Reversible interactions between plasminogen activators and plasminogen activator inhibitor-1.

We have shown that the urokinase (UK) kringle domain contains a high-affinity plasminogen activator inhibitor-1 (PAI-1) binding site, responsible for the 10-fold faster complex formation between UK and PAI-1 than between PAI-1 and low-molecular-weight urokinase (LMWUK). Complex formation between UK and PAI-1, but not between LMWUK and PAI-1, was suppressed 10-fold in the presence of peptide U-107 derived from the UK kringle domain. Peptide U-373 derived from the UK catalytic domain slowed complex formation between UK and PAI-1 and also LMWUK and PAI-1. Inactivation of tissue-type plasminogen activator (tPA) by PAI-1 was slowed 10-fold in the presence of peptides derived from the tPA finger and kringle-2 domains. DFP-inactivated (DIP) UK and both forms of DIP-tPA inhibited PAI-1 binding to U-107 and to U-373 whereas single-chain urokinase-type PA (scuPA) was unable to compete with either peptide for PAI-1 binding. These data suggest that the reversible PAI-1 binding site in the UK A-chain plays a role in the rapid association with PAI-1 as important as those that reside in the tPA A-chain and that reversible PAI-1 binding sites are expressed on the surface of UK upon conversion from scuPA, in contrast to tPA.     

Kinetic analysis of the interactions between plasminogen activator inhibitor 1 and both urokinase and tissue plasminogen activator

Plasminogen activator inhibitor 1 (PAI-1) was purified from medium conditioned by cultured bovine aortic endothelial cells by successive chromatography on concanavalin A Sepharose, Sephacryl S-200, Blue B agarose, and Bio-Gel P-60. As shown previously for conditioned media (C. M. Hekman and D. J. Loskutoff (1985) J. Biol. Chem. 260, 11581-11587) the purified PAI-1 preparation contained latent inhibitory activity which could be stimulated 9.4-fold by sodium dodecyl sulfate and 45-fold by guanidine-HCl. The specific activity of the preparation following treatment with 0.1% sodium dodecyl sulfate was 2.5 X 10(3) IU/mg. The reaction between purified, guanidine-activated PAI-1 and both urokinase and tissue plasminogen activator (tPA) was studied. The second-order rate constants (pH 7.2, 35 degrees C) for the interaction between guanidine-activated PAI-1 and urokinase (UK), and one- and two-chain tPA are 1.6 X 10(8), 4.0 X 10(7), and 1.5 X 10(8) M-1 S-1, respectively. The presence of CNBr fibrinogen fragments had no affect on the rate constants of either one- or two-chain tPA. Steady-state kinetic analysis of the effect of PAI-1 on the rate of plasminogen activation revealed that the initial UK/PAI-1 interaction can be competed with plasminogen suggesting that the UK/PAI-1 interaction may involve a competitive type of inhibition. In contrast, the initial tPA/PAI-1 interaction can be competed only partially with plasminogen, suggesting that the tPA/PAI-1 interaction may involve a mixed type of inhibition. The results indicate that PAI-1 interacts more rapidly with UK and tPA than any PAI reported to date and suggest that PAI-1 is the primary physiological inhibitor of single-chain tPA. Moreover, the interaction of PAI-1 with tPA differs from its interaction with UK, and may involve two sites on the tPA molecule.     

Catabolism of tissue-type plasminogen activator by the human hepatoma cell line Hep G2. Modulation by plasminogen activator inhibitor type 1

