Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UNIPROT:P00750 (PLA)
 16,800 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The potential contribution of serine/threonine-specific protein phosphatases in the transcriptional regulation of plasminogen activator and plasminogen activator inhibitor gene expression was explored in human HT-1080 fibrosarcoma and U-937 monocyte-like cells using okadaic acid, a potent and specific inhibitor of phosphatases 1 and 2A (PP1 and PP2A). In both cell types okadaic acid induced plasminogen activator type 2 (PAI-2) gene transcription and mRNA and potentiated induction mediated by phorbol-12-myristate-13-acetate and tumor necrosis factor. Okadaic acid-mediated induction of PAI-2 was inhibited by 8-bromo-cAMP in HT-1080 cells but not in U-937 cells. Okadaic acid had opposite effects on urokinase (u-PA) gene expression in the two cell lines; u-PA mRNA and gene transcription was suppressed in HT-1080 cells but transiently induced in U-937 cells. Tissue-type PA (t-PA) mRNA, although undetectable in U-937 cells, was also suppressed by okadaic acid in HT-1080 cells. This effect was selective, as constitutive and phorbol-12-myristate-13-acetate-mediated expression of plasminogen activator inhibitor type 1 mRNA was not modulated by okadaic acid in either cell type. These results indicate that PP1 and PP2A protein phosphatases are involved in signal transduction pathways modulating PAI-2, u-PA, and t-PA, and furthermore, that okadaic acid interaction with the protein kinase C and A pathways are gene- and cell type-specific.
 ...
PMID:Cell- and gene-specific interactions between signal transduction pathways revealed by okadaic acid. Studies on the plasminogen activating system. 131 13

Epidermal growth factor (EGF) domains are found in many proteins, particularly those of the coagulation/fibrinolytic system. We and others have demonstrated that tissue plasminogen activator (t-PA) and prourokinase are modified by the attachment of fucose to equivalent threonine residues within their EGF domains. Factor XII and protein C each contain two EGF domains; in both proteins, the EGF domain nearest the N terminus has a threonine residue in a position homologous to that which is fucosylated in t-PA. In protein C, this site is 3 residues from the position of another post-translational modification, beta-hydroxylation of Asp-71. We isolated peptides containing these sites to determine, primarily by mass spectrometric analysis, the presence of O-linked fucose and/or beta-hydroxyaspartate. We found that factor XII is fully fucosylated at Thr-90. Protein C is unmodified at the equivalent site (Thr-68) and is completely beta-hydroxylated at Asp-71. It has been recently reported that the first EGF domain of human factor VII has O-linked fucose at the equivalent position (Ser-60) (Bjoern, S., Foster, D. C., Thim, L., Wiberg, F. C., Christensen, M., Komiyama, Y., Pedersen, A. H., and Kisiel, W. (1991) J. Biol. Chem. 266, 11051-11057), while it is unmodified at Asp-63 despite having the consensus sequence for beta-hydroxylation at the latter site. These observations raise the possibility that O-linked fucosylation and beta-hydroxylation of EGF domains are mutually exclusive post-translational modifications.
 ...
PMID:O-linked fucose is present in the first epidermal growth factor domain of factor XII but not protein C. 154 94

Tissue-type plasminogen activator (t-PA) induces synthesis of a rapidly acting inhibitor, plasminogen activator inhibitor type-1 (PAI-1) in human umbilical vein endothelial cells (HUVEC) in culture. In vivo, an analogous process may induce negative feedback on the fibrinolytic system. To define specific determinants in the t-PA molecule contributing to the induction, PAI-1 synthesis was characterized in 35S-methionine labeled HUVEC in response to several molecular variants of t-PA. Catalytically active variants devoid of several specific structural domains in the A-chain retained the capacity to form complexes with PAI-1 and to induce increased concentrations of total PAI-1 (free and complexed) in conditioned media without depleting PAI-1 from the extracellular matrix. Surprisingly, a mutant t-PA with markedly reduced catalytic activity reflecting replacement of the active site serine with threonine (S478T) formed complexes with PAI-1 and induced increased PAI-1 synthesis as well. However, in contrast to wild-type t-PA and A-chain variants, it did not release 35S-methionine labeled PAI-1 from the extracellular matrix. Thus, its effects appeared to reflect increased secretion exclusively. Our results suggest that induction of PAI-1 synthesis in HUVEC by t-PA depends on its protease domain but that an active site serine is not a requirement.
 ...
PMID:Determinants of induction of increased synthesis of plasminogen activator inhibitor type-1 in human endothelial cells by t-PA. 162 Dec 43

