Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.73 (urokinase-type plasminogen activator)
 10,685 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Neonatal human foreskin obtained at circumcision was cut into 2 x 2-mm pieces and placed in organ culture. Culture medium consisted of a serum-free, growth factor-free basal medium containing either 0.15 mmol/L Ca2+ or 1.4 mmol/L Ca2+. Some cultures were left as control, whereas others were treated with 3 mumol/L all-trans retinoic acid (RA). In the presence of RA, epidermal cohesion was disrupted and the upper layers separated from the viable epidermis beneath. This effect was observed under both low Ca2+ and high Ca2+ conditions. At 2-day intervals, culture fluids were collected and analyzed for serine and metalloproteinase activities. Serine proteinase activity was detected in the culture fluids and virtually all of the detected activity was dependent on the presence of plasminogen. Activity was elevated in the RA-treated tissues and this was due to increased amounts of both urokinase-type plasminogen activator (u-PA) and tissue-type plasminogen activator (t-PA). Elastase and cathepsin G were not detected in either control or RA-treated cultures. Increased plasminogen activator levels were also detected in RA-treated keratinocytes and fibroblasts in monolayer culture. Significant amounts of t-PA (though not u-PA) were found in fibroblast culture fluids, whereas both t-PA and u-PA were detected in culture fluids from keratinocytes. Metalloproteinase activity was also detected in the culture fluids of control and RA-treated tissues but in contrast to plasminogen activator, metalloproteinase activity decreased in the presence of RA. Casein and gelatin zymographic studies indicated the presence of both 92- and 72-kd gelatinases and stromelysin-1 and suggested that the decreased activity was primarily due to reduction in the 92- and 72-kd gelatinases. When serine proteinase inhibitors (aprotinin and soybean trypsin inhibitor) were included in the culture medium throughout the incubation period, epidermal discohesion was reduced. A metalloproteinase inhibitor, tissue inhibitor of metalloproteinase-2, did not have this effect. Taken together, these data show that a number of proteolytic enzymes are produced during organ culture of human skin. They suggest that these proteases may influence the structural integrity of the tissue.
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PMID:Expression of serine proteinases and metalloproteinases in organ-cultured human skin. Altered levels in the presence of retinoic acid and possible relationship to retinoid-induced loss of epidermal cohesion. 808 40

In vitro activation of pro-cathepsin B purified from ascitic fluid of ovarian carcinomas by serine proteinases was studied. Both elastase and cathepsin G from human leucocytes were found to be activators, on the basis of generation of cathepsin B activity and processing of the precursor. These results represent a new cooperative pathway between cancer cells and host cells. The urokinase-type plasminogen activator activated pro-cathepsin B faster than leucocyte proteinases. A new relationship is emerging between the cysteine proteinases and the plasmin-activation system. Both pathways suggest an important role of cathepsin B in the proteolytic cascade associated with tumour invasion.
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PMID:In vitro activation of pro-cathepsin B by three serine proteinases: leucocyte elastase, cathepsin G, and the urokinase-type plasminogen activator. 840 67

The myxoma and malignant rabbit fibroma poxviruses are lethal tumorigenic viruses of rabbits whose virulence is modulated by the production of a virus-encoded secreted serine proteinase inhibitor, SERP-1. This viral protein was detected in medium harvested from myxoma and malignant rabbit fibroma virus-infected cells, and its inhibitory profile has been characterized by gel and kinetic analysis. SERP-1 forms complexes with and inhibits the human fibrinolytic enzymes plasmin, urokinase, and two-chain tissue-type plasminogen activator (association rate constants 3.4 x 10(4), 4.3 x 10(4), and 3.6 x 10(4) M-1 s-1 respectively). It is also able to inhibit C1S, the first enzyme in the complement cascade with an association rate constant which was unaffected by the addition of heparin (1.3 x 10(3) M-1 s-1). SERP-1 acts as a substrate for and is cleaved by thrombin, porcine trypsin, human neutrophil elastase, porcine pancreatic elastase, thermolysin, subtilisin, bovine alpha-chymotrypsin, and factor Xa. Incubation with kallikrein and cathepsin G had no effect. The structure of SERP-1 has been modeled on other members of the serpin family which revealed the characteristic serpin architecture apart from the absence of the D-helix. Structural analysis and kinetic assays demonstrate that the absence of this region does not prevent inhibitory activity and furthermore allow the identification of cysteine residues involved in internal and intermolecular disulfide bonding.
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PMID:Inhibition of plasmin, urokinase, tissue plasminogen activator, and C1S by a myxoma virus serine proteinase inhibitor. 841 56

