Affinity purification of active plasminogen activator inhibitor-1 (PAI-1) using immobilized anhydrourokinase. Demonstration of the binding, stabilization, and activation of PAI-1 by vitronectin

Human Hep G2 hepatoma and HT 1080 fibrosarcoma cells were cultured in large scale under conditions which allowed enhanced secretion of plasminogen activator inhibitor-1 (PAI-1). A modified urokinase was obtained by reacting urokinase with phenylmethylsulfonyl fluoride followed by alkali treatment. The resulting product, called anhydrourokinase, was found to reversibly bind the PAI-1 when immobilized on cyanogen bromide-activated Sepharose 4B beads. Using this affinity absorbent, we have purified PAI-1 from the cell-conditioned media. A number of differences have been observed during Hep G2 and HT 1080 PAI purification. 1) The PAI activity in Hep G2 medium concentrate is more stable, and the concentrate depleted of active PAI-1 showed spontaneous regeneration of PAI-1 activity. In contrast, the PAI activity in HT 1080 medium concentrate declines rapidly on standing. 2) Hep G2 PAI-1 invariably copurified with an adhesive protein, vitronectin or its NH2-terminal fragment, while pure HT 1080 PAI-1 alone was obtained by affinity purification on anhydrourokinase-Sepharose 4B. 3) Based on specific activity measurement and complex formation analysis using a sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis technique, the purified Hep G2 PAI-1 appears completely active while the HT 1080 PAI-1 is only one-fourth as active. SDS was found to exert dual effects on purified PAI-1s. SDS treatment partially inactivated a fully active Hep G2 PAI-1 and a moderately active HT 1080 PAI-1 but partially activated an HT 1080 PAI-1 whose activity had previously been allowed to decay to a very low level. Purified vitronectin was found to enhance and stabilize the PAI-1 activity of the partially active HT 1080 PAI-1. It is concluded that fully active PAI-1 in association with vitronectin can be isolated by anhydrourokinase-Sepharose 4B chromatography and that vitronectin is a binding protein for PAI-1 which activates and stabilizes PAI-1.     

Identification of a PAI‐1‐binding site within an intrinsically disordered region of vitronectin

The serine protease inhibitor, plasminogen activator inhibitor Type‐1 (PAI‐1) is a metastable protein that undergoes an unusual transition to an inactive conformation with a short half‐life of only 1–2 hr. Circulating PAI‐1 is bound to a cofactor vitronectin, which stabilizes PAI‐1 by slowing this latency conversion. A well‐characterized PAI‐1‐binding site on vitronectin is located within the somatomedin B (SMB) domain, corresponding to the first 44 residues of the protein. Another PAI‐1 recognition site has been identified with an engineered form of vitronectin lacking the SMB domain, yet retaining PAI‐1 binding capacity (Schar, Blouse, Minor, Peterson. J Biol Chem. 2008;283:28487–28496). This additional binding site is hypothesized to lie within an intrinsically disordered domain (IDD) of vitronectin. To localize the putative binding site, we constructed a truncated form of vitronectin containing 71 amino acids from the N‐terminus, including the SMB domain and an additional 24 amino acids from the IDD region. This portion of the IDD is rich in acidic amino acids, which are hypothesized to be complementary to several basic residues identified within an extensive vitronectin‐binding site mapped on PAI‐1 (Schar, Jensen, Christensen, Blouse, Andreasen, Peterson. J Biol Chem. 2008;283:10297–10309). Steady‐state and stopped‐flow fluorescence measurements demonstrate that the truncated form of vitronectin exhibits the same rapid biphasic association as full‐length vitronectin and that the IDD hosts the elusive second PAI‐1 binding site that lies external to the SMB domain of vitronectin.     

Purification of high and low molecular weight plasminogen activator inhibitor 1 from fibrosarcoma cell-line HT 1080 conditioned medium

Functionally active (high-Mr) and inactive (low-Mr) plasminogen activator inhibitor 1 (PAI) have been purified from fibrosarcoma cell-line HT 1080 conditioned medium, containing 1% fetal calf serum. The two forms were first purified by affinity chromatography on heparin-Sepharose and then separated from each other by gel filtration on Sephadex G-150. The final purification was achieved by affinity chromatography on insolubilized monoclonal antibodies towards human PAI. Alternatively, the low-Mr form was purified by chromatography on carboxymethyl-cellulose. Low-Mr PAI purified in this way, could be almost fully reactivated by treatment with guanidinium chloride. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis followed by immunoblotting revealed that the low-Mr form contained nothing but PAI at an Mr of about 50,000. In addition to PAI, the high-Mr form contained a component, which was not antigenically related to PAI. This compound had a molecular weight of about 75,000 and its NH2-terminal amino acid sequence corresponded to that of human vitronectin. We conclude that the high-Mr form of PAI constitutes a complex between 50,000 Mr PAI and vitronectin from fetal calf serum.     

