronectin-PAI-1 complex by adding tPA at early times (∼1 h) diminishes binding to the matrix, as shown in Fig. 5 A. Note that the binding observed is nevertheless enhanced relative to the binding of monomeric vitronectin. Indeed, analysis by HPLC gel filtration (Fig. 5 B) demonstrated that, by 1 h, a stable multimeric form of vitronectin that more readily associates with the matrix is present, and that it persists even after tPA-mediated release of PAI-1 from the complexes. The fact that tPA treatment reduces the amount of complexes observed indicates that active PAI-1 is present in these high molecular weight complexes formed at 1 h. From the decrease in peak height eluting at the void volume of the column, the findings also indicate that some of the complexes can disaggregate when tPA neutralizes PAI-1 and dissociates it from vitronectin. However, at longer times, adding tPA to the mixtures has little effect, indicating that the aged vitronectin aggregates exhibit enhanced matrix binding that is independent of PAI-1. Presumably, this reflects the multimeric nature of the large complexes that persist after PAI-1 dissociates. As shown in Fig.6, increasing ionic strength can reverse the association of vitronectin complexes with the matrix upon PAI-1 binding. At a 1 m NaCl concentration, the enhancement of vitronectin binding is decreased by ∼70%. These results indicate that the binding of vitronectin-PAI-1 complexes to components of the extracellular matrix is also partly dependent on ionic interactions, such as those known to occur between vitronectin and glycosaminoglycans. To ensure that these results are not because of an effect of ionic strength on the interaction between PAI-1 and vitronectin, the binding of PAI-1 and vitronectin was monitored at varying ionic strengths using a fluorescence-based assay with NBD-labeled PAI-1. The binding of the two proteins has been previously measured using this approach with PAI-1 mutants containing fluorescent labels within the reactive center loop (18Gibson A. Baburaj K. Day D.E. Verhamme I. Shore J.D. Peterson C.B. J. Biol. Chem. 1997; 272: 5112-5121Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). These former measurements were challenging because of low changes in fluorescence quantum yield upon vitronectin binding. A more suitable form of PAI-1 for the fluorescence assay is the mutant with an NBD label at a cysteine substituted for serine 119, situated near the vitronectin-binding site on PAI-1 (22Lawrence D.A. Berkenpas M.B. Palaniappan S. Ginsburg D. J. Biol. Chem. 1994; 269: 15223-15228Abstract Full Text PDF PubMed Google Scholar). Binding isotherms for NBD-S119C-PAI-1 and vitronectin, in the presence of both 150 and 500 mm NaCl, are shown in Fig. 7. Cooperative binding isotherms were observed, reflecting a complex binding/association phenomenon consistent with the ultracentrifugation data that demonstrate a nonstoichiometric binding of the two proteins accompanied by self-association of vitronectin (8Podor T.J. Shaughnessy S.G. Blackburn M.N. Peterson C.B. J. Biol. Chem. 2000; 275: 25402-25410Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Data fits to the Hill equation were used to calculate K 0.5, which can be correlated with an average binding affinity for the interaction. Under both salt conditions, a K 0.5 of ∼60 nm and a Hill coefficient of 2 were obtained. Differences observed in the maximal fluorescence change observed with the two salt concentrations are presumably because of intrinsic properties (e.g. quantum yield, exposure to solvent, quenching) of the fluorophores at the two ionic strengths. Salt-dependent quenches of fluorescence are a well established phenomenon in photobiophysics. These findings demonstrate that similar complexes form regardless of the ionic strength of the solution. Furthermore, they argue that the ionic-strength dependent effects observed in Fig. 6reflect dissoc" @default.
• W1991421191 created "2016-06-24" @default.
• W1991421191 creator A5000590070 @default.
• W1991421191 creator A5091044424 @default.
• W1991421191 date "2002-03-01" @default.
• W1991421191 modified "2023-10-11" @default.
• W1991421191 title "Plasminogen Activator Inhibitor Type 1 Promotes the Self-association of Vitronectin into Complexes Exhibiting Altered Incorporation into the Extracellular Matrix" @default.
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• W1991421191 doi "https://doi.org/10.1074/jbc.m109564200" @default.
• W1991421191 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/11796716" @default.
• W1991421191 hasPublicationYear "2002" @default.
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