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• W2074955423 abstract "Vitronectin and plasminogen activator inhibitor-1 (PAI-1) are important physiological binding partners that work in concert to regulate cellular adhesion, migration, and fibrinolysis. The high affinity binding site for PAI-1 is located within the N-terminal somatomedin B domain of vitronectin; however, several studies have suggested a second PAI-1-binding site within vitronectin. To investigate this secondary site, a vitronectin mutant lacking the somatomedin B domain (rΔsBVN) was engineered. The short deletion had no effect on heparin-binding, integrin-binding, or cellular adhesion. Binding to the urokinase receptor was completely abolished while PAI-1 binding was still observed, albeit with a lower affinity. Analytical ultracentrifugation on the PAI-1-vitronectin complex demonstrated that increasing NaCl concentration favors 1:1 versus 2:1 PAI-1-vitronectin complexes and hampers formation of higher order complexes, pointing to the contribution of charge-charge interactions for PAI-1 binding to the second site. Furthermore, fluorescence resonance energy transfer between differentially labeled PAI-1 molecules confirmed that two independent molecules of PAI-1 are capable of binding to vitronectin. These results support a model for the assembly of higher order PAI-1-vitronectin complexes via two distinct binding sites in both proteins. Vitronectin and plasminogen activator inhibitor-1 (PAI-1) are important physiological binding partners that work in concert to regulate cellular adhesion, migration, and fibrinolysis. The high affinity binding site for PAI-1 is located within the N-terminal somatomedin B domain of vitronectin; however, several studies have suggested a second PAI-1-binding site within vitronectin. To investigate this secondary site, a vitronectin mutant lacking the somatomedin B domain (rΔsBVN) was engineered. The short deletion had no effect on heparin-binding, integrin-binding, or cellular adhesion. Binding to the urokinase receptor was completely abolished while PAI-1 binding was still observed, albeit with a lower affinity. Analytical ultracentrifugation on the PAI-1-vitronectin complex demonstrated that increasing NaCl concentration favors 1:1 versus 2:1 PAI-1-vitronectin complexes and hampers formation of higher order complexes, pointing to the contribution of charge-charge interactions for PAI-1 binding to the second site. Furthermore, fluorescence resonance energy transfer between differentially labeled PAI-1 molecules confirmed that two independent molecules of PAI-1 are capable of binding to vitronectin. These results support a model for the assembly of higher order PAI-1-vitronectin complexes via two distinct binding sites in both proteins. First identified as serum-spreading factor, vitronectin was recognized as a protein in serum capable of promoting cellular adhesion and spreading (1Holmes R. J. Cell Biol. 1967; 32: 297-308Crossref PubMed Scopus (100) Google Scholar). After these initial findings, further roles for vitronectin in maintaining hemostasis, wound healing, angiogenesis, and tumor metastasis have been elucidated based on the diverse number of ligands with which it interacts. Vitronectin works to regulate hemostasis via interactions with heparin and PAI-1. 2The abbreviations used are: PAI-1, plasminogen activator inhibitor type 1; uPA, urokinase-type plasminogen activator; uPAR, urokinase receptor; rVN, recombinant full-length vitronectin; rΔsBVN, recombinant vitronectin lacking the somatomedin B domain due to deletion of residues 1–40; mVN, multimeric vitronectin; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; BSA, bovine serum albumin; SPR, surface plasmon resonance; P1–P1′, Schecter and Berger nomenclature for the reactive center loop residues of PAI-1, where P1, P2, P3, P4,… and P1′, P2′, P3′, P4′,… denote those residues on the N-terminal and C-terminal sides of the scissile bond, respectively; 5-IAF, 5-iodoacetamidofluorescein; TMRIA, tetramethylrhodamine-5-iodoacetamide; PAI-1P1′-FL, PAI-1 labeled at the P1′ residue with 5-IAF; PAI-1P1′-TMR, PAI-1 labeled at the P1′ residue with TMRIA; GPI, glycosylphosphatidylinositol. 