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• W1990971755 abstract "Plasminogen activator inhibitor-1 is the main physiological regulator of tissue-type plasminogen activator in normal plasma. In addition to its critical function in fibrinolysis, plasminogen activator inhibitor-1 has been implicated in roles in other physiological and pathophysiological processes. To investigate structure-function aspects of mouse plasminogen activator inhibitor-1, the recombinant protein was expressed in Escherichia coli and purified. Five variant recombinant murine proteins (R76E, Q123K, R346A, R101A, and Q123K/R101A) were also generated using site-directed mutagenesis. The variant (R346A) was found to be defective in its inhibitory activity against tissue plasminogen activator relative to its wild-type counterpart. Enzyme-linked immunosorbent assay and surface plasmon resonance experiments demonstrated reduced vitronectin-binding affinity of the (Q123K) variant (KD = 1800 nm) relative to the wild-type protein (KD = 5.4 nm). Kinetic analyses indicated that the (Q123K) variant had a slower association (kon = 2.92 × 104m-1 s-1) to, and a faster dissociation from, vitronectin (koff = 5.3 × 10-2 s-1), (wild-type kon = 1.03 × 106m-1 s-1 and koff = 5.27 × 10-3 s-1). The Q123K/R101A variant demonstrated an even lower vitronectin-binding ability. Low density lipoprotein receptor-related protein binding was decreased for the (R76E) variant. It was also demonstrated that the plasminogen activator inhibitor-1/vitronectin complex decreased the interaction of plasminogen activator inhibitor-1 with low density lipoprotein receptor-related protein. These results indicate that the complex interactions traditionally associated with different plasminogen activator inhibitor-1 functions apply to the murine system, thus showing a commonality of subtle functions among different species and evolutionary conservation of this protein. Further, this study provides additional evidence that the human hemostasis system can be studied effectively in the mouse, which is a great asset for investigations with gene-altered mice. Plasminogen activator inhibitor-1 is the main physiological regulator of tissue-type plasminogen activator in normal plasma. In addition to its critical function in fibrinolysis, plasminogen activator inhibitor-1 has been implicated in roles in other physiological and pathophysiological processes. To investigate structure-function aspects of mouse plasminogen activator inhibitor-1, the recombinant protein was expressed in Escherichia coli and purified. Five variant recombinant murine proteins (R76E, Q123K, R346A, R101A, and Q123K/R101A) were also generated using site-directed mutagenesis. The variant (R346A) was found to be defective in its inhibitory activity against tissue plasminogen activator relative to its wild-type counterpart. Enzyme-linked immunosorbent assay and surface plasmon resonance experiments demonstrated reduced vitronectin-binding affinity of the (Q123K) variant (KD = 1800 nm) relative to the wild-type protein (KD = 5.4 nm). Kinetic analyses indicated that the (Q123K) variant had a slower association (kon = 2.92 × 104m-1 s-1) to, and a faster dissociation from, vitronectin (koff = 5.3 × 10-2 s-1), (wild-type kon = 1.03 × 106m-1 s-1 and koff = 5.27 × 10-3 s-1). The Q123K/R101A variant demonstrated an even lower vitronectin-binding ability. Low density lipoprotein receptor-related protein binding was decreased for the (R76E) variant. It was also demonstrated that the plasminogen activator inhibitor-1/vitronectin complex decreased the interaction of plasminogen activator inhibitor-1 with low density lipoprotein receptor-related protein. These results indicate that the complex interactions traditionally associated with different