Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.68 (tissue plasminogen activator)
 11,311 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Osteoblasts produce proteolytic enzymes and their production is regulated by osteotropic agents. It has been suggested that these proteases play a role in bone resorption by removing the superficial collagenous layer from the bone matrix and indirectly inducing migration of osteoclast precursors towards the bone matrix. We examined the effect of the plasminogen activator tPA on osteoclastic resorption using 17-day-old mouse embryonic long bone explants representing different stages of osteoclast development, that is, radii containing already mature osteoclasts and metacarpals containing no mature osteoclasts but only osteoclast precursors/progenitors which are still confined to the periosteum. Tissue type PA stimulated osteoclastic resorption (measured as 45Ca-release) in 17-day-old fetal metacarpals but not in radii of the same animal. Blocking the enzymatic activity of tPA did not inhibit its effect on osteoclastic resorption. Plasmin, the direct product of PA enzymatic activity, did not induce osteoclastic resorption. However, a tPA-mutant missing the growth-factor-like domain of the molecule, failed to stimulate 45Ca-release from the metacarpals. In addition, in both systems tPA and transforming growth factor alpha had similar effects on osteoclastic resorption. The finding that tPA stimulated 45Ca-release only in the metacarpals suggests that tPA has an effect on osteoclast formation rather than on the activity of already mature osteoclasts. Under the experimental conditions used this effect seems to be mediated by the growth factor domain of tPA rather than by the enzymatic activity of the molecule.
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PMID:The effect of tissue type plasminogen activator (tPA) on osteoclastic resorption in embryonic mouse long bone explants: a possible role for the growth factor domain of tPA. 153 5

We have studied the mechanism of a transforming growth factor-beta (TGF-beta)-stimulated production of type-1 plasminogen activator inhibitor (PAI-1) in WI-38 human lung fibroblasts. TGF-beta causes an early increase in the PAI-1 mRNA level which reaches a maximal 50-fold enhancement after 8 h. Blocking of protein synthesis with cycloheximide causes an equally strong increase in the level of PAI-1 mRNA. Quantitative studies of the effect of TGF-beta on PAI-1 protein levels in cell extracts and culture media by using monoclonal antibodies are consistent with the effect on PAI-1 mRNA. The results suggest a primary effect of TGF-beta on PAI-1 gene transcription, and also suggest the possibility that the transcription of this gene in non-induced cells may be suppressed by a short-lived negatively regulating protein. Urokinase-type (u-PA) and tissue-type (t-PA) plasminogen activators are decreased in the culture media of TGF-beta-treated cells concomitantly with the increase in PAI-1 accumulation. These findings show that a primary and important biological effect of TGF-beta may be an overall decreased extracellular proteolytic activity, and give an insight into the molecular mechanisms underlying TGF-beta action at the genetic level.
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PMID:Transforming growth factor-beta is a strong and fast acting positive regulator of the level of type-1 plasminogen activator inhibitor mRNA in WI-38 human lung fibroblasts. 311 44

The role of the RGDX putative receptor-recognition sites, which are present on the alpha chains of fibrin, in promoting platelet adhesion has been examined in flowing whole blood using the rectangular perfusion chamber at wall shear rates of 340 and 1,600/s. Platelets adhered to a comparable extent to surfaces coated with native fibrin and surfaces coated with fragment X-fibrin, a product of limited fibrinolysis that lacks the RGDS sites normally present at positions 572 to 575 of the alpha chains. The strengths of these adhesive interactions were comparable based on the concentrations of the antiadhesive peptide D-RGDW required to block platelet deposition to native and fragment X-fibrin at both low and high wall shear rate. Blocking either or both RGDX sequences with peptide-specific monoclonal antibodies did not inhibit platelet deposition in perfusion experiments performed with normal blood at 340/s, indicating that neither RGD motif is required for adhesion. However, adhesion was partly inhibited by anti-RGDX antibodies when perfusions were performed with blood from an afibrinogenemic patient, suggesting the RGDX sequences may play a limited role in platelet deposition. Exposure of fibrin surfaces to plasminogen/tissue-type plasminogen activator did cause a time-dependent loss of adhesiveness, but this effect was only weakly correlated with proteolysis of the fibrin alpha chains. These observations provide evidence that neither RGDX sequence is required for platelets to adhere avidly to fibrin in flowing blood. These results further suggest that incomplete fibrinolysis yields a highly thrombogenic surface.
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PMID:Evidence that fibrin alpha-chain RGDX sequences are not required for platelet adhesion in flowing whole blood. 762 Jan 53

