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Query: UNIPROT:P00750 (PLA)
 16,800 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The fibrinolytic activity of the new plasminogen activator, recombinant staphylokinase, was compared to that of streptokinase and tissue-type plasminogen activator in the fibrin plate assay. The pattern of fibrinolysis by staphylokinase on fibrin plates differs from the other plasminogen activators. A number of mutants of staphylokinase with various amino acids in position 26 substituted for methionine in wild-type staphylokinase were compared with respect to their fibrinolytic potencies. Only the mutants with cysteine or leucine in this position have a fibrinolytic activity comparable to wild-type staphylokinase. The results on the fibrinolytic activities in the fibrin plate assay correlate with those of a plasmin generation assay, the latter is, however, less sensitive.
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PMID:The fibrinolytic activity of staphylokinase mutants in the fibrin plate assay. 858 17

Variants of tissue-type plasminogen activator (t-PA) were constructed with selected cysteines replaced by alanine to evaluate the role of an unpaired cysteine, which has been presumed to be in the growth factor module. C75A, C83A, C84A and CC83-84AA variants of t-PA were expressed transiently in human embryonic kidney cells. The biochemical properties of these variants provided experimental evidence to identify the unpaired cysteine in t-PA. Assays of amidolytic activity, plasminogen activation (in the presence or absence of fibrinogen or fibrin), plasma clot lysis, fibrin binding, clearance in mice, and interaction with a panel of monoclonal antibodies were performed as the basis for comparing these variants with wild-type t-PA. In all assays, C83A t-PA was biochemically equivalent to wild-type t-PA. C75A t-PA, C84A t-PA and CC83-84AA t-PA variants exhibited reduced activities in a variety of functional assays. These variants displayed two-to threefold lower activity in fibrinogen or fibrin stimulated plasminogen activation, and fivefold reduced plasma clot lysis activity compared with that of wild-type t-PA. The affinity of C75A t-PA and C84A t-PA for fibrin was decreased more than two orders of magnitude compared with C83A t-PA or wild-type t-PA. Plasma clearance of C75A t-PA and C84A t-PA was reduced 2-fold in mice. The C75A, C84A and CC83-84AA variants displayed significantly decreased reactivity with anti-tPA monoclonal antibodies specific for finger/growth factor domain epitopes. These data are consistent with a disulfide linkage of Cys75 with Cys84 and that Cys83 exists as an unpaired sulfhydryl. The significance of the unpaired cysteine is as yet undetermined since C83A t-PA and wild-type t-PA are functionally equivalent.
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PMID:Locating the unpaired cysteine of tissue-type plasminogen activator. 873 95

Collagens of most connective tissues are subject to continuous remodelling and turnover, a phenomenon which occurs under both physiological and pathological conditions. Degradation of these proteins involves participation of a variety of proteolytic enzymes including members of the following proteinase classes: matrix metalloproteinases (e.g. collagenase, gelatinase and stromelysin), cysteine proteinases (e.g. cathepsin B and L) and serine proteinases (e.g. plasmin and plasminogen activator). Convincing evidence is available indicating a pivotal role for matrix metalloproteinases, in particular collagenase, in the degradation of collagen under conditions of rapid remodelling, e.g. inflammation and involution of the uterus. Under steady state conditions, such as during turnover of soft connective tissues, involvement of collagenase has yet to be demonstrated. Under these circumstances collagen degradation is likely to take place particularly within the lysosomal apparatus after phagocytosis of the fibrils. We propose that this process involves the following steps: (i) recognition of the fibril by membrane-bound receptors (integrins?), (ii) segregation of the fibril, (iii) partial digestion of the fibril and/or its surrounding non-collagenous proteins by matrix metalloproteinases (possibly gelatinase), and finally (iv) lysosomal digestion by cysteine proteinases, such as cathepsin B and/or L. Modulation of this pathway is carried out under the influence of growth factors and cytokines, including transforming growth factor beta and interleukin 1 alpha.
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PMID:Phagocytosis and intracellular digestion of collagen, its role in turnover and remodelling. 876 55

A variant of recombinant plasminogen with the plasmin active site serine (S741) replaced by cysteine was produced and labeled with fluorescein at this residue to provide the derivative Plg(S741C-fluorescein). Studies of cleavage, conformation, and fibrin-binding properties of the derivative showed it to be a good model substrate to study plasminogen activation. Both in solution and in a fully polymerized fibrin clot, cleavage of the single chain zymogen to the two-chain "plasmin" molecule was accompanied by a 50% quench of fluorescence intensity. This change allows facile, continuous monitoring of the kinetics of cleavage. Measurements of cleavage by single chain t-PA within intact, fully polymerized 3 microM fibrin yielded apparent kcat and Km values of (0.08 s-1, 0.52 microM) and (0.092 s-1, 0.098 microM) for [Glu1]- and [Lys78]Plg(S741C-fluorescein), respectively. These values are similar to those obtained by others with plasma plasminogen. The approach used here might generally be useful in simplifying the analysis of zymogen activation kinetics in cases where the product (protease) has a great influence on its own formation via positive or negative feedback loops.
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PMID:Production and characterization of recombinant human plasminogen(S741C-fluorescein). A novel approach to study zymogen activation without generation of active protease. 899 20