Cyclic AMP-dependent protein kinase phosphorylates serine378 in vitronectin.

We previously observed that Ser378 in the heparin-binding domain of vitronectin becomes phosphorylated by a protein kinase in plasma upon addition of ATP and divalent cations. We now report that purified plasma vitronectin contains approximately 2.5 mol of phosphate per mol of protein and that vitronectin becomes phosphorylated during biosynthesis in human hepatoma (HepG2) cells. In vitro, rabbit muscle cAMP-dependent protein kinase specifically phosphorylates Ser378 in single-chain (75 kDa) vitronectin but does not phosphorylate the two-chain (65/10 kDa) form cleaved at Arg379. Heparin affects neither the time course nor the extent of phosphorylation of Ser378 at neutral pH. The extent of phosphorylation of Ser378 achieved with cAMP-dependent protein kinase (greater than or equal to 0.3 mol phosphate per mol vitronectin) is greater than that obtainable in plasma and should enable comparisons to be made of the activities of the native and phosphorylated forms.     

The contributions of integrin affinity and integrin-cytoskeletal engagement in endothelial and smooth muscle cell adhesion to vitronectin

The serine proteinase inhibitor, plasminogen activator inhibitor type-1 (PAI-1), binds to the adhesion protein vitronectin with high affinity at a site that is located directly adjacent to the vitronectin RGD integrin binding sequence. The binding of PAI-1 to vitronectin sterically blocks integrin access to this site and completely inhibits the binding of purified integrins to vitronectin; however, its inhibition of endothelial and smooth muscle cell adhesion to vitronectin is at most 50-75%. Because PAI-1 binds vitronectin with approximately 10-100-fold higher affinity than purified integrins, we have analyzed the mechanism whereby these cells are able to overcome this obstacle. Our studies exclude proteolytic removal of PAI-1 from vitronectin as the mechanism, and show instead that cell adhesion in the presence of PAI-1 is dependent on integrin-cytoskeleton engagement. Disrupting endothelial or smooth muscle cell actin polymerization and/or focal adhesion assembly reduces cell adhesion to vitronectin in the presence of PAI-1 to levels similar to that observed for the binding of purified integrins to vitronectin. Furthermore, endothelial cell, but not smooth muscle cell adhesion to vitronectin in the presence of PAI-1 requires both polymerized microtubules and actin, further demonstrating the importance of the cytoskeleton for integrin-mediated adhesion. Finally, we show that cell adhesion in the presence of PAI-1 leads to colocalization of PAI-1 with the integrins alphavbeta3 and alphavbeta5 at the cell-matrix interface.     

Characterization of Vitronectin-Binding Proteins of Staphylococcus epidermidis

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

Purification and characterization of a plasminogen activator inhibitor 1 binding protein from human plasma. Identification as a multimeric form of S protein (vitronectin)

A binding protein for plasminogen activator inhibitor 1 (PAI-1-BP) was isolated from human plasma by a four-step procedure. 1) The 7 S globulin fraction of plasma was isolated by gel filtration on Sephacryl S-300. 2) Human endothelial cell-type plasminogen activator inhibitor (PAI-1), pretreated with 12 M urea, was added to this fraction (22 micrograms of PAI-1/ml of plasma), and a PAI-1 antigen peak with apparent mass 450 kDa (representing 65% of PAI-1 antigen and 85% of PAI activity) was isolated by gel filtration of this mixture. 3) The PAI-1.PAI-1-BP complex was further purified by immunoadsorption on an immobilized murine monoclonal antibody directed against PAI-1 (MA-7D4) and by elution with 4 M KSCN. 4) The complex was then dissociated by addition of excess human tissue-type plasminogen activator (t-PA), and t-PA and PAI-1 antigen (t-PA.PAI-1 complexes and free t-PA and PAI-1) were removed by immunoadsorption on monoclonal antibodies directed against t-PA (MA-62E8) and against PAI-1 (MA-7D4 and MA-12A4). Sodium dodecyl sulfate-gel electrophoresis of the purified material under nonreducing conditions revealed two bands with apparent mass approximately equal to 150 kDa and two bands with mass 74 and 68 kDa. Reduced sodium dodecyl sulfate-gel electrophoresis displayed two main bands with apparent masses of 73 and 64 kDa. The PAI-1-BP reacts with urea-treated, but not with inactive PAI-1. t-PA dissociates the complex between PAI-1 and PAI-1-BP. PAI-1 in complex with PAI-1-BP is 2-3-fold more stable at 37 degrees C than purified PAI-1, suggesting that PAI-1-BP may stabilize PAI-1 in blood. The concentration of PAI-1-BP in plasma determined by titration with PAI-1 is approximately 130 mg/liter. The isolated PAI-1-BP was shown to be identical to S protein (vitronectin) both by cross-reactivity with monospecific rabbit antisera and by NH2-terminal amino acid sequence analysis. The gel filtration behavior, mobility on sodium dodecyl sulfate-gel electrophoresis, and concentration in plasma suggest that PAI-1-BP is a multimer (presumably a dimer) of S protein accounting for approximately 35% of the S protein in plasma.     