An A alpha-arginine-141 to serine substitution has been identified in a homozygous dysfibrinogen, fibrinogen Lima, associated with impaired fibrin polymerization. The point mutation created an asparagine-X-serine-type glycosylation sequence, and indeed, extra, mainly disialylated biantennary oligosaccharides have been isolated from A alpha asparagine-139 of the patient's fibrinogen. This type of glycosylation sequence is unique for human fibrinogen, because the sequences shown for normal and abnormal fibrinogens are all asparagine-X-threonine types. The terminal sialic acids of the extra oligosaccharides seem to have largely contributed to the impaired fibrin gel formation, as evidenced by its correction to a near normal level by desialylation. Nevertheless, the polymerizing fibrin facilitated tissue-type plasminogen activator-catalyzed plasmin formation in a normal fashion, indicating that the initial two-stranded fibrin protofibrils had been constructed normally. Thus the impaired fibrin gel formation could be attributed to the delay in their subsequent lateral association, most probably because of the repulsive forces generated by the negative electric charge of the extra sialic acids. The substitution of a basic residue arginine to a noncharged residue serine may also have contributed to the impaired function in a similar manner or by steric hindrance in association with bulky extra oligosaccharide chains.
 ...
PMID:Fibrinogen Lima: a homozygous dysfibrinogen with an A alpha-arginine-141 to serine substitution associated with extra N-glycosylation at A alpha-asparagine-139. Impaired fibrin gel formation but normal fibrin-facilitated plasminogen activation catalyzed by tissue-type plasminogen activator. 163 21

An unusual type of glycosylation has been observed for tissue plasminogen activator (t-PA). The monosaccharide fucose is glycosidically linked to threonine-61 in the epidermal growth factor region of t-PA. The presence of O-linked fucose was demonstrated by carbohydrate analysis and mass spectrometry of tryptic and chymotryptic peptides that contain this site. The susceptibility of the fucose residue to alpha-fucosidase indicated that it was in the alpha-anomeric configuration. Fucosylation of threonine-61 was observed in t-PA isolated from the Bowes melanoma cell line and from recombinant expression systems using Chinese hamster ovary or human embryonic kidney cells. Fucosylation of the homologous residue in prourokinase has also been reported recently. Our results indicate that this novel type of glycosylation may be common to the epidermal growth factor domains found in coagulation and fibrinolytic proteins and, therefore, suggest that the modification may have functional significance.
 ...
PMID:Tissue plasminogen activator has an O-linked fucose attached to threonine-61 in the epidermal growth factor domain. 190 Apr 31