2The abbreviations used are: PAI-1, plasminogen activator inhibitor type 1; uPA, urokinase-type plasminogen activator; uPAR, urokinase receptor; rVN, recombinant full-length vitronectin; rΔsBVN, recombinant vitronectin lacking the somatomedin B domain due to deletion of residues 1–40; mVN, multimeric vitronectin; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; BSA, bovine serum albumin; SPR, surface plasmon resonance; P1–P1′, Schecter and Berger nomenclature for the reactive center loop residues of PAI-1, where P1, P2, P3, P4,… and P1′, P2′, P3′, P4′,… denote those residues on the N-terminal and C-terminal sides of the scissile bond, respectively; 5-IAF, 5-iodoacetamidofluorescein; TMRIA, tetramethylrhodamine-5-iodoacetamide; PAI-1P1′-FL, PAI-1 labeled at the P1′ residue with 5-IAF; PAI-1P1′-TMR, PAI-1 labeled at the P1′ residue with TMRIA; GPI, glycosylphosphatidylinositol. Vitronectin competes for heparin binding with both thrombin and antithrombin, effectively reducing the anticoagulant ability of heparin (2Preissner K.T. Muller-Berghaus G. Eur. J. Biochem. 1986; 156: 645-650Crossref PubMed Scopus (29) Google Scholar). Because PAI-1 inhibits plasminogen activators that convert plasminogen into the protease plasmin, vitronectin acts to prevent fibrinolysis by prolonging the functional lifespan of PAI-1 and localizing it to fibrin clots (3Podor T.J. Campbell S. Chindemi P. Foulon D.M. Farrell D.H. Walton P.D. Weitz J.I. Peterson C.B. J. Biol. Chem. 2002; 277: 7520-7528Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). A high resolution structure of vitronectin has not been determined, although a model of the domains of vitronectin was proposed based on computer-calculated structure predictions using threading and docking algorithms (4Xu D. Baburaj K. Peterson C.B. Xu Y. Proteins. 2001; 44: 312-320Crossref PubMed Scopus (31) Google Scholar). This model predicted one complete and one incomplete β-propeller for the central and C-terminal domains, respectively, that comprise the majority of vitronectin between amino acids 131 and 456. The structure of the N-terminal somatomedin B domain (residues 1–51) could not be reliably modeled with the threading algorithm. However, direct structural determinations using NMR (5Mayasundari A. Whittemore N.A. Serpersu E.H. Peterson C.B. J. Biol. Chem. 2004; 279: 29359-29366Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 6Kamikubo Y. De Guzman R. Kroon G. Curriden S. Neels J.G. Churchill M.J. Dawson P. Oldziej S. Jagielska A. Scheraga H.A. Loskutoff D.J. Dyson H.J. Biochemistry. 2004; 43: 6519-6534Crossref PubMed Scopus (33) Google Scholar, 7Kamikubo Y. Kroon G. Curriden S.A. Dyson H.J. Loskutoff D.J. Biochemistry. 2006; 45: 3297-3306Crossref PubMed Scopus (11) Google Scholar, 8Kjaergaard M. Gardsvoll H. Hirschberg D. Nielbo S. Mayasundari A. Peterson C.B. Jansson A. Jørgensen T.J.D. Poulsen F. Ploug M. Prot. Sci. 2007; 16: 1934-1945Crossref PubMed Scopus (30) Google Scholar) and x-ray crystallography (9Zhou A. Huntington J.A. Pannu N.S. Carrell R.W. Read R.J. Nat. Struct. Biol. 2003; 10: 541-544Crossref PubMed Scopus (220) Google Scholar) have elucidated the structure of the somatomedin B domain. Although there is some conflict among the structures that is rooted in the identification of disulfide bonds (6Kamikubo Y. De Guzman R. Kroon G. Curriden S. Neels J.G. Churchill M.J. Dawson P. Oldziej S. Jagielska A. Scheraga H.A. Loskutoff D.J. Dyson H.J. Biochemistry. 