Complex formation between plasminogen activator inhibitor 1 and vitronectin in purified systems and in plasma

Functionally active PAI-1 is bound to a discrete binding or carrier protein in plasma, which was recently identified as vitronectin. In the present study, the interaction between PAI-1 and vitronectin has been studied in purified systems and in plasma by agarose gel electrophesis using non-denaturing conditions and by crossed immunoelectrophoresis using an antiserum produced towards purified PAI-1/vitronectin complex. Both methods revealed a clearly distinguishable complex with electrophoretic mobility in between the parent molecules. Virtually all of the purified vitronectin, which did not contain any appreciable amounts of polymerized material, and almost all of the vitronectin in plasma, had the capacity to form a complex with PAI-1. The results suggested a stoichiometry of 1:1 as the most likely ratio between the two molecules in the complex. In contrast to functionally active PAI-1, latent or chloramine T-inactivated PAI-1 did not form such a complex with vitronectin.     

Complex formation between plasminogen activator inhibitor 1 and vitronectin in purified systems and in plasma.

Functionally active PAI-1 is bound to a discrete binding or carrier protein in plasma, which was recently identified as vitronectin. In the present study, the interaction between PAI-1 and vitronectin has been studied in purified systems and in plasma by agarose gel electrophoresis using non-denaturing conditions and by crossed immunoelectrophoresis using an antiserum produced towards purified PAI-1/vitronectin complex. Both methods revealed a clearly distinguishable complex with electrophoretic mobility in between the parent molecules. Virtually all of the purified vitronectin, which did not contain any appreciable amounts of polymerized material, and almost all of the vitronectin in plasma, had the capacity to form a complex with PAI-1. The results suggested a stoichiometry of 1:1 as the most likely ratio between the two molecules in the complex. In contrast to functionally active PAI-1, latent or chloramine T-inactivated PAI-1 did not form such a complex with vitronectin.     

The phosphorylation of the two-chain form of vitronectin by protein kinase A is heparin dependent.

In circulating blood, vitronectin occurs in two forms: a single-chain (75 kDa) and an endogenously clipped two-chain form (65 kDa and 10 kDa) held together by a disulfide bridge. The 75 kDa form was previously shown to be phosphorylated at Ser378 by protein kinase A, released by physiologically stimulated platelets. By contrast, at pH 7.5 the two-chain form is not phosphorylated at all. Heparin or heparan sulfate are shown here to modulate the conformation of clipped vitronectin at physiological pH, exposing Ser378 and allowing its stoichiometric phosphorylation by the kinase. At this pH the two-chain form of vitronectin in plasma exhibits a higher affinity for heparin, and behaves as a flexible molecule, which can conformationally respond to heparin and heparan sulfate, effectors involved in vitronectin function.     

Polymorphism of the human vitronectin gene causes vitronectin blood type.

