Inhibition of receptor-bound urokinase by plasminogen-activator inhibitors. J Biol Chem 265: 9904-8 (original) (raw)
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Journal of Biological …, 1986
An approximately 75% pure form of a human M,-54,000 plasminogen activator inhibitor from conditioned culture fluid of the fibrosarcoma cell line HT-1080 was obtained by a single step of chromatography on concanavalin A-Sepharose. The inhibitor inhibited human urokinase-type plasminogen activator (u-PA) and tissue-type plasminogen activator, but not plasmin. Rabbit antibodies against this plasminogen activator inhibitor also reacted with a plasminogen activator inhibitor with identical electrophoretic mobility in extracts of human blood platelets, indicating that the HT-1080-inhibitor is of the same type as the inhibitor of blood platelets. As revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by fibrin-agarose zymography, incubation of HT-1080inhibitor with the active form of human u-PA led to the formation of an equimolar sodium dodecyl sulfateresistant complex between them; in contrast, no complex formation was observed between the inhibitor and the proenzyme form of human u-PA (pro-u-PA). Likewise, using a column of anti-inhibitor antibodies coupled to Sepharose for removal of excess inhibitor and activator-inhibitor complexes, the potential enzymatic activity of pro-u-PA was found to be unaffected by incubation with inhibitor under conditions in which more than 95% of the active u-PA had formed complex with inhibitor. Plasminogen activators comprise a group of serine proteases, which by limited proteolysis convert the abundant extracellular proenzyme plasminogen to the active protease plasmin. Plasmin has a relatively broad trypsin-like specificity. Release of plasminogen activators from cells may thus initiate localized extracellular proteolysis. TWO types of plasminogen activators have been recognized in mammals, the urokinase-type (u-PA1) and the tissue-type (t-PA), with M I of-50,000 and-70,000, respectively. They are products of different genes (1-3). Several functions have been proposed
The urokinase plasminogen activator and its receptor
Thrombosis and Haemostasis, 2005
SummaryThe urinary-type plasminogen activator, or uPA, controls matrix degradation through the conversion of plasminogen into plasmin and is regarded as the critical trigger for plasmin generation during cell migration and invasion, under physiological and pathological conditions (such as cancer metastasis).The proteolytic activity of uPA is responsible for the activation or release of several growth factors and modulates the cell survival/apoptosis ratio through the dynamic control of cell-matrix contacts. The urokinase receptor (uPAR), binding to the EGF-like domain of uPA, directs membrane-associated extracellular proteolysis and signals through transmembrane proteins, thus regulating cell migration, adhesion and cytoskeletal status. However, recent evidence highlights an intricate relationship linking the uPA/uPAR system to cell growth and apoptosis.
The urokinase plasminogen activator system as a novel target for tumour therapy
2000
Substantial data have been collected for numerous types of solid cancer, including cancer of the breast, the gastrointestinal and urological tract, the lung, and the brain, demonstrating a strong clinical value of the plasminogen activation system in predicting disease recurrence and survival in cancer patients. Elevated levels of certain members of the plasminogen activation system, the serine protease uPA (urokinase-type plasminogen activator), its receptor (uPA-R; CD87), and inhibitor (PAI-1), in tumour tissue or blood emphasize their fundamental role in tumour invasion and metastasis and provide the rationale for novel therapeutic strategies. uPA, besides its proteolytic action toward the extracellular matrix, in concert with uPA-R, PAI-1, and integrins contributes to tumour cell proliferation, adhesion, and migration. Several technical methods of affecting tumour growth and metastasis by targeting the uPA-system in cancer patients at the gene and protein level have been explored: (1) antisense oligodeoxynucleotides to uPA, uPA-R, or PAI-1; (2) antisense oligonucleotides to signal transduction pathway components such as Rel (NF-κ B), affecting uPA but not PAI-1 synthesis; (3) viral vectors delivering genes for components of the plasminogen activation system; (4) soluble, recombinant uPA-R as a scavenger for uPA; (5) monoclonal antibodies directed to uPA or uPA-R blocking uPA/uPA-R interaction; (6) enzymatically inactive uPA to compete for active uPA binding to uPA-R; (7) linear and cyclic uPA-derived peptides to block uPA/uPA-R interaction; (8) toxins, coupled to uPA or fractions thereof to kill tumour cells; (9) naturally occurring inhibitors to uPA and its derivatives for inhibition of uPA proteolytic activity; and (10) synthetic inhibitors to uPA to inhibit uPA proteolytic activity. There is substantial hope that substances designed to affect or turn off the plasminogen activation system will eventually be administered to cancer patients thereby opening a new vista for tumour biology-based, individualized cancer therapy.