plasminogen activator inhibitor-1 functions apply to the murine system, thus showing a commonality of subtle functions among different species and evolutionary conservation of this protein. Further, this study provides additional evidence that the human hemostasis system can be studied effectively in the mouse, which is a great asset for investigations with gene-altered mice. Plasminogen activator inhibitor-1 (PAI-1) 1The abbreviations used are: PAI-1, plasminogen activator inhibitor-1; SERPIN, serine protease inhibitor; uPA, urokinase; tPA, tissue plasminogen activator; VN, vitronectin; uPAR, urokinase receptor; LRP, low density lipoprotein receptor-related protein; sctPA, single-chain tPA; ELISA, enzyme-linked immunosorbent assay; WT, wild-type; NTA, nickel-nitrilotriacetic acid; pNA, p-nitroaniline; SPR, surface plasmon resonance; RU, response units; RCL, reactive center loop. 1The abbreviations used are: PAI-1, plasminogen activator inhibitor-1; SERPIN, serine protease inhibitor; uPA, urokinase; tPA, tissue plasminogen activator; VN, vitronectin; uPAR, urokinase receptor; LRP, low density lipoprotein receptor-related protein; sctPA, single-chain tPA; ELISA, enzyme-linked immunosorbent assay; WT, wild-type; NTA, nickel-nitrilotriacetic acid; pNA, p-nitroaniline; SPR, surface plasmon resonance; RU, response units; RCL, reactive center loop. is a member of the serine protease inhibitor (SERPIN) superfamily and is the primary physiological regulator of urokinase (uPA) and tissue plasminogen activator (tPA) activity (1Von Mourik J.A. Lawrence D.A. Loskutoff D.J. J. Biol. Chem. 1984; 259: 14914-14921Abstract Full Text PDF PubMed Google Scholar). The inactivation of these latter proteases with PAI-1 is mediated through high affinity interactions, resulting in the formation of stable serpin-protease complexes (2Francis C.W. Marder V.J. Colman R.W. Hirsch J. Marder V.J. Salzman E.W. Hemostasis and Thrombosis: Basic Principles and Clinical Practice. J. B. Lippincott Company, Philadelphia, PA1987: 358Google Scholar). PAI-1 has two major conformations, an active and a latent form. PAI-1 is a labile protein unless complexed with the plasma protein, vitronectin (VN), which stabilizes PAI-1 in an active form (3Declerck P.J. DeMol 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). Otherwise, PAI-1 would be converted to a latent conformation and become inactivated. VN is also present in the extracellular matrix of many tissues and may serve to selectively localize PAI-1 function (3Declerck P.J. DeMol 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 urokinase receptor (uPAR) also binds to VN, and the N-terminal region of this matrix protein contains the binding domain for both of these proteins (4Deng G. Curriden S.A. Wang S. Rosenberg S. Loskutoff D.J. J. Cell Biol. 1996; 13: 1563-1571Crossref Scopus (430) Google Scholar). Occupancy of the receptor by uPA results in an increased affinity for VN (5Wei Y. Waltz D.A. Rao N. Drummond R.J. Rosenberg S. Chapman H.A. J. Biol. Chem. 1994; 269: 32380-32388Abstract Full Text PDF PubMed Google Scholar), as well as activation of intracellular signaling reactions that regulate focal adhesion turnover (6Tang H. Kerins D.M. Hao Q. Inagami T. Vaughan D.E. J. Biol. Chem. 1998; 273: 18268-18272Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Colocalization of this complex with integrins is the result of RGD-containing binding sites for αv integrins in VN, which are located adjacent to the N-terminal domain of VN (7Suzuki S. Oldberg A. Hayman E.G. Pierschbacher M.D. Ruoslahti E. EMBO J. 1985; 4: 2519-2524Crossref PubMed Scopus (277) Google Scholar). This complex can lead to local dissolution of extracellular matrix protein through activation of a proteolytic cascade. Regulation of this process occurs as a result of active PAI-1 sterically interfering with binding of either uPAR or integrin to VN (4Deng G. Curriden S.A. Wang S. Rosenberg S. Loskutoff D.J. J. Cell Biol. 1996; 13: 1563-1571Crossref Scopus (430) Google Scholar, 8Stefansson S. Lawrence D.A. Nature. 