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

Urokinase links plasminogen activation and cell adhesion by cleavage of the RGD motif in vitronectin

Components of the plasminogen activation system including urokinase (uPA), its inhibitor (PAI‐1) and its cell surface receptor (uPAR) have been implicated in a wide variety of biological processes related to tissue homoeostasis. Firstly, the binding of uPA to uPAR favours extracellular proteolysis by enhancing cell surface plasminogen activation. Secondly, it promotes cell adhesion and signalling through binding of the provisional matrix protein vitronectin. We now report that uPA and plasmin induces a potent negative feedback on cell adhesion through specific cleavage of the RGD motif in vitronectin. Cleavage of vitronectin by uPA displays a remarkable receptor dependence and requires concomitant binding of both uPA and vitronectin to uPAR. Moreover, we show that PAI‐1 counteracts the negative feedback and behaves as a proteolysis‐triggered stabilizer of uPAR‐mediated cell adhesion to vitronectin. These findings identify a novel and highly specific function for the plasminogen activation system in the regulation of cell adhesion to vitronectin. The cleavage of vitronectin by uPA and plasmin results in the release of N‐terminal vitronectin fragments that can be detected in vivo, underscoring the potential physiological relevance of the process.     

Association of thrombin-antithrombin III complex with vitronectin in serum

Purification of vitronectin by identical procedures from serum instead of plasma results in the coisolation of an additional protein component with mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of 82 kDa. We show that this component is the thrombin-antithrombin III complex based on the following evidence. Similar to a complex constructed using purified thrombin and antithrombin III, the 82-kDa component has a reduced molecular size of 69 kDa if it is not boiled prior to SDS-PAGE. Upon prolonged boiling in SDS it dissociates into 56- and 32-kDa components which co-migrate in SDS-PAGE with purified antithrombin III and thrombin, respectively. The 82- and 56-kDa components react with an antiserum against antithrombin III, and an antiserum prepared against the 82-kDa complex reacts with purified antithrombin III. Thrombin-antithrombin III complex, from either serum or recalcified clotted plasma, bound to vitronectin immobilized on Sepharose or plastic. However, purified antithrombin III which had not reacted with thrombin lacked affinity for vitronectin as did antithrombin III from citrated plasma. Purified antithrombin III acquired affinity for immobilized vitronectin if it was complexed with thrombin or was modified by radioiodination. Binding of vitronectin to antithrombin III coated on plastic was demonstrated using enzyme-linked immunosorbent assay. These results demonstrate that vitronectin binds thrombin-antithrombin III complexes through a cryptic site in antithrombin III which can be exposed when antithrombin III is radioiodinated, bound to plastic, or complexed with thrombin. Since vitronectin can interact with cells, the binding of vitronectin to the thrombin-antithrombin III complex may serve to facilitate the interaction of this complex with cell surfaces.     

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

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

Pseudomonas aeruginosa Uses Dihydrolipoamide Dehydrogenase (Lpd) to Bind to the Human Terminal Pathway Regulators Vitronectin and Clusterin to Inhibit Terminal Pathway Complement Attack

The opportunistic human pathogen Pseudomonas aeruginosa controls host innate immune and complement attack. Here we identify Dihydrolipoamide dehydrogenase (Lpd), a 57 kDa moonlighting protein, as the first P. aeruginosa protein that binds the two human terminal pathway inhibitors vitronectin and clusterin. Both human regulators when bound to the bacterium inhibited effector function of the terminal complement, blocked C5b-9 deposition and protected the bacterium from complement damage. P. aeruginosa when challenged with complement active human serum depleted from vitronectin was severely damaged and bacterial survival was reduced by over 50%. Similarly, when in human serum clusterin was blocked by a mAb, bacterial survival was reduced by 44%. Thus, demonstrating that Pseudomonas benefits from attachment of each human regulator and controls complement attack. The Lpd binding site in vitronectin was localized to the C-terminal region, i.e. to residues 354–363. Thus, Lpd of P. aeruginosa is a surface exposed moonlighting protein that binds two human terminal pathway inhibitors, vitronectin and clusterin and each human inhibitor when attached protected the bacterial pathogen from the action of the terminal complement pathway. Our results showed insights into the important function of Lpd as a complement regulator binding protein that might play an important role in virulence of P. aeruginosa.     

Subunit structure of a yeast site-specific endodeoxyribonuclease, endo SceI. A study using monoclonal antibodies

Endo SceI is a eucaryotic site-specific endoDNase of 120 kDa that causes double-stranded scission at well-defined sites, but is distinguishable from procaryotic restriction endonucleases by its mode of sequence recognition and lack of related specific DNA modification. In purified preparations of endoSceI, only two polypeptide species of 75 kDa (75-kDa peptide) and 50 kDa (50-kDa peptide) are detected in apparently equal amounts. We prepared mouse monoclonal IgGs that bound specifically to the 75-kDa peptide (but not the 50-kDa peptide) without inhibiting the endoSceI activity. Immunoprecipitation experiments with these IgGs revealed that the 75-kDa peptide and the 50-kDa peptide are physically associated with each other and with the endonucleolytic activity. Full endoSceI activity was recovered by mixing the purified 75-kDa peptide and the partially purified 50-kDa peptide, each of which exhibited little or no endonuclease activity alone. These observations indicate that endoSceI consists of two non-identical subunits of 75 kDa and 50 kDa, and that both subunits are required for full enzyme activity.     