Enhanced thrombin activity has been associated with coronary thrombosis and with acute and long-term complications following coronary balloon angioplasty. Blocking thrombin activity with specific inhibitors is proposed as a promising antithrombotic therapy. We describe the anticoagulant and antithrombotic properties of hirunorm, a novel synthetic 26-aminoacid peptide thrombin inhibitor, in comparison with r-hirudin and hirulog-1. Hirunorm was equipotent to hirulog-1 and 1/30 as potent as r-hirudin in blocking alpha-thrombin amidolytic activity (IC50 = 10 +/- 2, 15 +/- 1 and 0.3 +/- 0.1 nM, respectively), but it did not affect trypsin, plasmin and t-PA activities at 10 microM. All the compounds inhibited clot-bound thrombin to clots prepared by thrombin hydrolysis of purified fibrinogen in buffer. Hirunorm and hirulog-1 showed similar species-dependent potency in doubling basal in vitro clotting times of human, rat and rabbit plasma (EC200 varied 70 to 200 nM for TT, 0.7 to 16 microM for aPTT and 0.8 to 17 microM for PT), while r-hirudin was always at least three times more active. When assayed by HPLC or by bioassay of the intact peptide, hirunorm was stable against alpha-thrombin and plasma hydrolases, but it was catabolized by rat liver and kidney enzymes. Venous thrombosis was produced in anaesthetized rats by vena cava ligation following a procoagulant serum injection. Intravenous and subcutaneous hirunorm inhibited venous thrombosis at doses (< or = 0.3 mg/kg) two-three times higher than those of r-hirudin. Hirulog-1 was as active as hirunorm only after i.v. infusion. Arterial thrombosis was obtained in the anaesthetized rat by chemical (FeCl2) stimulation of a common carotid and i.v. infused hirunorm (1-3 mg/kg/30 min) inhibited it dose-dependently; r-hirudin was partly active only at 3 mg/kg, but hirulog-1 was inactive at either dose. Full antithrombotic doses of hirunorm did not affect the bleeding time as measured from punctured mesenteric vessels, in anaesthetized rats. In conclusion, hirunorm is a potent peptide thrombin inhibitor endowed with antithrombotic activity in models of venous and arterial thrombosis.
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PMID:Experimental pharmacology of hirunorm: a novel synthetic peptide thrombin inhibitor. 888 75

Tissue plasminogen activator (tPA), the serine protease that converts inactive plasminogen to the protease plasmin, was recently shown to mediate neurodegeneration in the mouse hippocampus. Mice deficient in tissue plasminogen activator (tPA) display a dramatic resistance to a paradigm of excitotoxic neuronal death that involves intrahippocampal injection of the excitotoxin. This model is thought to reproduce the mechanism of neuronal death observed during acute (such as ischemic stroke) and degenerative (such as amyotrophic lateral sclerosis) diseases of the nervous system. The requirement for the proteolytic activity of tPA to mediate neuronal death is acute in the adult mouse. Serine protease inhibitors, specific for tPA or the tPA/plasmin proteolytic cascade, are effective in conferring extensive neuroprotection following the excitotoxic injection. These findings suggest possible new ways for interfering with the neuronal death observed in the hippocampus as a result of excitotoxicity. In addition, tPA is produced in the hippocampus primarily by microglial cells, which become activated in response to the neuronal injury. Blocking microglial activation has been shown in other injury paradigms to protect against neuronal death, therefore suggesting another way to retard neurodegeneration in the CNS. Furthermore, after the insult has been inflicted and in the presence of a compromised blood-brain barrier macrophages (cells deriving from the same lineage as microglia) migrate into the brain, where they are thought to contribute to the neuronal cell loss by secreting neurotoxic molecules. If these macrophages/microglia expressed, however, a tPA inhibitor, rather than the possibly neurotoxic tPA, they might be able to protect the neurons from dying.
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PMID:Clinical implications of the involvement of tPA in neuronal cell death. 918 75