Catalytic activity of tissue-type plasminogen activator (t-PA) in plasma is regulated in part by formation of complexes with specific inhibitors as well as by hepatic clearance. Potential interaction of these two regulatory mechanisms was examined in the human hepatoma cell line Hep G2. These cells secrete plasminogen activator inhibitor type-1 (PAI-1) and initiate catabolism of exogenous t-PA by receptor-mediated endocytosis. Specific binding of 125I-t-PA to cells at 4 degrees C results in dose-dependent formation of a 95-kDa species recognized by monospecific anti-PAI-1 and anti-t-PA antibodies and stable in the presence of low (0.2%) concentrations of sodium dodecyl sulfate (SDS). Specific binding of 125I-t-PA and formation of the 95-kDa SDS-stable species are inhibited in a concentration-dependent manner following preincubation of cells with anti-PAI-1 antibodies. High and low molecular weight forms of urokinase plasminogen activator (u-PA) capable of forming specific complexes with PAI-1 complete for 125I-t-PA binding sites. However, the proenzyme form of u-PA (scu-PA), incapable of forming complexes with PAI-1, does not compete for 125I-t-PA binding sites. The role of the serine protease active site of t-PA in mediating both interaction with PAI-1 and specific binding was examined using 125I-t-PA that had been functionally inactivated with D-phenylalanyl-L-propyl-L-arginyl-chloromethyl ketone (PPACK). 125I-t-PA-PPACK, despite a 6-fold lower affinity than active 125I-t-PA, exhibited specific binding to cells without detectable formation of SDS-stable complexes with PAI-1. Both surface-bound 125I-t-PA and 125I-t-PA-PPACK are internalized and degraded by cells at 37 degrees C. 125I-t-PA is internalized as a stable complex with PAI-1, whereas 125I-t-PA-PPACK is internalized with similar kinetics but without the presence of an SDS-stable complex. Thus, PAI-1 appears capable of modulating t-PA catabolism in the human hepatocyte.     

Tissue-type plasminogen activator binding to human endothelial cells. Evidence for two distinct binding sites

The endothelium may contribute to fibrinolysis through the binding of plasminogen activators or plasminogen activator inhibitors to the cell surface. Using a solid-phase radioimmunoassay, we observed that antibodies to recombinant tissue-type plasminogen activator (rt-PA) and plasminogen activator inhibitor type 1 (PAI-1) bound to the surface of cultured human umbilical vein endothelial cells (HUVEC). HUVEC also specifically bound added radiolabeled rt-PA with apparent steady-state binding being reached by 1 h at 4 degrees C. When added at low concentrations (less than 5 nM), rt-PA bound with high affinity mainly via the catalytic site, forming a sodium dodecyl sulfate-stable 105-kDa complex which dissociates from the cell surface over time and which could be immunoprecipitated by a monoclonal antibody to PAI-1. rt-PA bound to this high affinity site retained less than 5% of its expected plasminogen activator activity. At higher concentrations, binding did not require the catalytic site and was rapidly reversible. rt-PA initially bound to this site retained plasminogen activator activity. These studies suggest that tissue-type plasminogen activator and PAI-1 are expressed on the surface of cultured HUVEC. HUVEC also express unoccupied binding sites for exogenous tissue-type plasminogen activator. The balance between the expression of plasminogen activator inhibitors and these unoccupied binding sites for plasminogen activators on the endothelial surface may contribute to the regulation of fibrinolysis.     

Purification of active human plasminogen activator inhibitor 1 from Escherichia coli. Comparison with natural and recombinant forms purified from eucaryotic cells

Plasminogen activator inhibitor 1 (PAI-1) inhibits both tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) and, therefore, is an important regulator of plasminogen activation. We have developed eucaryotic and procaryotic expression systems for PAI-1 and characterized the recombinant glycosylated and non-glycosylated products, together with a non-recombinant natural control, produced in the histosarcoma cell line HT 1080. For eucaryotic expression, the PAI-1 cDNA was stably transfected into chinese hamster ovary cells (CHO cells), while procaryotic expression in Escherichia coli was examined after inserting the DNA sequence encoding the mature PAI-1 protein into an inducible expression vector. Recombinant PAI-1 from CHO cells was purified approximately 50-fold in two steps and was indistinguishable from natural PAI-1. Between 3% and 4% of total cellular protein in the procaryotic expression system consisted of PAI-1, from which it was purified approximately 30-fold, with yields of between 15% and 20%. This PAI-1 formed 1:1 complexes with uPA and also with the single- and two-chain forms of tPA. Kinetic analysis demonstrated that the procaryote-produced PAI-1 had an inhibitory activity towards all three forms of PA that resembled that of natural PAI-1 with association rate constants of approximately 10(7) M-1 s-1. In contrast to PAI-1 from eucaryotic cells, the PAI-1 from E. coli had an inherent activity equal to that of guanidine/HCl-activated natural PAI-1. The activity could not be increased by treatment with denaturants suggesting that the latent form of PAI-1 was absent. However, at 37 degrees C the procaryote-produced PAI-1 lost activity at the same rate as natural PAI-1, with approximately 50% of the activity remaining after 3 h. This activity could be partially restored by treatment with 4 M guanidine/HCl. E. coli-derived PAI-1, added to human plasma and fractionated by Sephacryl S-200 chromatography, eluted in two peaks that were similar to those obtained with guanidine-activated PAI-1 from eucaryotic cells, suggesting that it bound to the PAI-1-binding protein (vitronectin).     