2004; 43: 6519-6534Crossref PubMed Scopus (33) Google Scholar, 9Zhou A. Huntington J.A. Pannu N.S. Carrell R.W. Read R.J. Nat. Struct. Biol. 2003; 10: 541-544Crossref PubMed Scopus (220) Google Scholar, 10Kamikubo Y. Okumura Y. Loskutoff D.J. J. Biol. Chem. 2002; 277: 27109-27119Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 11Horn N.A. Hurst G.B. Mayasundari A. Whittemore N.A. Serpersu E.H. Peterson C.B. J. Biol. Chem. 2004; 279: 35867-35878Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 12Li X. Zou G. Yuan W. Lu W. J. Biol. Chem. 2007; 282: 5318-5326Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 13Zhou A. Prot. Sci. 2007; 16: 1502-1508Crossref PubMed Scopus (21) Google Scholar), in all of the structures on the somatomedin B domain, the most important feature is a single turn α-helix that defines the binding interface with PAI-1. Small-angle x-ray scattering on monomeric vitronectin generated a low resolution model that revealed a bi-lobed structure for vitronectin (14Lynn G.W. Heller W.T. Mayasundari A. Minor K.H. Peterson C.B. Biochemistry. 2005; 44: 565-574Crossref PubMed Scopus (36) Google Scholar). Assembling the NMR and x-ray scattering data with the structure predicted from threading suggests that the C-terminal domain lies within a separate lobe from the N-terminal somatomedin B domain (14Lynn G.W. Heller W.T. Mayasundari A. Minor K.H. Peterson C.B. Biochemistry. 2005; 44: 565-574Crossref PubMed Scopus (36) Google Scholar). PAI-1, the primary inhibitor of the plasminogen activators uPA and tissue plasminogen activator belongs to the serine protease inhibitor (serpin) superfamily. PAI-1 shares the common structural features and mechanism of inhibition characteristic of the other inhibitory members of the serpin family (15Huber R. Carrell R.W. Biochemistry. 1989; 28: 8951-8966Crossref PubMed Scopus (828) Google Scholar, 16Stout T.J. Graham H. Buckley D.I. Matthews D.J. Biochemistry. 2000; 39: 8460-8469Crossref PubMed Scopus (97) Google Scholar). The tertiary structure of PAI-1 is composed of nine α-helices (hA–hI), three β-sheets (A, B, and C), and a solvent-exposed unstructured loop of ∼20 amino acids referred to as the reactive center loop, because it carries the inhibitory reactive peptide bond. The overall scaffold of the molecule is defined by the five-stranded central β-sheet A. PAI-1 spontaneously alters in conformation, converting from the active state to an energetically more favorable inactive latent state (16Stout T.J. Graham H. Buckley D.I. Matthews D.J. Biochemistry. 2000; 39: 8460-8469Crossref PubMed Scopus (97) Google Scholar, 17Mottonen J. Strand A. Symersky J. Sweet R.M. Danley D.E. Geoghegan K.F. Gerard R.D. Goldsmith E.J. Nature. 1992; 355: 270-273Crossref PubMed Scopus (520) Google Scholar, 19Baker D. Agard D.A. Biochemistry. 1994; 33: 7505-7509Crossref PubMed Scopus (225) Google Scholar), where the reactive center loop translocates into the central β-sheet. The interaction between vitronectin and PAI-1 has important consequences for the regulation of cellular adhesion to the extracellular matrix of the vasculature. Vitronectin binds a number of integrins as well as the GPI-linked urokinase receptor, uPAR, at sites in the region of the somatomedin B domain. When PAI-1 binds the somatomedin B domain of vitronectin, it directly competes for binding with uPAR at an overlapping binding site, thereby inhibiting uPAR-mediated cellular adhesion (20Waltz D.A. Natkin L.R. Fujita R.M. Wei Y. Chapman H.A. J. Clin. Invest. 