1996; 383: 441-443Crossref PubMed Scopus (605) Google Scholar, 9Loskutoff D.J. Curriden S.A. Hu G. Deng G. APMIS. 1999; 107: 54-61Crossref PubMed Scopus (144) Google Scholar). Additionally, the interaction of PAI-1 with the surface-associated uPA-uPAR complex induces internalization of the complex via interaction with low density lipoprotein receptor-related protein (LRP) (10Chapman H.A. Curr. Opin. Cell Biol. 1997; 9: 714-724Crossref PubMed Scopus (421) Google Scholar). This process alters cytoplasmic signaling via uPA-uPAR. Because cell proliferation relies on a cycle of cell attachment, spreading, flattening (to initiate DNA synthesis), and then rounding up (the latter property resulting from disengagement of cell contacts through dissolution of the matrix), it would seem that alterations in PAI-1 expression could have a profound effect upon these cellular events.Previous in vitro studies have elaborated structure/function relationships of human PAI-1, resulting in the delineation of functional domains that play a significant role in regulating its pathophysiology. Identification of critical sequences within specific domains has been achieved through structural and site-directed mutagenesis studies. For example, it is now known that human PAI-1 can inhibit VN-mediated cell adhesion and migration through interaction with VN. Sequences within human PAI-1, specifically encompassing the region 110-147, primarily localized in the α helix E, have been identified that mediate this interaction (11Lawrence D.A. Berkenpas M.B. Palanniappan S. Ginsburg D. J. Biol. Chem. 1994; 269: 15223-15228Abstract Full Text PDF PubMed Google Scholar, 12Arroyo De Prada N. Schroeck F. Sinner E.K. Muehlenweg B. Twellmeyer J. Speri S. Wilhelm O.G. Schmitt M. Magdolen V. Eur. J. Biochem. 2002; 269: 184-192Crossref PubMed Scopus (38) Google Scholar). Further, a mutation at amino acid 123 (Q → K) has been shown to maintain PAI-1 serpin activity, but to significantly reduce affinity for VN (11Lawrence D.A. Berkenpas M.B. Palanniappan S. Ginsburg D. J. Biol. Chem. 1994; 269: 15223-15228Abstract Full Text PDF PubMed Google Scholar). In contrast, an R → A mutation at position 346, a critical residue in the reactive center loop, leads to a variant that interacts with the active site of target proteases, binds VN normally, but does not inhibit plasminogen activation (8Stefansson S. Lawrence D.A. Nature. 1996; 383: 441-443Crossref PubMed Scopus (605) Google Scholar). In terms of LRP interactions, a cryptic site within the heparin-binding domain was identified (13Stefansson S. Muhammad S. Cheng X.F. Battey F.D. Strickland D.K. Lawrence D.A. J. Biol. Chem. 1998; 273: 6358-6366Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). An R → E mutation at amino acid position 76 of PAI-1 within this domain results in a protein with preserved inhibitor activity but diminished affinity for LRP. These targeted mutations of human PAI-1 indicate that functional changes at sites other than those specifically targeted are minor and, therefore, are ideal for delineating specific functional domains in select biological processes involving PAI-1. This study was aimed at an exploration of these functional domains in the murine PAI-1 through the expression and characterization of site-specific mutations in recombinant murine PAI-1. One practical value of this work is to evaluate the commonality of these subtle functions of PAI-1 across species, with special emphasis on the murine system, which is so widely employed for in vivo studies