Structural determinants of the noncatalytic chain of tissue-type plasminogen activator that modulate its association rate with plasminogen activator inhibitor-1

In order to identify the regions of recombinant (r) tissue plasminogen activator (tPA) that mediate its kinetically relevant interaction with r-plasminogen activator inhibitor-1 (rPAI-1), we have determined the second-order association rate (k1) constants of domain-altered variants of tPA with rPAI-1, at 10 degrees C. With two-chain, wild-type recombinant tPA (tcwt-rtPA), obtained by expression of the human cDNA for tPA in five different cell systems (viz. insect cells, human kidney 293 cells, Chinese hamster ovary cells, human melanoma cells, and mouse C127 cells), the average k1 was 1.45 x 10(7) M-1 s-1 (range, 1.34 10(7) M-1 s-1-1.68 x 10(7) M-1 s-1). Since this value was not significantly different for the different tcwt-rtPA preparations, it appears as though the nature of the glycosylation of tPA plays little role in its initial interaction with PAI-1. The k1 determined for tcwt-rtPA was slightly higher than that of 0.87 x 10(7) M-1 s-1, obtained for a similar inhibition of human urokinase by rPAI-1. The k1 value obtained for single-chain (sc) wt-rtPA was approximately 6-fold lower than that of the two-chain molecules, results consistent with previous conclusions on this matter. The k1 value for tcwt-rtPA was not influenced by the presence of epsilon-aminocaproic acid, suggesting that the lysine-binding site associated with the kringle 2 (K2) region of tPA does not modulate the rate of its initial interaction with rPAI-1. Removal of the K2 domain from tPA, by recombinant DNA technology, results in a protein, F-E-K1-P (tc-r delta K2-tPA), containing only the finger (F), growth factor (E), kringle 1 (K1), and serine protease (P) domains. This variant protein was more rapidly inhibited by rPAI-1 (k1 = 3.00 x 10(7) M-1 s-1) than its wild-type counterparts. Deletion of both the K1 and K2 domains resulted in a variant molecule, F-E-P (tc-r delta K1 delta K2-tPA), that was slightly more rapidly inhibited by rPAI-1 (k1 = 2.01 x 10(7) M-1 s-1).(ABSTRACT TRUNCATED AT 400 WORDS)     

Structure-function studies of the SERPIN plasminogen activator inhibitor type 1. Analysis of chimeric strained loop mutants.

Three chimeric mutants of plasminogen activator inhibitor 1 (PAI-1) have been constructed where the strained loop of wild type PAI-1 (wtPAI-1) has been replaced with a 19-amino acid region from either plasminogen activator inhibitor 2 (PAI-2), antithrombin III, or with an artificial serine protease inhibitor superfamily consensus strained loop. The inhibitors were expressed in Escherichia coli, and the purified proteins had specific activities toward urokinase-type plasminogen activator (uPA) or the single- and two-chain forms of tissue type plasminogen activator (tPA) that were similar to wtPAI-1. Experiments suggest that the strained loop of PAI-1 is not responsible for the transition between the latent and the active conformations or for binding to vitronectin. Second-order rate constants for the interactions with uPA and single- or two-chain tPA were similar to those of wtPAI-1. Values range from a low of 1.8 x 10(5) M-1 s-1 for the interaction of the PAI-2 chimera with single-chain tPA to a high value of 1.6 x 10(7) M-1 s-1 for the consensus mutant with two-chain tPA. This former value is 200 times higher than the reported rate constant for the interaction between PAI-2 and single-chain tPA, suggesting that structures outside of the strained loop are responsible for the major differences in specificity between PAI-1 and PAI-2.     

Thrombin increases proliferation and decreases fibrinolytic activity of kidney glomerular epithelial cells.