Plasminogen activator inhibitor-1 (PAI-1), the primary inhibitor of tissue-type plasminogen activator and urokinase, is known to convert readily to a latent form by insertion of the reactive center loop into a central beta-sheet. Interaction with vitronectin stabilizes PAI-1 and decreases the rate of conversion to the latent form, but conformational effects of vitronectin on the reactive center loop of PAI-1 have not been documented. Mutant forms of PAI-1 were designed with a cysteine substitution at either position P1' or P9 of the reactive center loop. Labeling of the unique cysteine with a sulfhydryl-reactive fluorophore provides a probe that is sensitive to vitronectin binding. Results indicate that the scissile P1-P1' bond of PAI-1 is more solvent exposed upon interaction with vitronectin, whereas the N-terminal portion of the reactive loop does not experience a significant change in its environment. These results were complemented by labeling vitronectin with an arginine-specific coumarin probe which compromises heparin binding but does not interfere with PAI-1 binding to the protein. Dissociation constants of approximately 100 nM are calculated for the vitronectin/PAI-1 interaction from titrations using both fluorescent probes. Furthermore, experiments in which PAI-1 failed to compete with heparin for binding to vitronectin argue for separate binding sites for the two ligands on vitronectin.
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PMID:The use of fluorescent probes to characterize conformational changes in the interaction between vitronectin and plasminogen activator inhibitor-1. 903 May 77

Angiostatin, a potent naturally occurring inhibitor of angiogenesis and growth of tumor metastases, is generated by cancer-mediated proteolysis of plasminogen. Human prostate carcinoma cells (PC-3) release enzymatic activity that converts plasminogen to angiostatin. We have now identified two components released by PC-3 cells, urokinase (uPA) and free sulfhydryl donors (FSDs), that are sufficient for angiostatin generation. Furthermore, in a defined cell-free system, plasminogen activators [uPA, tissue-type plasminogen activator (tPA), or streptokinase], in combination with one of a series of FSDs (N-acetyl-L-cysteine, D-penicillamine, captopril, L-cysteine, or reduced glutathione] generate angiostatin from plasminogen. An essential role of plasmin catalytic activity for angiostatin generation was identified by using recombinant mutant plasminogens as substrates. The wild-type recombinant plasminogen was converted to angiostatin in the setting of uPA/FSD; however, a plasminogen activation site mutant and a catalytically inactive mutant failed to generate angiostatin. Cell-free derived angiostatin inhibited angiogenesis in vitro and in vivo and suppressed the growth of Lewis lung carcinoma metastases. These findings define a direct mechanism for cancer-cell-mediated angiostatin generation and permit large-scale production of bioactive angiostatin for investigation and potential therapeutic application.
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PMID:The mechanism of cancer-mediated conversion of plasminogen to the angiogenesis inhibitor angiostatin. 938 Jul 26

Previously we observed that the finger/growth factor (FG) region of tissue-type plasminogen activator (t-PA) blocked the low-density-lipoprotein-receptor-related protein (LRP)-mediated clearance of t-PA by rat hepatocytes. However, the concentrations needed were much higher than those for intact t-PA. The FG region was expressed in yeast and lacked the fucose on Thr61, which was reported to be important for efficient clearance of t-PA by human hepatoma cells. At position 83 it had a serine, whereas human t-PA has a free cysteine and rodent t-PA an arginine at this position. To understand the reason for the low efficacy of the FG protein we produced in CHO cells chimeric molecules composed of two FG modules linked to the Fc portion of human IgG1 (FG2-Fc). Two variants were studied, one having Ser83, the other Arg83. The two fucosylated FG2-Fc chimeras were compared with each other, with non-fucosylated FG and with intact t-PA with regard to their effect on the clearance of t-PA and t-PA x plasminogen-activator inhibitor type 1 (PAI-1). For this comparison, LRP-specific clearance models were used. In rat hepatoma cells and in mouse embryonic fibroblasts (MEF-1) the clearance of t-PA and of t-PA x PAI-1 was inhibited more than 95% by receptor-associated protein, an inhibitor of LRP-mediated clearance, whereas no t-PA or t-PA x PAI-1 clearance was observed in LRP-deficient PEA-13 mouse embryonic fibroblasts. The Ser83 and Arg83 FG2-Fc chimeras were equally efficient inhibitors in these models. Their efficacies in inhibiting t-PA and t-PA x PAI-1 degradation (IC50 750 nM and 890 nM, respectively) were similar to those of non-fucosylated FG (IC50 1950 nM and 1560 nM) and 75-fold lower than that of intact t-PA (IC50 9.9 nM and 21.1 nM). The results indicate that the presence of a serine or an arginine at position 83 and the presence of a fucose on Thr61 are not of major importance for the LRP-mediated clearance of t-PA.
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PMID:Studies on the effect of fucosylated and non-fucosylated finger/growth-factor constructs on the clearance of tissue-type plasminogen activator mediated by the low-density-lipoprotein-receptor-related protein. 949 55