The interaction of novel series of synthetic inhibitors with various serine proteases (leukocyte elastase, thrombin, cathepsin G, chymotrypsin, plasminogen activators and plasmin) and an aspartic protease (HIV-1 protease) were studied. Various aspects were analyzed: mechanism of action, structure-activity relationships, and in some cases, molecular modelling and biological evaluation. Functionalized cyclopeptides and N-aryl azetidin-2-ones behaved as suicide substrates acting specifically on trypsin-like proteases (thrombin or urokinase) and elastases, respectively. Novel hydrazinopeptides acted as reversible inhibitors of elastases. Coumarin derivatives inactivated very efficiently chymotrypsin-like proteases (k(inact)/K(I) = 760,000 M(-1) .s(-1)). Inhibitors of HIV-1 protease acting either as inactivators or dimerization inhibitors are under investigation. The inhibitors described above are useful for elucidating the biological roles of the target enzymes and constitute potential drugs.
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PMID:[Synthetic inhibitors targeting serine and aspartic acid proteases]. 877 49

The kinetic parameters were determined for the hydrolysis of a peptide based on the activation site of the thrombin receptor (residues 38-60) by thrombin and 12 other proteases. The kcat and Km values for the cleavage of this peptide (TR39-40) by thrombin were 107 s-1 and 1.3 microM; the kcat/Km of TR39-40 is among the highest observed for thrombin. A model is presented that reconciles the parameters for cleavage of the peptide with the concentration dependence of cellular responses to thrombin. Cleavage of TR39-40 was not specific for thrombin. The pancreatic proteases trypsin and chymotrypsin hydrolysed TR39-40 efficiently (kcat/Km > 10(6) M-1.s-1). Whereas trypsin cleaved TR39-40 at the thrombin activation site (Arg41-Ser42), chymotrypsin hydrolysed the peptide after Phe43. This chymotryptic cleavage would result in inactivation of the receptor. The efficient cleavage of TR39-40 by chymotrypsin (kcat/Km approximately 10(6) M-1.s-1) was predominantly due to a low Km value (2.8 microM). The proteases factor Xa, plasmin, plasma kallikrein, activated protein C and granzyme A also hydrolysed TR39-40 at the Arg41-Ser43 bond, but exhibited kcat/Km values that were at least 10(3)-fold lower than that observed with thrombin. Both tissue and urokinase plasminogen activators as well as granzyme B and neutrophil elastase were unable to cleave TR39-60 at appreciable rates. However, neutrophil cathepsin G hydrolysed the receptor peptide after Phe55. Like the chymotryptic cleavage, this cleavage would lead to inactivation of the receptor, but the cathepsin G reaction was markedly less efficient; the kcat/K(m) value was almost four orders of magnitude lower than that for thrombin. In addition to the above cleavage sites, a secondary site for thrombin and other arginine-specific proteases was identified at Arg46, but the cleavage at this site only occurred at very low rates and is unlikely to be significant in vivo.
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PMID:Cleavage of the thrombin receptor: identification of potential activators and inactivators. 894 6