of PAI-1. The results of this investigation are described herein.EXPERIMENTAL PROCEDURESMaterials—Restriction enzymes, T4 DNA ligase, reverse transcriptase, and TaqDNA polymerase were purchased from Promega (Madison, WI), unless otherwise specified. Oligonucleotides were synthesized by MWG Biotech (High Point, NC). The gel purification, PCR product purification, and plasmid mini/midi preparation kits were purchased from Qiagen (Valencia, CA). The chromogenic substrates H-d-Ile-Pro-Arg-pNA (S-2288) and pyro Glu-Phe-Lys-pNA (S-2403) were purchased from Chromogenix AB (West Chester, OH). Human VN, purified under non-denaturing conditions, was obtained from Promega. Human recombinant single-chain tPA (sctPA) was obtained from Genentech (San Francisco, CA). Human LRP was a gift from Dr. Dudley Strickland of the American Red Cross, and human PAI-1 and PAI-1 mutants were generated as described earlier (8Stefansson S. Lawrence D.A. Nature. 1996; 383: 441-443Crossref PubMed Scopus (605) Google Scholar, 11Lawrence D.A. Berkenpas M.B. Palanniappan S. Ginsburg D. J. Biol. Chem. 1994; 269: 15223-15228Abstract Full Text PDF PubMed Google Scholar, 13Stefansson S. Muhammad S. Cheng X.F. Battey F.D. Strickland D.K. Lawrence D.A. J. Biol. Chem. 1998; 273: 6358-6366Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). All additional reagents were purchased from Fisher Scientific (Pittsburgh, PA). The data were analyzed by either Origin version 7.0 (Origin Lab, Northampton, MA) or GraphPad Prism version 3.0 (GraphPad Software Inc., San Diego, CA) software.Construction of Mouse PAI-1 Variant Expression Vectors—The coding region of WT mouse PAI-1 (amino acid region 24-402, Swiss-Prot primary accession no. P22777) was cloned in-frame with an N-terminal His6 tag into the Escherichia coli expression vector pET15-b (Novagen, Madison, WI) using Nde-I/BamH-I sites. This construct lacks nucleotides encoding the first 23 amino acids that are thought to be the secretory signal region. The forward primer was 5′-GGAATTCCATCATATGTTTACCCCTCCGAGAA-3′. The reverse primer was 5′-CGCGGATCCTCAAGGCTCCATCAC-3′. The plasmids were transformed into DH5α competent E. coli cells (Invitrogen, Carlsbad, CA). The sequences were verified by DNA sequencing performed by the W. M. Keck Biotechnology Center (University of Illinois, Urbana-Champaign, IL). Mouse PAI-1 variants were generated with the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) as described in the manual, using the following oligonucleotides: R76E mutation forward primer, 5′-GGCACAGCTCATGCCCTCGAGCAGCTCTCCAAGGAG-3′, and reverse primer, 5′-GCTCCTTGGAGAGCTGCTCGAGGGCATGAGCTGTGC-3′; Q123K mutation forward primer, 5′-TTCCAGACTATGGTGAAAAAGGTGGACTTCTCAGAG-3′, and reverse primer, 5′-CCTCTGAGAAGTCCACCTTTTTCACCATAGTCTGGA-3; R346A mutation forward primer, 5′-GCCTTTGTCATCTCAGCCGCCATGGCCCCCACGGAG-3′, and reverse primer, 5′-CTCCGTGGGGGCCATGGCTGAGATGACAAAGG-3′; R101A mutation forward primer, 5′-CTTTGTCCAGGCCGATCTAGAGCTGGTCCAGGGCTT-3′, and reverse primer, 5′-GAAGCCCTGGACCAGCTCTAGATCGGCCTGGACAAA-3′. The Q123K/R101A double mutation was generated by using Q123K as a template. A new restriction site was incorporated into the primers to facilitate screening. The integrity of all mutant clones selected for protein expression was confirmed by DNA sequencing.Expression and Purification of Recombinant Murine PAI-1 Proteins—Vectors containing mouse WT or variant murine PAI-1 were transformed into the E. coli expression strain BL21/DE3 (Novagen, Madison, WI). Expression of recombinant proteins was induced by adding isopropyl thio-b-d-galactoside (final concentration, 1 mm), into the cell suspension pregrown in Luria-Bertani medium supplemented with 100 μg/ml ampicillin to A600 = 0.4-0.5. The induced cells were grown at 37 °C for another 