The urokinase-type plasminogen activator (UPA) and its receptor are expressed in the vasculature and are involved in cell migration and remodeling of the extracellular matrix in the neointima. Vessels with atherosclerosis or neointimal hyperplasia, when compared with normal vessels, contain high UPA activity as well as increased levels of UPA receptor. In this study, we have identified the stimulation of vascular smooth muscle cell proliferation as a novel activity for UPA in the vessel wall. High-molecular-weight-UPA (12-200 nmol/L range) stimulated DNA synthesis and cell proliferation, which was half that induced by fetal calf serum or by platelet-derived growth factor-BB. UPA did not induce growth of endothelial cells, and tissue-type plasminogen activator showed no activity on either cell type. Induction of proliferation required the complete UPA molecule but was independent of the proteolytic activity of UPA, whereas neither the amino-terminal fragment nor the catalytic domain by itself was mitogenic. UPA also stimulated c-fos/c-myc mRNA expression and mitogen-activated protein kinase activity in smooth muscle cells. Blocking monoclonal antibodies against the UPA receptor and the enzymatic removal of receptors were ineffective in inhibiting the mitogenic effect of UPA, suggesting a UPA receptor-independent mechanism. Thus, we provide evidence for a novel function of UPA on vascular smooth muscle cell proliferation that, together with its previously documented involvement in regulating pericellular proteolysis-related events and cell migration, provides additional evidence for a role in the pathogenesis of atherosclerosis/restenosis.
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PMID:Induction of vascular SMC proliferation by urokinase indicates a novel mechanism of action in vasoproliferative disorders. 940 65

Recent studies have implicated tissue-type plasminogen activator (tPA) in neurodegeneration. We studied multiple sclerosis (MS) brain tissue for tPA gene and protein expression in comparison with reference tissue, by in situ hybridisation and immunohistochemistry. MS is characterised by demyelination in the central nervous system. In this study, neuronal cell bodies in MS brain showed high expression of tPA mRNA and protein, while in reference brains, staining for protein and mRNA expression were very low in neurons and mostly restricted to blood vessel walls. In MS, there was an additional staining of mononuclear cells within perivascular cuffs and foamy macrophages within demyelinating plaques. In view of evidence that the final process of demyelination in MS is thought to be enzyme-mediated, our work suggests the involvement of tPA and by inference plasmin, in the demyelinating process. Blocking tPA or plasmin activity may be a potentially beneficial therapeutic approach in MS.
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PMID:Tissue plasminogen activator gene expression in multiple sclerosis brain tissue. 1042 51

Michaelis complex, acylation, and conformational change steps were resolved in the reactions of the serpin, plasminogen activator inhibitor-1 (PAI-1), with tissue plasminogen activator (tPA) and trypsin by comparing the reactions of active and Ser 195-inactivated enzymes with site-specific fluorescent-labeled PAI-1 derivatives that report these events. Anhydrotrypsin or S195A tPA-induced fluorescence changes in P1'-Cys and P9-Cys PAI-1 variants labeled with the fluorophore, NBD, indicative of a substrate-like interaction of the serpin reactive loop with the proteinase active-site, with the P1' label but not the P9 label perturbing the interactions by 10-60-fold. Rapid kinetic analyses of the labeled PAI-1-inactive enzyme interactions were consistent with a single-step reversible binding process involving no conformational change. Blocking of PAI-1 reactive loop-beta-sheet A interactions through mutation of the P14 Thr --> Arg or annealing a reactive center loop peptide into sheet A did not weaken the binding of the inactive enzymes, suggesting that loop-sheet interactions were unlikely to be induced by the binding. Only active trypsin and tPA induced the characteristic fluorescence changes in the labeled PAI-1 variants previously shown to report acylation and reactive loop-sheet A interactions during the PAI-1-proteinase reaction. Rapid kinetic analyses showed saturation of the reaction rate constant and, in the case of the P1'-labeled PAI-1 reaction, biphasic changes in fluorescence indicative of an intermediate resembling the noncovalent complex on the path to the covalent complex. Indistinguishable K(M) and k(lim) values of approximately 20 microM and 80-90 s(-1) for reaction of the two labeled PAI-1s with trypsin suggested that a diffusion-limited association of PAI-1 and trypsin and rate-limiting acylation step, insensitive to the effects of labeling, controlled covalent complex formation. By contrast, differing values of K(M) of 1.7 and 0.1 microM and of k(lim) of 17 and 2.6 s(-1) for tPA reactions with P1' and P9-labeled PAI-1s, respectively, suggested that tPA-PAI-1 exosite interactions, sensitive to the effects of labeling, promoted a rapid association of PAI-1 and tPA and reversible formation of an acyl-enzyme complex but impeded a rate-limiting burial of the reactive loop leading to trapping of the acyl-enzyme complex. Together, the results suggest a kinetic pathway for formation of the covalent complex between PAI-1 and proteinases involving the initial formation of a Michaelis-type noncovalent complex without significant conformational change, followed by reversible acylation and irreversible reactive loop conformational change steps that trap the proteinase in a covalent complex.
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PMID:Resolution of Michaelis complex, acylation, and conformational change steps in the reactions of the serpin, plasminogen activator inhibitor-1, with tissue plasminogen activator and trypsin. 1157 Aug 75