This study demonstrated the profile of the neutral proteinases, i) matrix metalloproteinases (MMP)-1, -2, -3, and -9, and ii) serine proteinases, elastase, cathepsin G, urokinase and tissue type plasminogen activators (uPA and tPA) as well as their inhibitors, namely, tissue inhibitor of matrix metalloproteinases (TIMP)-1, alpha 1-antitrypsin, alpha 1-antichymotrypsin, plasminogen activator inhibitor (PAI)-1 & 2, around loose hip prostheses to clarify the step in the cascade of biological host response in the loosening of replaced total hip joints. Immunohistochemical analysis showed the presence of MMPs (MMP-1, -2, -3, and -9) and serine proteinases (elastase, cathepsin G, uPA and tPA) both in the interface tissues and pseudocapsular tissues. Functional biochemical analysis revealed elevated proteolytic activities of MMPs, especially, MMP-2 and MMP-9, and also elastase and cathepsin G, which were not inhibited in loco, although the inhibitors, TIMP-1, alpha 1-antitrypsin and alpha 1-antichymotrypsin were detected. The results suggested the imbalance of neutral proteinase-inhibitor levels around loose hip prostheses. The proteolytic enzyme in the interface tissues could directly weaken periprosthetic tissues. The pseudocapsular tissues may induce cellular host response and proteolytic activation. Thus, the pseudocapsular tissues could contribute to the loosening via production of MMPs and serine proteinases into the synovial fluid. Pseudosynovial fluid, which showed high contents of inhibitors (TIMP-1, alpha 1-antitrypsin and alpha 1-antichymotrypsin) associated with low proteolytic potentials, could be produced to prevent the unfavorable elevation of proteolytic enzymes in loco as a local host response to implants.
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PMID:Neutral proteinases and their inhibitors in the loosening of total hip prostheses. 897 33

A large body of literature supports the role of interleukin-8 (IL-8) in inflammatory lung disease. Numerous factors induce the local synthesis and secretion of this potent chemokine leading to the recruitment and activation of polymorphonuclear leukocytes. However, little is currently known about the fate of IL-8 secreted at sites of inflammatory injury. We have found that incubation of recombinant human IL-8 with purified human neutrophil elastase (HNE) results in the loss of IL-8 chemotactic activity in a dose- and time-dependent fashion. This loss in bioactivity is accompanied by a similar loss of IL-8 immunoreactivity. Western blot analysis revealed that IL-8 chemotactic activity is lost by proteolysis of the parent molecule into undetectable small fragments. The terminal digestion of IL-8 was specific to HNE as no loss of bioactivity was observed with equimolar concentrations of the serine proteases urokinase, plasmin, thrombin, or cathepsin G. This effect on chemotactic activity is not limited to recombinant IL-8 because HNE also digested IL-8 secreted by human monocytes. HNE-mediated proteolysis offers a novel mechanism for down-regulating the inflammatory cascade initiated by IL-8.
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PMID:Human neutrophil elastase abolishes interleukin-8 chemotactic activity. 906 Apr 60

Human endothelial cells express thrombin receptors and PAR-2, the two known members of the family of protease-activated G protein-coupled receptors. Because previous studies have shown that the biology of the human thrombin receptor varies according to the cell in which it is expressed, we have taken advantage of the presence of both receptors in endothelial cells to examine the enabling and disabling interactions with candidate proteases likely to be encountered in and around the vascular space to compare the responses elicited by the two receptors when they are present in the same cell and to compare the mechanisms of thrombin receptor and PAR-2 clearance and replacement in a common cellular environment. Of the proteases that were tested, only trypsin activated both receptors. Cathepsin G, which disables thrombin receptors, had no effect on PAR-2, while urokinase, kallikrein, and coagulation factors IXa, Xa, XIa, and XIIa neither substantially activated nor noticeably disabled either receptor. Like thrombin receptors, activation of PAR-2 caused pertussis toxin-sensitive phospholipase C activation as well as activation of phospholipase A2, leading to the release of PGI2. Concurrent activation of both receptors caused a greater response than activation of either alone. It also abolished a subsequent response to the PAR-2 agonist peptide, SLIGRL, while only partially inhibiting the response to the agonist peptide, SFLLRN, which activates both receptors. After proteolytic or nonproteolytic activation, PAR-2, like thrombin receptors, was cleared from the endothelial cell surface and then rapidly replaced with new receptors by a process that does not require protein synthesis. Selective activation of either receptor had no effect on the clearance of the other. These results suggest that the expression of both thrombin receptors and PAR-2 on endothelial cells serves more to extend the range of proteases to which the cells can respond than it does to extend the range of potential responses. The results also show that proteases that can disable these receptors can distinguish between them, just as do most of the proteases that activate them. Finally, the residual response to SFLLRN after activation of thrombin receptors and PAR-2 raises the possibility that a third, as yet unidentified member of this family is expressed on endothelial cells, one that is activated by neither thrombin nor trypsin.
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PMID:Endothelial cell thrombin receptors and PAR-2. Two protease-activated receptors located in a single cellular environment. 911 Oct 10