Plasminogen contains a unique disulphide bond, Cys558-Cys566, responsible for the cyclic nature of the peptide sequence surrounding the activation site at Arg561-Val562. A recombinant [Ser558, Ser566]-Lys78-plasminogen variant was produced in which the two cysteine residues were replaced by serine residues. The variant was used to study the functional implications of removing the structural restrains imposed to the activation loop by this disulphide bond. Elimination of the Cys558-Cys566 bond attenuated activation by urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA), but resulted in an increased susceptibility to cleavage by trypsin and plasma kallikrein. Two opposite effects on the interaction of plasminogen with streptokinase were produced by modification of this bond; (a) attenuation of the rate at which the active complex with streptokinase was formed and (b) a 7.5-fold increase in plasminogen activation catalysed by this complex. Activation by tPA in the presence of fibrin, in contrast to activation in its absence, was not attenuated by elimination of this disulphide bond. However, the activation rate as a function of plasminogen concentration followed a different saturation curve, and the fibrin degradation pattern was changed. The results suggest that the Cys558-Cys566 disulphide bond is of importance for the specificity of plasminogen. This applies to its activation and also to its role in subsequent fibrin clot degradation.
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PMID:Elimination of the Cys558-Cys566 bond in Lys78-plasminogen--effect on activation and fibrin interaction. 949 20

Much attention has been paid to proteinases derived from not only neurons but also microglia in relation to neuronal death. There is accumulating evidence that intra- and extracellular proteinases in these cells are part of the basic machinery of neuronal death pathways. Some members of the ced-3/interleukin-1 beta converting enzyme (ICE) (caspase) family of cysteine proteinases have been thought to play a major role in apoptosis of not only non-neuronal cells but also neurons. Calpain has also been demonstrated to be a mediator of the neurodegenerative response. Recent studies have shown that excitotoxic and ischemic neuronal injury could be attenuated by inhibitors of caspases and calpain. Several recent studies have suggested the involvement of endosomal/lysosomal proteinases, including cathepsins B, D and E, in neuronal death induced by excitotoxins and ischemia. Furthermore, it has been reported that the extracellular tissue-type plasminogen activator/plasmin proteolytic cascade is involved in excitotoxic injury of the hippocampal neurons. In addition to such neuronal proteinases, microglial proteinases are believed to be important for the modification of neuronal functions positively or negatively. Cathepsins E and S derived from microglia have been suggested to contribute to neuronal survival through degradation and removal of beta-amyloid, damaged neurons and cellular debris. On the other hand, 6-hydroxydopamine-induced microglial cell death was inhibited by inhibitors of aspartic proteinases and caspases, suggesting the involvement of cathepsins E and D and caspases in microglial cell death. Therefore, identification of which proteinases play a causative role in neuronal death execution and clarification of the regulators and substrates for such proteinases is very important for understanding the molecular basis of the neuronal death pathways and to develop novel neuroprotective agents.
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PMID:[Involvement of proteinases produced by both neurons and microglia in neuronal lesion and death pathways]. 978 98

Betaseron, an analogue of human beta-interferon where serine was genetically engineered to substitute for cysteine at position 17, is produced in E. coli. The molecule is a small polypeptide of 165 amino acids with a single disulphide bond, and is non-glycosylated. The site-specific substitution was made to obtain a product that is more stable upon storage. Similar to native IFN-beta, Betaseron is hydrophobic in nature and has been shown to have the same panel of biological activities which includes antiviral activity against a variety of viruses, inhibition of cell growth, activation of natural killer cells, and binding to interferon receptors on the cell surface. Betaseron has been tested in a wide variety of clinical settings since 1983. The pivotal trial for the treatment of relapsing-remitting multiple sclerosis began in 1988. A PLA was filed for this indication in 1992 by Berlex and Chiron, and FDA approval was received in 1993. Betaseron is synthesized in E. coli and deposited as inclusion bodies. The manufacturing process involves solubilization and reduction of the insoluble protein, followed by purification by organic extraction, cystine oxidation and size exclusion chromatographic steps. The purified Betaseron is formulated with human serum albumin to maintain solubility at neutral pH. The complete primary sequence of Betaseron was verified by amino and carboxy-terminal sequence analysis, peptide mapping, amino acid analysis and fragment analysis after chemical cleavages. Overlapping amino acid sequence information confirmed that the amino acid sequence is the same as predicted by the DNA sequence. The amino-terminal methionine of Betaseron is removed after synthesis in E. coli. An intramolecular disulphide bond between Cys 31 and Cys 141 formed during the manufacturing process is routinely confirmed by peptide map analysis. The purity of Betaseron is assessed using a panel of analytical methods including non-reducing and reducing SDS-PAGE and reversed phase HPLC analysis where minor product-related components can be identified. These minor species were characterized with respect to their biological and biochemical properties, and identified using a variety of approaches including construction of additional, beta-interferon analogs. There is significant redundancy in the release testing of Betaseron. The amount of characterization information available on this relatively simple molecule along with the extensive manufacturing experience would suggest that some redundant testing could be eliminated for this well-characterized protein.
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PMID:Betaseron. 989 May 22


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