1997; 100: 58-67Crossref PubMed Scopus (260) Google Scholar, 21Deng G. Curriden S.A. Wang S. Rosenberg S. Loskutoff D.J. J. Cell Biol. 1996; 134: 1563-1571Crossref PubMed Scopus (429) Google Scholar). Integrin-mediated cellular attachment may also be affected by PAI-1 binding to the somatomedin B domain, presumably by blocking the RGD sequence (22Stefansson S. Lawrence D.A. Nature. 1996; 383: 441-443Crossref PubMed Scopus (604) Google Scholar), which is found at residues 45–47. PAI-1 has also been shown to detach cells by disrupting the binding of integrin-uPAR complexes to vitronectin, fibronectin, and collagen type-1 (23Czekay R.P. Aertgeerts K. Curriden S.A. Loskutoff D.J. J. Cell Biol. 2003; 160: 781-791Crossref PubMed Scopus (269) Google Scholar). Because of the diverse roles of these proteins, investigating the nature of PAI-1 binding to vitronectin has been an important area of research. Much effort has been focused on defining the PAI-1-binding site within vitronectin. A large body of work has characterized the somatomedin B domain of vitronectin as the principal site of PAI-1 binding (21Deng G. Curriden S.A. Wang S. Rosenberg S. Loskutoff D.J. J. Cell Biol. 1996; 134: 1563-1571Crossref PubMed Scopus (429) Google Scholar, 24Royle G. Deng G. Seiffert D. Loskutoff D.J. Anal. Biochem. 2001; 296: 245-253Crossref PubMed Scopus (10) Google Scholar, 25Seiffert D. Loskutoff D.J. J. Biol. Chem. 1991; 266: 2824-2830Abstract Full Text PDF PubMed Google Scholar, 26Okumura Y. Kamikubo Y. Curriden S.A. Wang J. Kiwada T. Futaki S. Kitagawa K. Loskutoff D.J. J. Biol. Chem. 2002; 277: 9395-9404Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). The PAI-1-binding site within the somatomedin B domain has been restricted to amino acids 24–30 using monoclonal antibodies and mutagenesis (24Royle G. Deng G. Seiffert D. Loskutoff D.J. Anal. Biochem. 2001; 296: 245-253Crossref PubMed Scopus (10) Google Scholar). Recent crystallographic (9Zhou A. Huntington J.A. Pannu N.S. Carrell R.W. Read R.J. Nat. Struct. Biol. 2003; 10: 541-544Crossref PubMed Scopus (220) Google Scholar) and NMR (5Mayasundari A. Whittemore N.A. Serpersu E.H. Peterson C.B. J. Biol. Chem. 2004; 279: 29359-29366Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 6Kamikubo Y. De Guzman R. Kroon G. Curriden S. Neels J.G. Churchill M.J. Dawson P. Oldziej S. Jagielska A. Scheraga H.A. Loskutoff D.J. Dyson H.J. Biochemistry. 2004; 43: 6519-6534Crossref PubMed Scopus (33) Google Scholar, 7Kamikubo Y. Kroon G. Curriden S.A. Dyson H.J. Loskutoff D.J. Biochemistry. 2006; 45: 3297-3306Crossref PubMed Scopus (11) Google Scholar) structural studies have provided details about the PAI-1-binding site in the somatomedin B domain in the vicinity of a single-turn α-helix between residues 26 and 30. Conversely, the complementary vitronectin binding site within PAI-1 has been thoroughly investigated. A region between helices D, E, and F in PAI-1 termed the flexible joint region was proposed to comprise the primary binding site for vitronectin by Lawrence et al. (27Lawrence D.A. Berkenpas M.B. Palaniappan S. Ginsburg D. J. Biol. Chem. 1994; 269: 15223-15228Abstract Full Text PDF PubMed Google Scholar) in 1994 and was finally fully mapped to this region by Jensen et al. (28Jensen J.K. Wind T. Andreasen P.A. FEBS Lett. 2002; 521: 91-94Crossref PubMed Scopus (54) Google Scholar) in 2002. The epitope was later confirmed by x-ray crystallography studies of the PAI-1-somatomedin B complex (9Zhou A. Huntington J.A. Pannu N.S. Carrell R.W. Read R.J. Nat. Struct. Biol. 2003; 10: 541-544Crossref PubMed Scopus (220) Google Scholar). However, there is evidence that PAI-1 can bind to a region other than the somatomedin B domain of vitronectin. Competition experiments using synthetic peptides or monoclonal antibodies (29Gechtman Z. Sharma R. Kreizman T. Fridkin M. Shaltiel S. FEBS Lett. 1993; 315: 293-297Crossref PubMed Scopus (35) Google Scholar, 30Kost