Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.7 (plasmin)
9,023 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Evidence has accumulated that invasion and metastasis in solid tumors require the action of tumor-associated proteases, which promote the dissolution of the surrounding tumor matrix and the basement membranes. Receptor-bound urokinase-type plasminogen activator (uPA) appears to play a key role in these events. uPA converts plasminogen into plasmin and thus mediates pericellular proteolysis during cell migration and tissue remodeling under physiological and pathophysiological conditions. uPA is secreted as an enzymatically inactive proenzyme (pro-uPA) by tumor cells and stroma cells. uPA exerts its proteolytic function on normal cells and tumor cells as an ectoenzyme after having bound to a high-affinity cell surface receptor. After binding, pro-uPA is activated by serine proteases (e.g. plasmin, trypsin or plasma kallikrein) and by the cysteine proteases cathepsin B or L, resp. Receptor-bound enzymatically active uPA converts plasminogen to plasmin which is bound to a different low-affinity receptor on tumor cells. Plasmin then degrades components of the tumor stroma (e.g. fibrin, fibronectin, proteoglycans, laminin) and may activate procollagenase type IV which degrades collagen type IV, a major part of the basement membrane. Hence receptor-bound uPA will promote plasminogen activation and thus the dissolution of the tumor matrix and the basement membrane which is a prerequisite for invasion and metastasis. Tissues of primary cancer and/or metastases of the breast, ovary, prostate, cervix uteri, bladder, lung and of the gastrointestinal tract contain elevated levels of uPA compared to benign tissues. In breast cancer uPA and PAI-1 antigen in tumor tissue extracts are independent prognostic factors for relapse-free and overall survival.
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PMID:Tumor-associated urokinase-type plasminogen activator: biological and clinical significance. 151 91

When alpha 2-macroglobulin (alpha 2M) is reacted with proteinases including trypsin, plasmin, alpha-thrombin, or with CH3NH2, each resulting alpha 2M derivative is precipitated by Zn2+ in a similar manner. By contrast, unreacted alpha 2M is not precipitated over the same Zn2+ concentration range. Zn2+-induced precipitation of alpha 2M-CH3NH2 or alpha 2M-trypsin is prevented by acylation of the protein employing the histidine-specific reagent diethylpyrocarbonate (DEP). The Zn2+-induced precipitation of alpha 2M-trypsin is prevented by acylation of the preformed alpha 2M-trypsin complex or by the reaction of acylated native alpha 2M with trypsin. Acylation of alpha 2M by treatment with DEP results in the modification of 13.5 histidyl residues per subunit of either native alpha 2M or alpha 2M-CH3NH2. Subsequent treatment with hydroxylamine reverses the modification of 10.5 histidyl residues per subunit in each protein preparation. These results indicate that histidyl residues are involved in the Zn2+-induced precipitation of alpha 2M-proteinase or alpha 2M-CH3NH2 complexes, and that these residues are accessible to extensive protein-metal interactions only after alpha 2M has undergone a major conformational change. These appear to be the same histidyl residues which, upon acylation by DEP, are responsible for recognition of alpha 2M-proteinase complexes by the acyl-low-density-lipoprotein cell surface receptor (S. V. Pizzo, P. A. Roche, S. R. Feldman, and S. L. Gonias (1986) Biochem. J. 238, 217-225).
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PMID:The role of histidyl residues in zinc-induced precipitation of alpha 2-macroglobulin-proteinase complexes. 244 Mar 83