In breast stroma urokinase plasminogen activator (uPA) is predominantly expressed by fibroblasts located in the near vicinity of tumor cells, and fibroblast-derived insulin-like growth factor-1 (IGF-1) may be involved in inhibiting the expression of uPA in these fibroblasts. To investigate a possible role for fibroblast growth factors (FGFs), we evaluated the expression of components of the PA system and the IGF system in normal and tumor-tissue-derived human breast fibroblasts exposed to various FGFs in vitro. mRNA analysis revealed that FGF-1, FGF-2 and FGF-4 induced the mRNA expression levels of uPA, tPA, uPAR, PAI-1 and PAI-2, and reduced those of IGF-1, IGF-1R, IGF-2R and IGFBP-4, without significantly affecting the levels of IGFBP-3, IGFBP-5 and IGFBP-6 mRNA. Concerning the expression of IGF-2 mRNA, the effects mediated by FGF-1, FGF-2 and FGF-4 were divergent. In general, the effects elicited by FGF-1 on the various mRNA levels studied were rapid and short-term. Those mediated by FGF-2 overall lagged behind but were longer-lasting. For FGF-4 an in between pattern was observed. Blocking transcription and translation demonstrated that a) both the FGF-1 and FGF-2 induced effects were the result of altered gene transcription or mRNA stability, b) the short-term effects mediated by FGF-1 and FGF-2 required de novo protein synthesis, and c) the long-term effects elicited by FGF-2 did not depend on de novo protein synthesis during the first 24 h, but were triggered by proteins produced or made available thereafter. The data presented propose that of the FGFs studied (FGF-1, -2, -4, -5, and -7), FGF-2 is the most attractive target for therapeutical strategies aimed at diminishing the contribution of stromal fibroblasts in the PA-directed breast tumor proteolysis.
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PMID:Differential effects of fibroblast growth factors on expression of genes of the plasminogen activator and insulin-like growth factor systems by human breast fibroblasts. 1200 51

Glucocorticoid receptor (GR) activation has recently been shown to inhibit apoptosis in breast epithelial cells. We have previously described a group of genes that is rapidly up-regulated in these cells following dexamethasone (Dex) treatment. In an effort to dissect the mechanisms of GR-mediated breast epithelial cell survival, we now examine the molecular events downstream of GR activation. Here we show that GR activation leads to both the rapid induction of MAPK phosphatase-1 (MKP-1) mRNA and its sustained expression. Induction of the MKP-1 protein in the MCF10A-Myc and MDA-MB-231 breast epithelial cell lines was also seen. Paclitaxel treatment resulted in MAPK activation and apoptosis of MDA-MB-231 breast cancer cells, and both processes were inhibited by Dex pretreatment. Furthermore, induction of MKP-1 correlated with the inhibition of extracellular signal-regulated kinase (ERK1/2) and c-Jun N-terminal kinase (JNK) activity, whereas p38 activity was minimally affected. Blocking Dex-induced MKP-1 induction using small interfering RNA increased ERK1/2 and JNK phosphorylation and decreased cell survival. ERK1/2 and JNK inactivation was associated with Ets-like transcription factor-1 (ELK-1) dephosphorylation. To explore the gene expression changes that occur downstream of ELK-1 dephosphorylation, we used a combination of temporal gene expression data and promoter element analyses. This approach revealed a previously unrecognized transcriptional target of ELK-1, the human tissue plasminogen activator (tPA). We verified the predicted ELK-1--> tPA transcriptional regulatory relationship using a luciferase reporter assay. We conclude that GR-mediated MAPK inactivation contributes to cell survival and that the potential transcriptional targets of this inhibition can be identified from large scale gene array analysis.
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PMID:Glucocorticoid receptor-induced MAPK phosphatase-1 (MPK-1) expression inhibits paclitaxel-associated MAPK activation and contributes to breast cancer cell survival. 1559 Jun 93


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