C. Stuber W. Ehrlich H.J. Pannekoek H. Preissner K.T. J. Biol. Chem. 1992; 267: 12098-12105Abstract Full Text PDF PubMed Google Scholar, 31Preissner K.T. Grulich-Henn J. Ehrlich H.J. Declerck P. Justus C. Collen D. Pannekoek H. Muller-Berghaus G. J. Biol. Chem. 1990; 265: 18490-18498Abstract Full Text PDF PubMed Google Scholar, 32Podor 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 cleavage of vitronectin with proteases (33Chain D. Kreizman T. Shapira H. Shaltiel S. FEBS Lett. 1991; 285: 251-256Crossref PubMed Scopus (50) Google Scholar) have identified a PAI-1-binding site in the C-terminal region of vitronectin or a site in the connecting region (34Mimuro J. Muramatsu S. Kurano Y. Uchida Y. Ikadai H. Watanabe S. Sakata Y. Biochemistry. 1993; 32: 2314-2320Crossref PubMed Scopus (20) Google Scholar). Studying vitronectin-PAI-1 complexes with analytical ultracentrifugation revealed a 2:1 PAI-1-vitronectin binding stoichiometry and the formation of higher order complexes at a PAI-1-vitronectin ratio of 4:2 (32Podor 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, 35Minor K.H. Schar C.R. Blouse G.E. Shore J.D. Lawrence D.A. Schuck P. Peterson C.B. J. Biol. Chem. 2005; 280: 28711-28720Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). These reports of alternative PAI-1-binding sites, coupled with the unusual stoichiometry of the vitronectin-PAI-1 complex, have caused us to pursue a deletion mutagenesis approach to address this problem. The studies reported here were designed to evaluate directly whether there is a second site for the binding of PAI-1 to vitronectin that lies outside the well characterized N-terminal site in the somatomedin B domain. A mutant form of vitronectin, rΔsBVN, was engineered omitting the first 40 amino acids, so that the mature peptide begins at lysine 41 upon secretion and removal of the signal sequence. These experiments confirm a second PAI-1-binding site within vitronectin that lies outside of the somatomedin B region. Materials—Native vitronectin was purified from human plasma using a modified protocol of the method developed by Dahlback and Podack (36Bittorf S.V. Williams E.C. Mosher D.F. J. Biol. Chem. 1993; 268: 24838-24846Abstract Full Text PDF PubMed Google Scholar, 37Dahlback B. Podack E.R. Biochemistry. 1985; 24: 2368-2374Crossref PubMed Scopus (147) Google Scholar). Multimeric vitronectin was prepared by denaturation of the protein in 8 m urea for 2 h at room temperature followed by dialysis into PBS (140 mm NaCl, 3 mm KCl, 10 mm Na2HPO4, 2 mm KH2PO4, pH 7.4). Recombinant human wild-type PAI-1 and recombinant stable PAI-1 mutant, 14-1B (38Berkenpas M.B. Lawrence D.A. Ginsburg D. EMBO J. 1995; 14: 2969-2977Crossref PubMed Google Scholar), used in the ELISA assays were both purchased from Molecular Innovations, Inc. PAI-1 used in the Biacore experiments and latency transition assays was produced according to the method outlined in Jensen et al. (28Jensen J.K. Wind T. Andreasen P.A. FEBS Lett. 2002; 521: 91-94Crossref PubMed Scopus (54) Google Scholar). The PAI-1 protein preparations were tested for activity using a urokinase inhibition assay; all behaved similarly and fully inhibited urokinase. The numbering used to denote the amino acid positions in PAI-1 throughout the presented work is Ser1-Ala2-Val3-His4-His5, according to Andreasen et al. (39Andreasen P.A. Riccio A. Welinder K.G. Douglas R. Sartorio R. Nielsen L.S. Oppenheimer C. Blasi F. Dano K. FEBS Lett. 1986; 209: 213-218Crossref PubMed Scopus (169) Google Scholar). All other reagents were of analytical reagent grade or better. Cell Culture—Cultures of Spodoptera frugiperda (Sf9) or Trichoplusnia ni (Hi5) cells (Invitrogen) were routinely grown as monolayers in Corning Ti 75-cm2 culture flasks at 27 °C or as suspension