Human glomerular epithelial cells (GECs) in culture synthesize single-chain, urokinase-type plasminogen activator (SC-uPA), tissue-type plasminogen activator (t-PA), and plasminogen activator inhibitor 1 (PAI-1) and possess specific membrane-binding sites for u-PA. Using purified 125I-alpha thrombin, we demonstrate here the presence of two populations of specific binding sites for thrombin on GECs (1.Kd = 4.3 +/- 1.0 x 10(-10) M, 5.4 +/- 1.4 x 10(4) M sites per cell, 2. Kd = 1.6 +/- 0.5 x 10(-8) M, 7.9 +/- 1.8 x 10(5) sites per cell). Purified human alpha thrombin promoted the proliferation of GECs and induced a time- and dose-dependent increase of SC-uPA, t-PA, and PAI-1 antigens released by GECs. Thrombin-mediated increase in antigen was paralleled by an increase in the levels of corresponding u-PA and PAI-1 messenger RNA. In contrast, thrombin decreased u-PA activity in conditioned medium. This discrepancy between u-PA antigen and u-PA activity was explained by a limited proteolysis of SC-uPA by thrombin, leading to a two-chain form detected by immunoblotting and that could not be activated by plasmin. Thrombin also decreased the number of u-PA binding sites on GECs (p less than 0.05) without changing receptor affinity. Hirudin inhibited the binding and the cellular effects of thrombin, whereas thrombin inactivated by diisopropylfluorophosphate had no effect, indicating that both membrane binding and catalytic activity of thrombin were required. We conclude that thrombin, through specific membrane receptors, stimulates proliferation of GECs and decreases the fibrinolytic activity of GECs both at the cell surface and in the conditioned medium. These results suggest that thrombin could be involved in the pathogenesis of extracapillary proliferation and persistency of fibrin deposits in crescentic glomerulonephritis.     

Interactions between the finger and kringle-2 domains of tissue-type plasminogen activator and plasminogen activator inhibitor-1.

We have shown that plasminogen activator inhibitor-1 (PAI-1) inhibits the fibrin binding of both the single chain and two chain forms of tissue-type plasminogen activator (tPA) through two different mechanisms. PAI-1 inhibits the finger domain-dependent fibrin binding of diisopropylfluorophosphate-inactivated single chain tPA and the kringle-2 domain-dependent fibrin binding of diisopropylfluorophosphate-inactivated two chain tPA. In accordance with the data, preformed complexes of single chain tPA/PAI-1 and of two chain tPA/PAI-1 lost the fibrin binding abilities mediated by the finger and kringle-2 domains, respectively. These effects of PAI-1 appear to be mediated by steric hindrance of the fibrin binding sites after PAI-1 binding to adjacent regions in the functional domains of tPA. We thus propose a model in which a PAI-1 binding site resides in the finger domain of a single chain, and plays a role in the reversible association of single chain tPA and PAI-1. Conformational changes may take place during the conversion of single chain tPA to two chain tPA, resulting in burying of the original PAI-1 binding site and exposure of an alternate PAI-1 binding site on the surface of the kringle-2 domain.     

Dehydroepiandrosterone therapy in men with angiographically verified coronary heart disease: the effects on plasminogen activator inhibitor-1 (PAI-1), tissue plasminogen activator (tPA) and fibrinogen plasma concentrations