3 h and then harvested by centrifugation at 10,000 × g for 10 min. The pellets were stored at -70 °C. For purification of the recombinant proteins, the cell pellets were re-suspended in BugBuster™ reagent (5 ml/gram pellet weight) (Novagen). Benzonase (1 μl/ml) was added to reduce viscosity and to remove nucleic acids. The insoluble cell debris was removed by centrifugation at 16,000 × g for 20 min at 4 °C. The soluble PAI-1-containing supernatants were applied to an affinity chromatography column using nickel-nitrilotriacetic acid (NTA) agarose (Qiagen) resin. The column was then extensively washed with 0.5 m NaCl, 50 mm sodium phosphate, 0.1% Tween 80 (v/v), 10 mm imidazole, pH 7.0. The adsorbed proteins were eluted with 0.5 m NaCl, 50 mm sodium phosphate, 0.1% Tween 80 (v/v), 10-150 mm imidazole gradient, pH 7.0. Fractions containing PAI-1 were pooled and subjected to gel-filtration chromatography using Sephadex G-25 (Sigma) to desalt proteins and to change the buffer to 10 mm phosphate buffer, pH 7.0. Pooled mouse PAI-1 preparations (except for the R76E variant) were further purified by heparin-agarose (Sigma) chromatography. Proteins were eluted with 10 mm sodium phosphate, pH 7.0, with a 0-1 M NaCl gradient. (R76E)-PAI-1 was purified by a second NTA chromatography. The purified recombinant proteins were denatured with 4 m guanidinium-HCl and then refolded by extensive dialysis against 0.5 m NaCl, 50 mm sodium phosphate, 0.1% Tween 80 (v/v), pH 7.0. The proteins were subsequently concentrated by Centricon centrifugal filter devices (Millipore, Bedford, MA). Protein concentrations were determined by Micro BCA™ protein assay kit (Pierce, Rockford, IL). Proteins were assessed for purity and size by SDS-10% PAGE under non-reducing conditions and stained with Coomassie Brilliant Blue.Expression and purification of mouse PAI-1 variants were confirmed by Western blotting employing a polyclonal rabbit-anti-rat-PAI-1-IgG (American Diagnostica, Greenwich, CT) as the primary antibody and an alkaline phosphatase-conjugated goat-anti-rabbit-IgG (H+L) (Bio-Rad, Hercules, CA) as the secondary antibody. The chromogen, 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium was used for detection. The molecular weights of purified proteins were determined by ProteinChip (Ciphergen, Fremont, CA) using the Au chip.Inhibitory Activity Assay—The inhibitory activity of PAI-1 toward tPA was determined by the Coatest™ PAI-1 assay (Chromogenix, West Chester, OH). Briefly, a fixed amount of tPA was added in excess to undiluted plasma, in which a portion rapidly forms an inactive complex with PAI-1. Plasminogen was then activated to plasmin by the residual tPA in the presence of a stimulator (fibrinogen degradation products). The amount of plasmin formed was directly proportional to the residual tPA activity and, hence, inversely proportional to the PAI-1 activity in the sample. The amount of plasmin was determined by measuring the amidolytic activity with the chromogenic substrate, S-2403. The release of p-nitroaniline (pNA) was then determined by the absorbancy at 405 nm.ELISA Assays—The capturing ligand, VN or LRP, was diluted to a concentration of 1 μg/ml in phosphate-buffered saline buffer (137 mm NaCl, 2.7 mm KCl, 10 mm Na2HPO4,2mm KH2PO4, pH 7.4). To coat the plates, 100 μl/well of ligand was added to a NuncMaxiSorp 96-well microtiter plate (Fisher) and incubated overnight at 4 °C. After three washes with phosphate-buffered saline buffer, the plate was blocked with 5% dried milk dissolved in washing buffer (phosphate-buffered saline, 0.1% Tween 80 (v/v)) and incubated for 2 h at room temperature. The plates were then washed 3× with washing buffer, and PAI-1 variants were added and incubated at room temperature for 2 h. For studies involving analyses