cultures in side-arm spinner flasks with gentle stirring at room temperature. Sf9 cells were grown in serum-free Ex-Cell 420 medium, whereas Hi5 cells were grown in Ex-Cell 401 (both from J. R. H. Biosciences) without the addition of serum. Rabbit smooth muscle cells (the kind gift of Dr. Daniel Lawrence, University of Michigan) were grown in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum. U937 cells (American Type Culture Collection) were grown in RPMI 1640 media supplemented with 10% fetal bovine serum. U937 cells were stimulated for 24 h with 1 ng/ml human transforming growth factor β-1 (Sigma) and 50 nm 1α25-dihydroxyvitamin D3 (Calbiochem) (40Waltz D.A. Chapman H.A. J. Biol. Chem. 1994; 269: 14746-14750Abstract Full Text PDF PubMed Google Scholar). Generation of Recombinant Vitronectin—Both full-length vitronectin and a deletion mutant of vitronectin lacking the first 40 amino acids were expressed in the baculovirus FastBac system (Invitrogen) as in the method described earlier (41Gibson A.D. Peterson C.B. Biochim. Biophys. Acta. 2001; 1545: 289-304Crossref PubMed Scopus (8) Google Scholar). Both constructs were subcloned into the pFastBac-1 vector (Invitrogen) and utilized the endogenous vitronectin signal sequence to direct protein secretion. The deletion mutant, rΔsBVN, was generated using the following primers: ΔsB sense, 5′-GGCTGTCGACAAGCCCCAAGTGACTCGCGG-3′ and ΔsB antisense, 5′-GGGGCTTGTCGACAGCCAGAGCAACCCATG-3′. The C termini of both recombinant vitronectin and rΔsBVN have an additional 28 amino acids, containing an myc epitope and a 6× histidine tag. The manufacturer's guidelines were followed to transfect the insect cells for virus generation and subsequent protein expression. Both expressed recombinant vitronectin (rVN) and rΔsBVN were secreted into the medium, and clarified spent medium was passed over Chelating Sepharose-Fast Flow resin (Amersham Biosciences). The column was washed with binding buffer (5 mm imidazole, 0.5 m NaCl, 20 mm Tris, pH 7.9) followed by wash buffer (60 mm imidazole, 0.5 m NaCl, 20 mm Tris, pH 7.9). The bound protein was eluted with elution buffer containing 1 m imidazole. Fractions were analyzed by Western blotting and Coomassie staining. Fractions containing protein were pooled, dialyzed, and concentrated. Protein concentration was determined as described previously (41Gibson A.D. Peterson C.B. Biochim. Biophys. Acta. 2001; 1545: 289-304Crossref PubMed Scopus (8) Google Scholar) by quantitative ELISA and the Pierce BCA Protein Assay. The protein was also subjected to N-terminal protein sequencing, confirming the deletion of the somatomedin B domain as well as cleavage of all but the last amino acid (valine) of the signal sequence. Over the course of these studies, rΔsBVN was expressed from the initial viral stock multiple times. All expressed protein was tested using PAI-1 and heparin binding assays to confirm there was no significant preparation to preparation variability. Heparin Binding Assays—A direct heparin binding assay was used to measure vitronectin binding to heparin-coated microtiter plates as previously described (41Gibson A.D. Peterson C.B. Biochim. Biophys. Acta. 2001; 1545: 289-304Crossref PubMed Scopus (8) Google Scholar). Briefly, wells were coated with a 1 mg/ml solution of heparin (Sigma) then blocked using 3% casein in PBS. The plates were incubated with serial dilutions of native, multimeric, and recombinant vitronectin. Bound vitronectin was detected using polyclonal antibodies to vitronectin and peroxidase-labeled anti-rabbit secondary antibodies (Vector Laboratories). Kinetic analysis of the rate of inactivation of thrombin by antithrombin in the presence of heparin and the recombinant proteins was measured by continuously monitoring the cleavage