Journal of Histochemistry & Cytochemistry, 1997
The urokinase-type plasminogen activator (uPA) is a serine protease that plays a crucial role in blood coagulation and in tumor invasion and metastasis. uPA is a relatively large polypeptide and binds the uPA receptor (uPAR) with high affinity and specificity. Therefore, it was a good candidate for direct labeling with a fluorochrome for detection of the uPAR. We have produced a fluorescein (FITC)-labeled human uPA using a conjugation procedure that did not significantly alter its binding characteristics to the uPAR. Thirty nM FITC-uPA efficiently stains 2 ϫ 10 5 uPAR-transfected mouse cells in suspension, as determined by flow cytometric analysis. One g of FITC-uPA efficiently stains 2 ϫ 10 5 uPAR transfectants grown on slides and analyzed by fluorescence optical microscopy. Human cell lines expressing the endogenous uPAR were stained with similar efficiency. Fixation in paraformaldehyde only slightly reduced the efficiency of staining of both transfectants and cell lines. These characteristics allow the use of FITC-uPA in both static and dynamic morphological studies of uPAR-expressing cells. (J Histochem Cytochem 45:
Functional Analysis of the Cellular Receptor for Urokinase in Plasminogen Activation
Journal of Biological Chemistry
1 The abbreviations used are: DFP, diisopropyl fluorophosphate; uPA, urokinase-type plasminogen activator, both the activated two-chain protease and as a generic term; pro-uPA, single-chain form of uPA; uPAR, uPA receptor; s-uPAR, soluble recombinant uPAR residues 1-277; PAI-1, plasminogen activator inhibitor type-1; 6-AHA, 6-aminohexanoic acid; pNA, para-nitroaniline; AMC, 7-amido-4-methylcoumarin.
A Cellular Receptor for Urokinase-Type Plasminogen Activator
XIth International Congress on Thrombosis and Haemostasis, 1987
Recent cell biological and biochemical studies on the urokinase-type plasminogen activator (u-PA) have revealed an unsuspected property of this protein: it binds with high affinity and specificity to the plasma membrane of a number of cell types. Hence, while the interaction of tissue-type plasminogen activator (t-PA) with fibrin suggests a preferred role for this enzyme in the maintenance of fluidity of the extracellular milieu, the cellular binding of u-PA results in the focalisation of plasmin generation to the close environment of the cell surface; this appears as an optimal configuration if u-PA is to participate in the enzymatic events required for cell migration.The available information on the cellular binding of u-PA can be summarized as follows:1. Human monocytes-macrophages, monocyte-like cell lines, fibroblasts, and a variety of other cell lines all express u-PA binding sites. The number of u-PA binding sites on a given cell type may vary as a function of the functional ...
Accessibility of receptor-bound urokinase to type-1 plasminogen activator inhibitor
Proceedings of the National Academy of Sciences, 1989
Urokin plasminogen activator (uPA) interacts with a surface receptor and with specifi inhibitors, such as plasminogen activator inhibitor type 1 (PAM-). These interactions are mediated by two functionally independent domains of the molecule: the catalytic domain (at the carboxyl terminus) and the growth factor domain (at the amino terminus). We have now investigated whether PAMcan bind and inhibit receptor-bound uPA. Binding of 125I-labeled ATF (amino-terminal fragment of uPA) to human U937 monocyte-like cells can be competed for by uPA-PAI-l complexes, but not by PAMalone. Preformed '2SI-labeled uPA-PAI-1 complexes can bind to uPA receptor with the same binding specificity as uPA. PAMalso binds to, and inhibits the activity of, receptor-bound uPA in U937 cells, as shown in U937 cells by a caseinolytic plaque assay. Plasminogen activator activity of these cells is dependent on exogenous uPA, is competed for by receptorbinding diisopropyl fluorophosphate-treated uPA, and is inhibited by the addition ofPAM-. In conclusion, in U937 cells the binding to the receptor does not shield uPA from the action of PAI-i. The possibility that in adherent cells a different localization ofPAI-i and uPA leads to protection of uPA from PAMis to be considered.
European Journal of Biochemistry, 1997
The intrinsic activity of single-chain pro-urinary-type plasminogen activator (pro-uPA) and whether its receptor (uPAR) potentiates this activity remains controversial. In this report, the pro-uPA/uPAR-(1-281)-peptide complex in solution is shown to have equivalent plasniinogen-activator activity to that of active two-chain uPA (tc-uPA). However, the activity of the complex was dependent on a synthetic tripeptide, Spectrozyme plasmin (Spl, H-~-2-aminohexanoic acid(Ahx)-hexatyrosyl-lysine-p-nitroanilide), which can also be used as a chromogenic substrate for plasmin. Furthermore, this activity could be completely suppressed by commonly used carrier proteins and detergents. The pro-uPA/uPAR-(1-281)peptide complex at 1 nM displayed similar activity to that of tc-uPA for either [Clul]plasminogen or [Lys77]plasminogen in chromogenic assays with Spl present as the plasmin substrate. When assayed with another plasmin substrate, S22.51, the pro-uPA/uPAR-(1-281)-peptide complex was unable to activate plasminogen. The pro-uPAluPAR-(1-2Sl)-peptide complex and tc-uPA also showed a similar extent of plasminogen activation as measured by SDSPAGE, when incubated with plasminogen and Spl in the presence of 100 M aprotinin, and plasminogen activation by pro-uPA alone was also stimulated in the presence of Spl in this assay. Activation of plasminogen by the pro-uPA/uPAR-(I-281)-peptide strictly required the presence of Spl, and pro-uPA remained in single-chain form during these assays. This activity of the pro-uPA/uPAR-(1-281)-peptide complex but not that of tc-uPA was completely inhibited by human serum albumin, bovine serum albumin, Tween-80, Triton X-100, and Pluronic-F68. Taken together, the data indicates that uPAR-(1-281)-peptide itself is not sufficient to augment pro-uPA activity and the presence of an effector molecule (e.g. Spl) is required to elicit the full plasminogen-activator activity of the pro-uPA/uPAR-(I-281)-peptide complex. It remains to be seen whether there is a physiological counterpart to this phenomenon.