of PAI-1/tPA or PAI-1/VN complex interactions with LRP, 20 nm of each were incubated for 15 min at room temperature prior to addition to the LRP-coated plates. After three additional washings, the amount of PAI-1 captured by immobilized VN or LRP was visualized by addition of the primary antibody, polyclonal rabbit-anti-rat-PAI-1-IgG, followed by alkaline phosphatase-conjugated goat-anti-rabbit-IgG (H+L) secondary antibody. The chromogen, p-nitrophenyl phosphate (Sigma), was used for detection. The absorbancy was measured at 405 nm and normalized against readings from noncoated wells.Characterization of Mouse PAI-1 Variants by Surface Plasmon Resonance (SPR)—The binding of label-free PAI-1 variants to VN was analyzed by SPR using a BIAcore™ 3000 (BIAcore AB, Uppsala, Sweden). All experiments were performed at 25 °C employing HBS-EP (10 mm Hepes, 0.15 m NaCl, 3 mm EDTA, 0.005% polysorbate 20, pH 7.4) as running buffer at a flow rate of 20 μl/min. VN (20 μg/ml in 10 mm phosphate buffer, pH 4.0) was coupled to sensor chip CM-5 utilizing an amine-coupling kit (BIAcore AB) through the BIAcore flow cell at a rate of 5 ml/min for 6 min, resulting in ∼3,000 response units (RUs) immobilized. The binding reactions were then carried out by injecting various concentrations of PAI-1 (diluted with running buffer) to the immobilized chip surface. Binding levels were determined by monitoring the resulting signal expressed as RUs. Between experiments, the chip surfaces were regenerated by washing with 30 μl of 0.1 mm HCl, pH < 2.0, which did not alter the binding properties of the VN-immobilized chips. The data from sensorgrams were subtracted from those of a reference flow cell prepared by the same method, with the exception that VN was not immobilized on the chip. Kinetic constants were calculated from the association and dissociation rate curves generated by applying different concentrations of analytes onto ligand-immobilized surfaces, using a 1:1 binding model. All of the sensorgrams were analyzed utilizing the BioEvaluation software package version 3.0 (BIAcore AB).Measurement of the Functional Half-life—PAI-1 was diluted to 20 μg/ml in buffer containing 100 mm NaCl, 100 mm Hepes, 1 mm EDTA, 0.1% PEG-8000, 0.1 mg/ml bovine serum albumin, pH 7.4. The inhibitory activity against sctPA, at t = 0, was designated as 100% activity. The proteins were then incubated at 37 °C. Aliquots were removed at various times and the remaining sctPA inhibitory activity was determined and converted to percent relative to time zero.RESULTSPurification of PAI-1 Proteins—Typical final yields for recombinant WT PAI-1 were 3.8 mg/liter of culture. Yields for the variants were slightly better at 4.0-4.8 mg/liter of culture. Fig. 1 represents SDS-PAGE and Western blotting analyses of the WT and the five variant (R76E, Q123K, R101A, Q123K/R101A, and R346A) proteins.Plasminogen Activator Inhibitor Function—Analyses were performed on the purified rPAI-1 proteins with respect to their plasminogen activator inhibitory activity. The human and murine homologues of Q123K, R76E, and murine R101A and R101A/Q123K retain inhibitory activity, whereas murine and human (R346A)-PAI-1 demonstrated a significant reduction in activity (Fig. 2), confirming a conserved functional domain between the human and murine molecules with respect to inhibitory activity.Fig. 2Comparative analyses of plasminogen activator inhibitory activity of WT murine and human rPAI-1 and variants. Inhibitory activity was determined using single-chain tPA in a chromogenic assay. The relative levels of inhibitory activities of WT murine and human recombinant PAI-1 were considered to be 100%. The inhibitory activity of the variants was expressed as a percentage of WT PAI-1 activity. Assays were performed in four