Very-low-density lipoprotein receptor (VLDLR) and alpha2-macroglobulin receptor/low-density-lipoprotein-receptor-related protein (alpha2MR/LRP) are multifunctional endocytosis receptors of the low-density lipoprotein receptor family. Both have been shown to mediate endocytosis and degradation of complex between plasminogen activators and type-1 plasminogen-activator inhibitor (PAI-1) by cultured cells. We have now studied the specificity of binding and endocytosis by VLDLR and alpha2MR/LRP among a variety of serine proteinase/serpin complexes, including various combinations of the serine proteinases urokinase-type and tissue-type plasminogen activators, plasmin, thrombin, human leukocyte elastase, cathepsin G, and plasma kallikrein with the serpins PAI-1, horse leukocyte elastase inhibitor, protein C inhibitor, C1-inhibitor, alpha2-antiplasmin, alpha1-proteinase inhibitor, alpha1-antichymotrypsin, protease nexin-1, heparin cofactor II, and antithrombin III. Binding was estimated with radiolabelled ligands in ligand blotting analysis and microtiter well assays. Endocytosis was estimated by measuring receptor-associated protein (RAP)-sensitive degradation of radiolabelled complexes by Chinese hamster ovary cells transfected with VLDLR cDNA and by COS-1 cells, which have a high endogenous expression of alpha2MR/LRP. We found that the receptors bind with high affinity to some, but not all, combinations of plasminogen activators and thrombin with PAI-1, protease nexin-1, protein C inhibitor, and antithrombin III, while complexes of many serine proteinases with their primary inhibitor, i.e. plasmin/alpha2-antiplasmin complex, do not bind, or bind with a very low affinity. Both the serine proteinase and the serpin moieties contribute to the binding specificity. The binding specificities of VLDLR and alpha2MR/LRP are overlapping, but not identical. The results suggest that VLDLR and alpha2MR/LRP have different biological functions by having different binding specificities as well as by being expressed by different cell types.
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PMID:Specificity of serine proteinase/serpin complex binding to very-low-density lipoprotein receptor and alpha2-macroglobulin receptor/low-density-lipoprotein-receptor-related protein. 934 78

Heparan sulfate is rapidly degraded by an endoglycosidase (heparanase) secreted by activated platelets. Since the cleavage and release of heparan sulfate would profoundly alter the local physiology of the endothelium, platelet heparanase activity should be tightly regulated. Consistent with this hypothesis, platelet heparanase was found to degrade endothelial cell heparan sulfate at pH 6.0 but not at pH 7.4, even though 25% of maximum activity was detected at pH 7.4. Loss of heparanase activity occurred rapidly (t1/2 is approximately equal to 20 min) and reversibly at physiologic pH but did not occur at acidic pH (<7.0). Inactivation of heparanase at pH 7.4 did not affect heparin binding and was reversed by 0.5 M NaCl or by heparan sulfate but not by chondroitin sulfate, suggesting inactive heparanase could be tethered on cell surfaces and the function regulated by heparan sulfate. Heparanase was gradually inactivated by trypsin and urokinase (t1/2 = 5 h) but resisted cleavage by leukocyte cathepsin G, leukocyte elastase, plasmin, and thrombin. These findings are consistent with a model in which platelet heparanase is active at the low pH of inflammation but inactive under physiologic conditions preventing inadvertent cleavage of heparan sulfate and loss of physiologic functions of endothelial cells.
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PMID:Regulation of platelet heparanase during inflammation: role of pH and proteinases. 957 70


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