of the chromogenic thrombin substrate Chromozym-TH (Roche Applied Science) over time (42Peterson C.B. Morgan W.T. Blackburn M.N. J. Biol. Chem. 1987; 262: 7567-7574Abstract Full Text PDF PubMed Google Scholar). Thrombin was diluted in reaction buffer (PBS with 0.1% polyethylene glycol 8000) to a concentration of 44 nm. A final concentration of 45 nm antithrombin was used in the assay, resulting in 70–80% inhibition of thrombin. Chromozym-TH and heparin (average molecular weight, Mr 6000) were included in the reaction at a final concentration of 0.19 nm and 0.9 μg/ml, respectively. The reactions were monitored at 405 nm for 3 min at 30 °C in 1-ml acrylic cuvettes (Sarstedt). The data were analyzed using the IGOR software package (Wavemetrics) to determine the pseudo-first-order reaction rates. Cell Adhesion Assays—Two different cell lines, from muscular and monocyte origin, were used to assess the effect of deleting the N-terminal of vitronectin on binding to integrins and uPAR. Adherence of rabbit smooth muscle cells to recombinant vitronectin was determined by a method adapted from Stefansson and Lawrence (22Stefansson S. Lawrence D.A. Nature. 1996; 383: 441-443Crossref PubMed Scopus (604) Google Scholar), who showed this interaction to be primarily integrin-mediated. The wells of a 24-well tissue culture plate were coated overnight at 4 °C with a 10 nm solution of vitronectin or recombinant vitronectin diluted in PBS. After rinsing with PBS, the wells were blocked with 500 μl of a 3.5% solution of BSA in PBS at room temperature for 1 h. Rabbit smooth muscle cells were resuspended in serum-free medium containing 1.5% BSA to a concentration of 75,000 cells/well and allowed to adhere for 45 min. Bound cells were quantified by measuring cell surface acid phosphatase. After rinsing the wells to remove any unbound cells, 10 mg/ml p-nitrophenyl phosphate in 0.1 m sodium acetate, pH 5.0, was added to the wells. The reaction was stopped after 1 h with the addition of an equal volume of 1 m Tris, pH 9.0, and the absorbance was measured at 405 nm. A human lymphoma cell line, U937, was also tested for uPAR-mediated adhesion to wild-type vitronectin and rΔsBVN. For the U937 cell binding assay, 140 nm vitronectin or a recombinant vitronectin derivative was added to the wells of a FluoroNunc plate with Maxisorb Surface microtiter plate (Nunc) suitable for fluorescent applications. Wells were blocked with a solution of 3% BSA in PBS. Stimulated U937 cells were fluorescently labeled by uptake of 10 μm calcein AM (Molecular Probes) for 30 min. Cells were washed two times with serum-free RPMI medium and resuspended at a concentration of 2 × 105 cells/well in serum-free RPMI plus 20 nm uPA (Calbiochem). After an incubation of 1 h at 37 °C in 5% CO2, the plate was rinsed with PBS to remove unbound cells. Cell binding was determined by measuring the fluorescence (ex 485/em 535) of the adhered cells in a Wallac Victor2 1420 Multilabel Counter. Integrin Binding Assay—Microtiter plates were coated with 100 μl of a 2.5 μg/ml solution of the integrin GPIIbIIIa in binding buffer (50 mm Tris, pH 7.4, 0.1 m NaCl, 1 mm MgCl2, 1 mm CaCl2) according to the protocol described in a previous study (43Minor K.H. Peterson C.B. J. Biol. Chem. 2002; 277: 10337-10345Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Wells were blocked with 3.5% BSA in binding buffer for 1 h at room temperature. Recombinant vitronectin was diluted to 500 nm in binding buffer plus 0.1% Tween and 0.2% BSA and serially diluted down the plate. After incubating for 1 h at 37 °C, the wells were rinsed and anti-vitronectin monoclonal antibody (Quidel, 1:10,000) was added to the wells for 1 h at room temperature. Peroxidase-labeled anti-mouse secondary antibody was added for 1 h at room