replicates, and values are expressed as the means ± S.E.View Large Image Figure ViewerDownload Hi-res image Download (PPT)VN Binding by ELISA—The VN-binding capacity of the Q123K, R101A, and Q123K/R101A variants of PAI-1 were compared with the WT protein and the other two PAI-1 variants (R76E and R346A) by ELISA. (Q123K)-PAI-1 demonstrated impaired binding capacity to VN relative to the WT protein and the other two variants (Fig. 3), which is similar to the human protein studies. Additionally, (R101A)-PAI-1 and the double mutation (R101A/Q123K)-PAI-1 demonstrated even lower VN-binding abilities.Fig. 3VN-binding ability of WT murine rPAI-1 and its variants. The VN-binding ability was examined by ELISA. (Q123K)PAI-1 variant demonstrated impaired VN-binding ability compared with WT PAI-1 (p < 0.005, n = 20). (R101A)PAI-1 and (Q123K/R101A)PAI-1 demonstrated even lower VN-binding abilities. Assays were performed at least in triplicate and are expressed as the mean ± S.E.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Kinetic Characterization of Murine rPAI-1 WT and Q123K Binding to VN by SPR—Approximately 3000 RUs of VN were immobilized onto a CM5 chip using an amine-coupling method. Various amounts of rPAI-1 were then injected onto the surface, and the binding levels were detected in real time (Fig. 4A). A KD value for the interaction of murine WT PAI-1 with VN (KD = 5.4 nm) was determined by nonlinear regression fitting of steady-state binding (Fig. 4B). The association progress curves of WT PAI-1 binding were fit by a nonlinear single-exponential function to obtain the value of kobs. The kon was then determined by linear fitting of kobs as a function of PAI-1 concentrations (Fig. 4C). Murine (Q123K)-PAI-1/VN binding at steady state was used to determine the KD value (KD = 1800 nm) (Fig. 4D). The kon was determined from the association phase of (Q123K)-PAI-1/VN binding curves and then used to calculate the koff value (Fig. 4E). Murine (Q123K)-PAI-1 demonstrated slower association (kon = 2.92 × 104m-1 s-1) and faster dissociation (koff = 5.3 × 10-2 s-1) compared with WT murine PAI-1 (kon = 1.03 × 106m-1 s-1; koff = 5.27 × 10-3 s-1). Similar values were obtained for the equivalent human PAI-1 variants (Fig. 4F).Fig. 4Kinetic analyses of WT and (Q123K)PAI-1/VN interactions by SPR analyses. 20 μg/ml of VN was injected onto a CM5 sensor chip at a speed of 5 μl/min for 6 min. Approximately 3000 RUs of VN were immobilized by using an amine-coupling method. Various amounts of rPAI-1 were then injected onto the surface of the chip at a speed of at least 20 μl/min, and the binding levels were detected in real time by SPR. A, kinetic characterization of WT murine rPAI-1 binding to VN. WT murine PAI-1 demonstrated a dose-dependent effect of binding. B, KD value of WT murine PAI-1/VN binding was determined by nonlinear regression fitting of the steady-state binding. C, the association progress curves were nonlinearly fit by a single-exponential function to obtain the value of kobs. The kon constant was then determined by linear fitting of kobs as a function of PAI-1 concentrations. Insert, association progress curves at different PAI-1 concentrations. D, (Q123K)PAI-1/VN binding at various protein concentrations. The KD value of murine (Q123K)PAI-1/VN binding demonstrated more than a 300-fold difference relative to WT murine PAI-1. E, the kon value was determined from the association phase of (Q123K)PAI-1/VN binding and then used to calculate the koff value. These values demonstrated slower association and faster dissociation relative to WT murine rPAI-1. F, kinetic constants of the PAI-1/VN interaction. Murine (Q123K)PAI-1 demonstrated a slower association and a faster dissociation compared with the WT protein. Furthermore, these values are comparable