Insulin-like growth factor (IGF)-I is markedly induced after balloon injury in the rat aorta, where it may serve to mediate vascular repair. Because the bioavailability of IGF-I is modulated by IGF-binding proteins (IGFBPs), we examined the regulation of IGFBPs by IGFs in primary cultures of rat aortic smooth muscle cells (SMCs). Serum-deprived SMC-conditioned medium contains IGFBPs of 38 to 45 kD (only in confluent cultures), 30 kD (possibly IGFBP-2), 28 kD, and 24 kD (IGFBP-4), the latter being the most abundant. IGF-I and IGF-II but not insulin evoked a marked decrease of IGFBP-4 as early as 4 hours after treatment. IGFBP-4 mRNA abundance, however, was entirely unaffected by IGF-I for up to 48 hours. IGF-I analogues with high affinity for the IGF-I receptor and weak affinity for IGFBP paradoxically evoked a small increase in IGFBP-4, probably through a general increase in protein synthesis. IGF-I only minimally decreased IGFBP-4 content in medium of sparse cultures, whereas it completely abolished IGFBP-4 content in conditioned medium of superconfluent SMCs. IGF-I also evoked a concentration-dependent increase in the abundance of IGFBP-3 in confluent, but not sparse, SMCs without affecting IGFBP-3 mRNA. Addition of IGF-I to cell-free medium conditioned by confluent, but not by sparsely cultured, SMCs led to rapid degradation of IGFBP-4. Interestingly, IGFBP-4 mRNA was markedly induced in confluent relative to sparsely grown SMCs in an IGF-I independent fashion. Thus, both biosynthesis and IGF-dependent proteolysis of IGFBP-4 are increased in confluent SMCs. Proteolysis was maximal at 37 degrees C and was abrogated by EDTA and by benzamidine. Phenylmethylsulfonyl fluoride and the plasmin inhibitor bdellin had minor inhibitory activity, whereas aprotinin, angiotensin-converting enzyme inhibitors, and N-ethylmaleimide were without effect. The protease does not affect the structure of IGF-I as determined by reverse-phase high-performance liquid chromatography and size-exclusion chromatography of 125I-IGF-I incubated for up to 24 hours with SMC-conditioned medium containing IGFBP-4. In summary, SMCs elaborate a cation-dependent protease in a confluence-dependent fashion, which degrades bound IGFBP-4 and likely releases free structurally intact IGF-I, presumably to interact with the cell surface receptor and/or other IGFBPs.
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PMID:Expression and insulin-like growth factor-dependent proteolysis of insulin-like growth factor-binding protein-4 are regulated by cell confluence in vascular smooth muscle cells. 751 Oct 71

Tumor cells avidly secrete various proteinases, and cascades of proteolytic activation occur around the cells. Therefore, cell surface receptors of tumor cells are under the constant influence of proteinases. In this study, the effects of serine proteinases on integrin-medicated cell-matrix interactions were studied in C32TG and Mewo human melanoma cells. These melanoma cells were pretreated with proteinases and their adhesive properties on various substrata were evaluated by cell adhesion assays. Paradoxically, appropriate cell surface proteolysis enhanced the RGD-sensitive cell adhesion on fibrinogen and vitronectin, but not the RGD-insensitive adhesion on type I collagen or laminin. Pretreatment of these cells with 0.1 to 1 microM of trypsin, chymotrypsin, or plasmin for 30 min at 37 degrees C increased the number of spread cells on fibrinogen and vitronectin by 200-300%. The enhancement of cell spreading was not accompanied by up-regulation of the relevant RGD-sensitive integrin expression. Analysis of the cell surface receptor by GRGDSPK-Sepharose affinity chromatography showed that trypsin treatment did not up-regulate alpha v beta 3 integrin, an RGD-sensitive receptor for fibrinogen and vitronectin in the melanoma cells, nor the induced appearance of novel receptors. Treatment of cells with 100 nM proteinases increased cell binding of both monoclonal and polyclonal antibodies against alpha v beta 3 integrin subunits by 70%, but not that of monoclonal antibody against alpha 2, alpha 3, or alpha 6 subunit, indicating that cell surface proteolysis exposed more alpha v beta 3 integrin on the cell surface. These results suggest that exposure of alpha v beta 3 integrin is a part of the mechanisms underlying the serine proteinase-induced enhancement of melanoma cell adhesion on fibrinogen and vitronectin.
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PMID:Cell surface proteolysis by serine proteinases enhances RGD-sensitive melanoma cell adhesion on fibrinogen and vitronectin. 754 29

Proteolytic enzymes such as urokinase-type plasminogen activator (uPA), plasmin, and collagenase mediate proteolysis by a variety of tumor cells. uPA secreted by tumor cells can be bound to a cell surface receptor via a growth factor-like domain within the amino-terminal fragment (ATF) of the uPA molecule with high affinity. Urinary trypsin inhibitor (UTI) efficiently inhibits the soluble and the tumor cell-surface receptor-bound plasmin and subsequently reduces tumor cell invasion and the formation of metastasis. The anti-invasive effect is dependent on the anti-plasmin activity of the UTI molecule, domain II in particular. We synthesized a conjugate between ATF of human uPA and a native UTI molecule or domain II of UTI (HI-8). The effect of the conjugates (ATF.UTI or ATF.HI-8) on tumor cell invasion in vitro was investigated. ATF.UTI and ATF.HI-8 bound to U937 cells in a rapid, saturable, dose-dependent, and reversible manner. A large part of receptor-bound ATF-UTI and ATF.HI-8 remains on the cell surface for at least 5 h at 37 degrees C. Inhibition of tumor cell-surface receptor-bound plasmin by ATF.UTI and ATF.HI-8 was markedly enhanced when compared with tumor cells treated either with ATF, UTI, or HI-8. Results of a cell invasion assay showed that ATF.UTI and ATF.HI-8 is very effective at targeting HI-8 specifically to uPA receptor-expressing tumor cells, whereas tumor cells devoid of uPA receptor may be less affected by the conjugates. Our results indicate that cell surface uPA and plasmin activity is essential to the invasive process and that the conjugates exhibit plasmin inhibition to the close environment of the cell surface and subsequently inhibit the tumor cell invasion through Matrigel in an in vitro invasion assay.
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PMID:Inhibitory effect of a conjugate between human urokinase and urinary trypsin inhibitor on tumor cell invasion in vitro. 771 45