Secretion of urokinase-type plasminogen activator (uPA) by chicken embryo fibroblasts (CEF) is increased approximately 50-fold following transformation by Rous sarcoma virus (RSV). Using a cloned and fully sequenced chicken uPA cDNA probe, we have established that this increase in plasminogen activator production can be largely accounted for by an increase in cellular uPA mRNA. CEF contained on average less than 1 molecule of uPA mRNA/cell, whereas RSV-CEF contained 25-60 molecules/cell. The increase in cellular uPA mRNA levels was dependent on the activity of the RSV-encoded transforming protein, protein-tyrosine kinase pp60v-src. Cells infected with an RSV mutant encoding a temperature-sensitive form of the src protein (ts-NY68) contained low uPA mRNA levels when cultured at the nonpermissive temperature and high uPA mRNA levels when maintained at the permissive temperature. Temperature shift studies with tsNY68-CEF demonstrated that changes in pp60v-src activity rapidly altered uPA mRNA levels; the uPA mRNA content of total RNA extracts increased and decreased with half-time kinetics of 3-5 h. Serine/threonine-specific protein kinases also appear to modulate uPA mRNA levels in CEF cultures. Exposure of CEF and RSV-CEF for 24 h to the protein kinase C activating agent phorbol myristate acetate (PMA) increased cellular uPA mRNA levels to 20 and 260 molecules/cell, respectively. These data are consistent with the previously observed synergism between RSV and PMA in increasing plasminogen activator secretion. Nuclear run-on transcription analyses established that both RSV and PMA increase cellular uPA mRNA levels by way of increased uPA gene expression.
 ...
PMID:Plasminogen activator gene expression is induced by the src oncogene product and tumor promoters. 215 28

Complexes between tissue-type plasminogen activator (t-PA) and its rapidly acting inhibitor plasminogen activator inhibitor type 1 (PAI-1) are bound, internalized, and degraded by HepG2 cells. The mechanism involves endocytosis mediated by a specific high-affinity receptor. However, the particular domains of the complex that are recognized by the receptor have not been elucidated. To identify the determinants involved in ligand binding to the receptor, several variants of t-PA were assessed for their ability to form complexes with PAI-1 and thereby to inhibit specific cellular binding of complexes between structurally unmodified 125I-t-PA and PAI-1. Catalytically active variants lacking selected structural domains form complexes with PAI-1 and inhibit 125I-t-PA.PAI-1 binding to HepG2 cells. In addition, several forms of the plasminogen activator urokinase (u-PA), which shares partial structural homology with t-PA, were evaluated as competitors of cellular binding. The catalytically active two-chain forms of u-PA, but not the inactive proenzyme single-chain form, complex with PAI-1 and inhibit specific binding of 125I-t-PA.PAI-1, suggesting that the serine protease domain, rather than other domains, may confer the determinants required for cellular binding. However, a mutant t-PA with markedly reduced catalytic activity, resulting from replacement of the active site serine with threonine, not only forms complexes with PAI-1 but also inhibits specific cellular binding of unmodified 125I-t-PA.PAI-1. These data indicate that specific binding of t-PA.PAI-1 to HepG2 cells does not require a serine-containing catalytic site in the protease domain. To determine whether binding of the complex is mediated through other components of t-PA or through structural elements of PAI-1, both t-PA and PAI-1 were examined separately for capacity to bind directly to HepG2 cells. To exclude potential interactions with components of the extracellular matrix which contains binding sites for PAI-1, ligand binding to HepG2 cells in suspension was assessed. Although neither t-PA nor PAI-1 alone binds specifically to HepG2 cells, the preformed t-PA.PAI-1 complexes do. These findings suggest that specific binding of t-PA.PAI-1 requires elements of the PAI-1 moiety and/or parts of the protease domain of t-PA.
 ...
PMID:Identification of determinants involved in binding of tissue-type plasminogen activator-plasminogen activator inhibitor type 1 complexes to HepG2 cells. 216 6

To define determinants of interactions of tissue-type plasminogen activator (t-PA) with plasminogen activator inhibitor type-1 (PAI-1), we utilized site-directed mutagenesis to substitute either threonine or glycine for the active-site serine of tissue-type plasminogen activator. Assays of conditioned media of transfected cells demonstrated that the threonine substitution markedly decreased but did not entirely abolish plasminogen activating activity. In contrast, the glycine substitution yielded a mutant with absolutely no detectable plasminogen activating activity. Wild-type t-PA formed stable complexes with PAI-1. However, even when exogenous inhibitor was present in the medium or purified mutant was added to plasma that had been rendered PAI-1-rich in vivo, the mutants were present in the free form exclusively judging from results of fibrin autography and Western blot analysis. Thus, despite maintenance of some residual plasminogen-activating activity associated with preservation of the hydroxyl group at the active site, the threonine mutant did not form stable complexes with inhibitor. The glycine mutant, developed so that steric hindrance or other unfavorable interactions at the modified active site would be minimal, was similarly incapable of forming complexes with PAI-1. These results show that the presence of an active site serine residue is necessary for formation of stable complexes between t-PA and PAI-1.
 ...
PMID:Characterization of interaction of active-site serine mutants of tissue-type plasminogen activator with plasminogen activator inhibitor-1. 249 85