INTRODUCTION: The aim of this study was to analyze the influence of DHEA therapy on fibrinogen, plasminogen activator inhibitor-1 (PAI-1) and tissue plasminogen activator (tPA) plasma concentrations in men with decreased serum DHEA-S levels and angiographically verified coronary heart disease (CHD). MATERIAL AND METHODS: The study included thirty men aged 41-60 years (mean age 52 +/- 0.90 yr) with serum DHEA-S concentration < 2000 mg/l, who were randomized into a double-blind, placebo-controlled, cross-over trial. Subjects completed the 80 days study of 40 days of 150 mg oral DHEA daily or placebo, and next groups were changed after 30 days of wash-out. Fasting early morning blood samples were obtained at baseline and after each treatment to determine serum hormones levels (testosterone, DHEA-S, LH, FSH and estradiol) and also fibrinogen, plasminogen activator inhibitor-1 (PAI-1) and tissue plasminogen activator (tPA) plasma concentrations. RESULTS: Administration of DHEA was associated with 4.5-fold increase in DHEA-S levels. Estrogen levels significantly increased after DHEA from 22.1 +/- 0.7 pg/ml to 26.4 +/- 1.6 pg/l (mean +/- SEM; p < 0.05), while testosterone levels did not changed. Fibrinogen concentrations significantly decreased in DHEA group from 4.5 +/- 0.3 g/l to 3.83 +/- 0.2 g/l (p < 0.05 vs. placebo). Changes of tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) were not statistical significant (respectively: 8.37 +/- 0.4 ng/ml vs. 8.93 +/- 0.5 ng/ml and 82.3 +/- 6.3 ng/ml vs. 92.7 +/- 9.1 ng/ml (mean +/- SEM; NS vs. placebo). Tolerance of the treatment was good and no adverse effects were observed. CONCLUSIONS: DHEA therapy in dose of 150 mg daily during 40 days in men with DHEAS levels < 2000 mg/l and angiographically verified coronary heart disease (CHD) was connected with significant decreasing of fibrinogen concentration and increasing of estradiol levels, and did not influence on plasminogen activator inhibitor-1 (PAI-1) and tissue plasminogen activator (tPA) plasma concentrations.     

Characterization of the binding of human tissue-type plasminogen activator to platelets

Cell surface binding sites for the constituent proteins of the fibrinolytic system may play a role in the localization and regulation of fibrinolysis. In the present study, specific binding of recombinant human tissue-type plasminogen activator (rt-PA) to human blood platelets was identified and characterized. 125I-labeled rt-PA was found to bind specifically, saturably, and reversibly to the surface of gel-filtered platelets, reaching equilibrium within 5 min at 22 degrees C. Scatchard analysis revealed a single class of binding sites. Unstimulated platelets bound 120,000 +/- 24,000 (mean +/- S.D.) molecules/platelet with an apparent Kd of 340 +/- 25 nM, whereas thrombin-stimulated platelets bound 290,000 +/- 32,000 molecules/platelet with an apparent Kd of 800 +/- 60 nM. Binding of 0.1 microM 125I-rt-PA was greater than 90% reversible by a 50-fold excess of unlabeled rt-PA. Binding was not inhibited by fibrinogen or single chain urokinase-type plasminogen activator, but plasminogen partially competed for binding of 125I-rt-PA to platelets (up to 40% displacement). These findings indicate that the platelet surface possesses a large number of specific, low affinity binding sites for t-PA and provide further evidence for the role of platelets in localization and regulation of fibrinolysis.     

Expression of plasminogen activator and plasminogen activator inhibitor mRNA in human fibroblasts grown on different substrates

mRNA levels for urokinase type plasminogen activator (uPA), tissue type plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1) and plasminogen activator inhibitor-2 (PAI-2) were examined in human diploid (neonatal foreskin) fibroblasts grown in 200-ml microcarrier suspension culture. Four different substrates were used. These included gelatin-coated polystyrene plastic, DEAE-dextran, glass-coated polystyrene plastic and uncoated polystyrene plastic. Our previous studies have shown that culture fluids from diploid fibroblasts grown on DEAE-dextran contained higher levels of plasminogen-dependent fibrinolytic activity than culture fluids from the same cells grown on other substrates. The increased plasminogen activator activity was due largely to elevated amounts of tPA (In Vitro Cell. Develop. Biol. 22: 575–582, 1986). The present study shows that there is a corresponding elevation of tPA mRNA in diploid fibroblasts cultured on DEAE-dextran relative to the other substrates. There does not appear to be any difference in uPA mRNA or in mRNA for PAI-1 or PAI-2 produced by the same cells on the four substrates. These data suggest that the influence of the substrate on plasminogen activator production is mediated at the genetic level.     

The plasminogen activator inhibitor-1 binding site in the kringle-2 domain of tissue-type plasminogen activator

We have shown that synthetic peptides containing the amino acid sequence Asn-Arg-Arg-Leu, derived from the amino acid sequence of the inner loop of the kringle-2 domain of tissue-type plasminogen activator (tPA), inhibited complex formation between two chain tPA and plasminogen activator inhibitor-1 (PAI-1) by binding to PAI-1. This binding was reversible and was inhibited by not only tPA but also by enzymatically inactive tPA. Quantitative analyses of the interaction of PAI-1 with the peptide containing the Asn-Arg-Arg-Leu sequence indicated that the PAI-1 binding site residues in the inner loop of the kringle-2 domain and is preferentially expressed in two chain tPA.     