The urokinase plasminogen activator (uPA) is a proteolytic enzyme which converts the proenzyme plasminogen to the active serine protease plasmin. A cell surface receptor for uPA (uPAR) is attached to the cell membrane by a glycosyl-phosphatidylinositol anchor. Binding of uPA to uPAR leads to an enhanced plasmin formation and thereby an amplification of pericellular proteolysis. We have shown previously that uPAR is expressed on normal blood monocytes and granulocytes, but is deficient on affected blood monocytes and granulocytes in patients with paroxysmal nocturnal haemoglobinuria (PNH), and that uPAR is present in plasma from these patients. In this study a newly established sensitive enzyme-linked immunosorbent assay (ELISA) has been applied for quantitation of uPAR in plasma. Unexpectedly, we found that uPAR is not only present in PNH plasma but also in plasma from healthy individuals. In 39 healthy individuals the mean plasma-uPAR value +/- SD was 31 +/- 15 pM, median 28 (range 11-108), and the corresponding value for six PNH patients was 116 +/- 67 pM, median 90 (range 61-228). The elevated uPAR-level in PNH patients was highly significant (Mann-Whitney test; P < 0.0001), and may possibly contribute to the propensity for thrombosis in PNH by inhibition of the fibrinolytic system. Binding of pro-uPA by uPAR in plasma may interfere with the appropriate binding of pro-uPA to cell-bound uPAR and therefore inhibit cell-associated plasmin generation and fibrinolysis. It is likely that the uPAR in normal plasma reflects the overall level of activity of the uPAR-mediated cell surface proteolysis. The present ELISA may be used for studies of uPAR levels in plasma from patients with conditions in which this activity might be increased, such as cancer and inflammatory disorders. Future studies will determine if uPAR in plasma is a parameter of clinical importance in these diseases.
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PMID:The receptor for urokinase plasminogen activator is present in plasma from healthy donors and elevated in patients with paroxysmal nocturnal haemoglobinuria. 773 57

The receptor for urokinase-type plasminogen activator (uPA-R) localizes uPA to the cell surface. The receptor-bound uPA converts plasminogen to the trypsin-like endopeptidase plasmin. Thus uPA is involved in the initiation of pericellular proteolysis. Pericellular proteolysis is assumed to facilitate the cellular infiltration into surrounding tissue. The uPA-R has recently been identified as a surface antigen of activated human T lymphocytes. We have characterized the uPA-R of the human CD4 T cell line Jurkat by immunological (flow cytometry), biochemical (ligand blotting), and physico-chemical (Scatchard blotting) methods. The collective data suggest that the human CD4+ T cell line Jurkat expresses a cell surface receptor for uPA similar to that of myelo/monocytes and normal T cells with regard to size, affinity, ligand specificity, and antigenicity. Binding studies using exogenous uPA and subsequent functional assays revealed that receptor-bound uPA retains its enzymatic activity. Saturation of the Jurkat cell uPA-R with exogenous uPA facilitated cellular invasion into fibrin matrices in vitro. uPA-dependent invasion was inhibited in the presence of an anti-catalytic monoclonal anti-uPA antibody. We propose that uPA-R-bound uPA may facilitate the invasiveness of uPA-R-positive T lymphocytes.
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PMID:Urokinase-type plasminogen activator enhances invasion of human T cells (Jurkat) into a fibrin matrix. 791 95

The plasminogen activator urokinase promotes tumor invasion by converting plasminogen into plasmin, which degrades several extracellular matrix components. Urokinase can bind to a specific cell surface receptor, which leads to accelerated plasmin production. While there is good evidence indicating a role for this binding site in tumor invasion/metastasis, there is little information concerning the regulation of urokinase receptor expression in invasive cancer. To address this question a series of colon cancer cell lines, which demonstrate either a high or low ability to invade an extracellular matrix-coated porous filter, was characterized for receptor expression at the transcriptional and post-transcriptional levels. The invasive cell lines possessed 10-fold more receptors than their non-invasive counterparts as shown by cross-linking experiments and by Western blotting. Northern blotting indicated that this disparity in receptor number could be largely accounted for by a different amount of steady-state mRNA encoding the binding site. However, neither gene amplification nor enhanced mRNA stability could account for the augmented receptor protein observed for the invasive colon cancer cell types. In contrast, nuclear run-on experiments with representative cell lines revealed that the 10-fold difference in receptor display between the invasive-competent and invasive-deficient cells could be largely accounted for by differences in transcription rates. Transcription of the u-PAR gene in the receptor-deficient GEO cells, but not in the receptor-rich RKO cells, could be augmented by protein kinase C stimulation. These findings provide a clear rationale for studies to determine if the urokinase receptor promoter in invasive colon cancer is activated in cis or in trans.
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PMID:Transcriptional activation of the urokinase receptor gene in invasive colon cancer. 807 48