Mutant urokinase-type plasminogen activator (u-PA) genes and hybrid genes between tissue-type plasminogen activator (t-PA) and u-PA have been designed to direct the synthesis of new plasminogen activators and to investigate the structure-function relationship in these molecules. The following classes of constructs were made starting from cDNA encoding human t-PA or u-PA: 1) u-PA mutants in which the Arg156 and Lys158 were substituted with threonine, thus preventing cleavage by thrombin and plasmin; 2) hybrid molecules in which the NH2-terminal regions of t-PA (amino acid residues 1-67, 1-262, or 1-313) were fused with the COOH-terminal region of u-PA (amino acids 136-411, 139-411, or 195-411, respectively); and 3) a hybrid molecule in which the second kringle of t-PA (amino acids 173-262) was inserted between amino acids 130 and 139 of u-PA. In all cases but one, the recombinant proteins, produced by transfected eukaryotic cells, were efficiently secreted in the culture medium. The translation products have been tested for their ability to activate plasminogen after in situ binding to an insolubilized monoclonal antibody directed against urokinase. All recombinant enzymes were shown to be active, except those in which Lys158 of u-PA was substituted with threonine. Recombination of structural regions derived from t-PA, such as the finger, the kringle 2, or most of the A-chain sequences, with the protease part or the complete u-PA molecule did not impair the catalytic activity of the hybrid polypeptides. This observation supports the hypothesis that structural domains in t-PA and u-PA fold independently from one to another.
 ...
PMID:Mutant and chimeric recombinant plasminogen activators. Production in eukaryotic cells and preliminary characterization. 311 52

rt-PA-K, a variant of recombinant tissue-type plasminogen activator (rt-PA) with substitution of amino acids 296 to 299 with alanine (KHRR296-299AAAA) has increased fibrin-specificity and reduced sensitivity to plasminogen activator inhibitor-1; rt-PA-T, with threonine 103 replaced by asparagine has an additional glycosylation site and a reduced clearance; and rt-PA-N, with asparagine 117 mutagenized to glutamine lacks the high mannose carbohydrate side chain. We have investigated whether combination of these properties in a single molecule might yield an improved thrombolytic agent. The thrombolytic potency and fibrin-specificity of the combination mutant rt-PA-TNK was compared with that of rt-PA in a combined venous whole blood clot model and platelet-rich arterial eversion graft thrombosis model in dogs given intravenous heparin and aspirin. Infusion of 0.125 to 1.0 mg/kg over 60 min in groups of 4 to 5 dogs produced dose-dependent fibrin-specific venous clot lysis. The thrombolytic potency (percent lysis per mg compound administered per kg body weight) of rt-PA-TNK was significantly higher than that of rt-PA as evidenced by a higher maximal rate of lysis of 480 +/- 100% versus 140 +/- 40% within the 2 h observation period per mg of compound administered per kg body weight (mean +/- SEM, p = 0.004) and a significantly lower dose of 0.08 +/- 0.01 versus 0.21 +/- 0.04 mg/kg body weight at which the maximal rate of lysis was obtained (p = 0.004).(ABSTRACT TRUNCATED AT 250 WORDS)
 ...
PMID:Comparative thrombolytic properties of tissue-type plasminogen activator and of a plasminogen activator inhibitor-1-resistant glycosylation variant, in a combined arterial and venous thrombosis model in the dog. 797 84


1 2 3 Next >>