Association of a plasminogen activator inhibitor (PAI-1) with the growth substratum and membrane of human endothelial cells

We have studied the distribution of the plasminogen activator inhibitor type 1 (PAI-1) in cultures of confluent human umbilical vein endothelial cells. Plasminogen activator inhibitor activity measured by the 125I-fibrin plate assay was detected in the cytosol (2.85 +/- 0.16 U), 100,000 g particulate fraction (1.26 +/- 0.30 U), and in the growth substratum (9.82 +/- 1.80 U). Characterization of the protein responsible for this activity by reverse fibrin autography, immunoprecipitation, and immunoblotting demonstrated that it had an Mr of 46,000 and was antigenically related to PAI-1. Only the active form of the inhibitor was found in all three fractions. Inhibitor in the cytosol and particulate fraction converted to the latent form during 37 degrees C incubation while the substratum inhibitor remained fully active. Extracellular PAI-1 was detected in the growth substratum before its appearance in conditioned medium and represented the major protein deposited beneath the cells. The inhibitor was only transiently localized in the substratum, disappearing within 6 h and concomitantly appearing in the culture medium. Incubation of isolated metabolically labeled substratum with tissue plasminogen activator (tPA) resulted in the appearance and release of an immunologically related inactive 44,000 Mr form as well as the tPA-PAI-1 complex (110,000 Mr). PAI-1 was also converted into its 44,000-Mr form and released by treatment of the substratum with human leukocyte elastase. The rapid deposition and predominance of PAI-1 in the underlying compartment of endothelial cells may explain how the basement membrane is protected from proteolytic degradation by plasmin-generating enzymes.     

Glucocorticoid induction of plasminogen activator and plasminogen activator-inhibitor messenger RNA in rat hepatoma cells

HTC rat hepatoma cells synthesize and secrete both tissue-type plasminogen activator (tPA) and type 1 plasminogen activator-inhibitor (PAI-1). Incubation with the synthetic glucocorticoid dexamethasone causes a rapid decrease in tPA activity which is secondary to a 5-fold increase in PAI-1 antigen and activity. Paradoxically, dexamethasone increases tPA antigen by 50%. We have analyzed HTC cell RNA by Northern and slot blot analysis, using as probes radiolabeled human PAI-1 and rat tPA cDNAs. HTC cells have a single species of PAI-1 mRNA of approximately 3.2 kilobases, which is increased 4-fold upon incubation with dexamethasone. Maximal induction occurs after 8-10 h of incubation. Half-maximal induction occurs at 5 nM dexamethasone. Dexamethasone also transiently increases the 2.8 kilobase tPA mRNA. The protein synthesis inhibitor cycloheximide does not affect accumulation of PAI-1 mRNA and does not block its induction by dexamethasone. In contrast, cycloheximide alone causes an increase in tPA mRNA, and in combination with dexamethasone, no further increase is observed. Induction of both mRNAs is prevented by actinomycin D. We conclude that the dexamethasone-induced increase in HTC cell PAI-1 activity and antigen is the result of a direct effect on accumulation of PAI-1 mRNA.     

Oncostatin M induces tissue-type plasminogen activator and plasminogen activator inhibitor-1 in Calu-1 lung carcinoma cells

Oncostatin M (OSM) is a glycoprotein cytokine that is produced by activated T-lymphocytes, monocytes, and macrophages. In a DNA synthesis assay, OSM reduced tritiated thymidine incorporation by 53% in Calu-1 lung carcinoma cells. Radiolabeled cDNAs from untreated Calu-1 cells and 30-h OSM-treated cells were used to probe duplicate nylon membrane cDNA expression arrays. This study revealed OSM-mediated expression of mRNAs encoding tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1). Northern blot analysis showed that the steady-state level of tPA mRNA is nearly undetectable in Calu-1 cells. Exposure of these cells to OSM for 30 h increased tPA mRNA expression by 20-fold and PAI-1 mRNA expression by 5-fold. Exposure of these cells to other gp130 receptor family cytokines, including leukemia inhibitory factor (LIF), interleukin-6 (IL-6), and IL-11, do not significantly affect DNA synthesis or induction of tPA/PAI-1. Western blot studies demonstrated that OSM mediates a marked increase in secretion of the tPA protein. Secreted tPA was present in the conditioned medium almost exclusively as tPA/PAI-1 complexes. Inhibitor studies demonstrated that OSM-mediated induction of tPA and PAI-1 mRNAs is largely dependent upon activation of the MEK1/2 pathway. The JAK3/STAT3 pathway potentially serves a secondary role in these regulatory events.     