Urokinase-type plasminogen activator (uPA) is an important mediator of cellular invasiveness. Specifically, cell surface receptor-bound uPA activates plasminogen to the potent general protease plasmin, which then degrades extracellular matrix or basement membrane either directly or via proteolytic activation of latent collagenases. Thus, cell surface uPA initiates an extracellular proteolytic cascade with which invasive cells eliminate barriers to movement. Since cellular invasiveness plays important roles in several disease states, including cancer metastasis and invasion, arthritis and inflammation, and diabetic retinal neovascularization, the development of synthetic uPA inhibitors is an attractive therapeutic goal. Here we show that 4-substituted benzo[b]thiophene-2-carboxamidines represent an important new class of potent and selective synthetic uPA inhibitor. Two compounds in this class, B428 and B623, inhibit human uPA in plasminogen-linked assays with median inhibition concentration (IC50) values of 0.32 and 0.07 microM, respectively. This level of inhibition represents 20- and 100-fold increases in potency, respectively, relative to the 6-7 microM potencies reported for amiloride and 4-chlorophenylguanidine, the two most potent selective synthetic uPA inhibitors previously described. Importantly, both compounds show > 300-fold selectivity for uPA relative to tissue-type plasminogen activator and > 1000-fold selectivity relative to plasmin. Lineweaver-Burk analyses show uPA inhibition by B428 and B623 to be competitive in nature with inhibition constants (Ki) of 0.53 and 0.16 microM, respectively. Since it is cell surface uPA and not free or secreted uPA that is primarily responsible for cellular invasiveness, biologically effective uPA inhibitors must be capable of inhibiting cell surface uPA. B428 and B623 meet this criterion by inhibiting cell surface uPA on HT1080 human fibrosarcoma cells with IC50 values of 0.54 and 0.20 microM, respectively. Moreover, degradation of [3H]fibronectin by HT1080 cells via cell surface uPA-mediated, plasminogen-dependent mechanisms is inhibited by B428 and B623, with IC50 values of 1.5 and 0.39 microM, respectively. In summary, 4-substituted benzo[b]thiophene-2-carboxamidines such as B428 and B623 represent the most potent class of competitive synthetic uPA inhibitors currently known. Their ability to selectively inhibit both free and cell surface uPA as well as cell surface uPA-mediated cellular degradative functions suggests that this class of compounds may hold significant promise for further development as antiinvasiveness drugs.
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PMID:Inhibition of urokinase by 4-substituted benzo[b]thiophene-2-carboxamidines: an important new class of selective synthetic urokinase inhibitor. 849 19

We have shown (Bizik et al., Cell Regul 1:895-905, 1990) that tPA can activate plasminogen on the surface of human melanoma cells in the presence of alpha 2-macroglobulin (alpha 2M) secretion. In the present study, we investigated the binding of tPA on the surface of Bowes melanoma cells, selected since they lacked production of PAI-1 and alpha 2M. Elution of tPA from the cell layers indicated that polylysine (5 micrograms/ml) and tranexamic acid (10 mM), an analog of lysine, were the most efficient agents for disrupting the interaction between tPA and cell surface component(s). Using a panel of monoclonal antibodies against individual domains of tPA revealed that an antibody directed to the kringle-2 domain of tPA interfered most significantly with cell-surface plasmin generation. As tPA is a glycoprotein, interactions between the tPA sugar moieties and cell surface were also tested by the use of a series of monosaccharides. N-acetyl-D-glucosamine (100 mM) was the most potent sugar to release tPA from melanoma cells, but the results indicated that the oligosaccharides of tPA play only a supportive role in the binding of tPA to the cell surface. Quantitative comparison of the cell surface localized tPA, which was eluted by tranexamic acid, with the total cellular tPA showed that cell surface bound tPA could represent up to 10%. We conclude that tPA interacts with the melanoma cell surface in a similar manner as has been described for binding of tPA to fibrin and to the putative endothelial cell surface receptor.
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PMID:Binding of tissue-type plasminogen activator to human melanoma cells. 850 Nov 35


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