with their human counterparts, indicating conservation of the VN-binding domain in murine PAI-1.View Large Image Figure ViewerDownload Hi-res image Download (PPT)LRP-binding Abilities of Murine PAI-1 Variants—The LRP-binding capacities of the altered PAI-1 forms were determined by ELISA experiments. (R76E)-PAI-1 demonstrated impaired LRP-binding ability, indicating that the LRP-binding domain was present in murine PAI-1 (Fig. 5A). Also, the PAI-1 variants were denatured by dialysis against 4 m guanidinium-HCl and then refolded by removing denaturant. At least a fraction of the PAI-1-binding activity was enhanced by this unfolding/refolding process (Fig. 5A, gray columns). Active PAI-1 demonstrated a higher LRP-binding ability than the latent form. PAI-1, in association with its protease substrate, tPA, also demonstrated enhanced association with LRP (Fig. 5B). VN decreased the LRP-binding ability of murine PAI-1 (Fig. 5C). This is most likely the result of steric hindrance due to the close proximity of the VN and LRP-binding sites.Fig. 5LRP-binding ability of WT murine PAI-1 and its variants. The LRP-binding capacity of WT PAI-1 and its variants was determined by ELISA experiments. A, (R76E)PAI-1 demonstrated impaired LRP-binding ability, indicating that LRP-binding domain was conserved in murine PAI-1. Also, PAI-1 variants were denatured by dialysis against 4 m guanidinium-HCl and then refolded by removing the denaturant. An increase in the PAI-1/LRP interaction was observed by this unfolding/refolding process (gray columns) relative to proteins that did not undergo the unfolding/refolding process (black columns). B, the LRP-binding ability of PAI-1 is increased if murine PAI-1 initially forms a complex with its protease substrates (gray columns) relative to PAI-1 alone (black columns). For the R346A mutant, the LRP-binding ability did not increase because its substrate-binding ability is impaired. C, VN decreases the LRP-binding ability of murine PAI-1 (gray columns) relative to PAI-1 alone (black columns). Because VN- and LRP-binding sites are in close proximity, this effect may be due to steric hindrance.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Stability of Murine rPAI-1 Variants—The inhibitory activity against sctPA at t = 0 was designated as 100%. The proteins were then incubated at 37 °C. Aliquots were removed at the indicated times, and the remaining sctPA inhibitory activity was determined" @default.
• W1990971755 created "2016-06-24" @default.
• W1990971755 creator A5006017457 @default.
• W1990971755 creator A5024436268 @default.
• W1990971755 creator A5038189350 @default.
• W1990971755 creator A5050593261 @default.
• W1990971755 creator A5063870060 @default.
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• W1990971755 date "2004-04-01" @default.
• W1990971755 modified "2023-10-18" @default.
• W1990971755 title "Conservation of Critical Functional Domains in Murine Plasminogen Activator Inhibitor-1" @default.
• W1990971755 cites W1484931749 @default.
• W1990971755 cites W1493779021 @default.
• W1990971755 cites W151540657 @default.
• W1990971755 cites W1536441019 @default.
• W1990971755 cites W1548286725 @default.
• W1990971755 cites W1550499229 @default.
• W1990971755 cites W1562197596 @default.
• W1990971755 cites W1589957034 @default.
• W1990971755 cites W1595622217 @default.
• W1990971755 cites W1670322156 @default.
• W1990971755 cites W1676144090 @default.
• W1990971755 cites W1926488772 @default.
• W1990971755 cites W1970314658 @default.
• W1990971755 cites W1973270637 @default.
• W1990971755 cites W1980095667 @default.
• W1990971755 cites W1995582010 @default.
• W1990971755 cites W2002811860 @default.
• W1990971755 cites W2013336992 @default.
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