temperature. The plates were developed using a 0.2 mg/ml solution of 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt in 50 mm sodium citrate, pH 5.5, containing 12 μl of 30% H2O2. Absorbance was read at 405 nm. All the following ELISA assays were developed and read in the same manner. uPAR Binding Assay—The binding of uPAR to recombinant vitronectin was measured as described by Sidenius et al. (44Sidenius N. Andolfo A. Fesce R. Blasi F. J. Biol. Chem. 2002; 277: 27982-27990Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) with slight modification. The wells of a microtiter plate were coated with multimeric vitronectin and recombinant vitronectin at a concentration of 15 nm in PBS. A 2:1 solution of uPAR (R & D Systems) and uPA (Calbiochem) in PBS containing 0.1% BSA and 0.1% Tween was mixed and allowed to incubate for 1 h at room temperature. Vitronectin-coated wells were blocked with 3% BSA in PBS, and the uPAR:uPA mixture (final concentration per well 60 and 30 nm, respectively) was added to the wells. Following the 1-h incubation, a polyclonal anti-uPAR antibody (Molecular Innovations) was added. The plate was developed as described above. PAI-1 Binding Assays—Competition experiments were used to compare interactions between PAI-1 and native vitronectin or the recombinant vitronectin samples, as described by Seiffert and Loskutoff (25Seiffert D. Loskutoff D.J. J. Biol. Chem. 1991; 266: 2824-2830Abstract Full Text PDF PubMed Google Scholar) with slight modification. Microtiter plates were coated with a 1 μg/ml solution of native vitronectin in PBS overnight at 4 °C. Wells were blocked with 3% BSA in PBS for 1 h at 37 °C then washed. PBS containing 0.1% BSA and 0.1% Tween 20 was used for the washes and for protein dilutions. Vitronectin or recombinant vitronectin was serially diluted on the plate following blocking. A constant concentration of either wild-type or stable PAI-1 (0.4 nm) was added to the wells, and the mixture was incubated at 37 °C for 2 h. The amount of PAI-1 that bound to the immobilized vitronectin was detected using polyclonal anti-PAI-1 antibodies. Analysis of PAI-1 Binding to rΔsBVN by Surface Plasmon Resonance—All SPR experiments were performed on a BIACORE 3000™ instrument. Recombinant ΔsBVN was coupled directly to the surface of a CM5™ chip by standard amine coupling protocol by applying a 30 μg/ml vitronectin, 10 mm sodium acetate, pH 4.5, until a density of 600 response units accumulated on the chip. Ethanolamine blocked empty flow cells were used as a reference. When necessary, regeneration of the flow cells between runs was achieved by injecting pulses of 10 mm glycine/HCl, pH 2.0, and/or 0.05% SDS in running buffer until baseline levels were acquired. To determine the binding affinity for PAI-1 binding to rΔsBVN, 120-μl samples of active PAI-1 diluted in running buffer were injected in an array of concentrations from 0.25 to 1500 nm at a flow rate of 30 μl/min. The equilibrium dissociation constant (KD) was calculated according to the method outlined by Rich and Myszka (45Rich R.L" @default.
• W2074955423 created "2016-06-24" @default.
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• W2074955423 date "2008-04-01" @default.
• W2074955423 modified "2023-09-27" @default.
• W2074955423 title "A Deletion Mutant of Vitronectin Lacking the Somatomedin B Domain Exhibits Residual Plasminogen Activator Inhibitor-1-binding Activity" @default.
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• W2074955423 doi "https://doi.org/10.1074/jbc.m708017200" @default.
• W2074955423 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2447658" @default.
• W2074955423 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/18174166" @default.
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