Identification of the collagen-binding domain of vitronectin using monoclonal antibodies

Vitronectin is a 75 kilodalton (kDa) cell-adhesive glycoprotein found in animal blood and connective tissue, also termed serum spreading factor, S-protein, and epibolin. It promotes attachment and spreading of animal cells on tissue culture dishes, and it also binds to collagen. We established four mouse hybridoma lines producing monoclonal antibodies (M1, M2, M4 and M5) to human vitronectin. By immunoblotting, both epitopes recognized by M4 and M5 were suggested to exist in the amino terminal 5 kDa portion of vitronectin, and both M1 and M2 bound to the adjacent 35 kDa portion. Cell spreading on vitronectin-coated dishes was inhibited by M4 = M5 greater than M1, but not by M2. Collagen binding to vitronectin was inhibited by M2 greater than M4 = M5, but not by M1. These results indicate that the collagen-binding site is located near the cell-binding site in the amino terminal half of vitronectin. Independent inhibition of vitronectin binding to the cell and to collagen by these monoclonal antibodies will provide a potential tool to dissect the structure and function of vitronectin.     

Purification and characterization of a DnaK-like and a GroEL-like protein from Porphyromonas gingivalis

Heat-shock proteins of Porphyromonas gingivalis were demonstrated and two of them were purified and further characterized. The amplified de novo synthesis of two different proteins, with apparent molecular weights of 75 kDa and 68 kDa, was observed by autofluorography when a P. gingivalis culture incubated in a 14C-labeled amino acid mixture was shifted from 37 degrees C to 44 degrees C. Both proteins possessed ATP-binding abilities and were purified to almost homogeneity employing affinity chromatography on ATP-agarose followed by preparative SDS-PAGE. Purified 75 kDa and 68 kDa proteins had isoelectric points of 4.4 and 4.6, respectively. They were shown to be immunoreactive with commercial anti-DnaK and anti-GroEL polyclonal antibodies, respectively. Immunoblotting analysis of whole cells using antiserum raised against each purified protein from P. gingivalis, confirmed elevated synthesis of both proteins during thermal shock. A GroEL protein reacted strongly with antiserum against the 68 kDa protein. However, a DnaK protein reacted weakly with antiserum to the 75 kDa protein. Analysis of the N-terminal amino acid sequence of the DnaK-like protein (75 kDa) showed a high degree of homology with those of the HSP70 family including both prokaryotic and eukaryotic cells. The N-terminal amino acid analysis of the GroEL-like protein (68 kDa) indicated that it was identical to those of cloned GroEL homologues from P. gingivalis.     

Purification of the serum acid-stable insulin-like growth factor binding protein from the pig (Sus scrofa)

1. An acid-stable IGF binding protein was isolated and purified from porcine serum. 2. The protein comprised two major species with Mrs of 45 and 41 kDa determined using SDS-PAGE under reducing conditions. 3. The IGFBP preparation specifically bound both IGF-I and II. 4. Four distinct protein bands (Mrs of 23, 45, 50 and 75 kDa) in the porcine IGFBP preparation specifically bound radiolabelled IGF-I. 5. The porcine IGFBP exhibited sequence homology with IGFBPs from human plasma and rat serum. 6. This is the first report of the purification and characterization of the acid-stable IGFBP from porcine serum.     

Characterization of the detergent solubilized receptor for gastrin-releasing peptide

Properties of detergent solubilized gastrin-releasing peptide receptor were investigated. Swiss 3T3 membranes were covalently labeled with [125I]GRP and homobifunctional cross-linkers. A major labeled protein of 75 kDa was resolved using SDS-polyacrylamide gel electrophoresis. When the same preparation was solubilized with zwitterionic detergent and analyzed under nondenaturing conditions the protein bound radioactivity was resolved in two different peaks, a major one of apparent molecular weight 220,000 (peak 1) and a minor one of 80,000 (peak 2) both containing the 75 kDa protein. Specific ligand binding activity also eluted with peak 1. These results indicate that the active form of bombesin/GRP receptor is a large complex containing the 75 kDa ligand binding domain.