Accelerated conversion of human plasminogen activator inhibitor-1 to its latent form by antibody binding.

The serpin plasminogen activator inhibitor-1 (PAI-1) slowly converts to an inactive latent form by inserting a major part of its reactive center loop (RCL) into its beta-sheet A. A murine monoclonal antibody (MA-33B8), raised against the human plasminogen activator (tPA).PAI-1 complex, rapidly inactivates PAI-1. Results presented here indicate that MA-33B8 induces acceleration of the active-to-latent conversion. The antibody-induced inactivation of PAI-1 labeled with the fluorescent probe N, N'-dimethyl-N-(acetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) ethylene diamine (NBD) at P9 in the RCL caused a fluorescence enhancement and shift identical to those accompanying the spontaneous conversion of the P9.NBD PAI-1 to the latent form. Like latent PAI-1, antibody-inactivated PAI-1 was protected from cleavage by elastase. The rate constants for MA-33B8 binding, measured by NBD fluorescence or inactivation, were similar (1.3-1.8 x 10(4) M-1 s-1), resulting in a 4000-fold faster inactivation at 4.2 microM antibody binding sites. The apparent antibody binding rate constant, at least 1000 times slower than one limited by diffusion, indicates that exposure of its epitope depends on an unfavorable equilibrium of PAI-1. Our observations are consistent with this idea and suggest that the equilibrium involves partial insertion of the RCL into sheet A: latent, RCL-cleaved, and tPA-complexed PAI-1, which are inactive loop-inserted forms, bound much faster than active PAI-1 to MA-33B8, whereas two loop-extracted forms of PAI-1, modified to prevent loop insertion, did not bind or bound much more weakly to the antibody.     

Extracellular matrix degradation by cultured mesangial cells: mediators and modulators

Decreased degradation of the glomerular extracellular matrix (ECM) is thought to contribute to the accumulation of glomerular ECM that occurs in diabetic nephropathy and other chronic renal diseases. Several lines of evidence indicate a key role for the plasminogen activator/plasminogen/plasmin system in glomerular ECM degradation. However, which of the two plasminogen activators (PAs) present in renal tissue, tissue plasminogen activator (tPA) or urokinase-type plasminogen activator (uPA), is responsible for plasmin generation and those factors that modulate the activity of this system remain unclear. This study utilized mesangial cells isolated from mice with gene deletions for tPA, uPA, and plasminogen activator inhibitor 1 (PAI-1) to further delineate the role of the PA/plasminogen/plasmin system in ECM accumulation. ECM degradation by uPA-null mesangial cells was not significantly different from controls (92% +/- 1%, n = 12). In contrast, ECM degradation by tPA-null mesangial cells was markedly reduced (-78 +/- 1%, n = 12, P < 0.05) compared with controls, whereas tPA/uPA double-null mesangial cells degraded virtually no ECM. Previous studies from this laboratory have established that transforming growth factor-beta1 (TGFbeta1) inhibits ECM degradation by cultured mesangial cells by increasing the production of PAI-1, the major physiological PA inhibitor. In keeping with this observation, TGFbeta1 (1 ng/ml) had no effect on ECM degradation by PAI-1-null MC. High glucose levels (30 mM) in the presence or absence of insulin (0.1 mM) caused a moderate increase in ECM degradation by normal human mesangial cells. In contrast, glycated albumin, whose concentration is known to increase in diabetes, produced a dose-dependent (0.2-0.5 mg/ml) inhibition of ECM degradation by normal human mesangial cells. Taken together, these results document the importance of tPA versus uPA in renal plasmin production and indicate that in contrast to elevated glucose, glycated albumin may contribute to ECM accumulation in diabetic nephropathy.