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:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Plasmin inhibited the biosynthesis of tissue-type plasminogen activator (tPA) antigen by human umbilical vein endothelial cells (HUVEC) in a dose-dependent manner. The amount of tPA antigen found in the 24-h conditioned medium of cells treated with 100 nM plasmin for 1 h was 20-30% of that in the control group. However, in contrast to tPA, such treatment led to a 3-fold increase in plasminogen activator inhibitor (PAI) activity, whereas the amount of PAI type 1 antigen was unchanged. The effects of plasmin on HUVEC were binding- and catalytic activity-dependent and were specifically blocked by epsilon-aminocaproic acid. Microplasmin, which has no kringle domains, was less effective in reducing tPA antigen biosynthesis or enhancing PAI activity in HUVEC. Kringle domains of plasmin affected neither tPA antigen nor PAI activity of the cells. Other proteases including chymotrypsin, trypsin, and collagenase at comparable concentrations did not have a significant effect on the biosynthesis of tPA antigen or PAI activity of HUVEC. Thrombin stimulated the biosynthesis of tPA and PAI-1 antigens by HUVEC. Thrombin also stimulated an increase in the protein kinase activity in HUVEC, whereas plasmin inhibited the protein kinase activity of the cells. It is possible that plasmin regulates the biosynthesis of tPA in HUVEC through the signal transduction pathway involving protein kinase.
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PMID:Plasmin and the regulation of tissue-type plasminogen activator biosynthesis in human endothelial cells. 138 68

Migrating cells degrade pericellular matrices and basement membranes. For these purposes cells produce a number of proteolytic enzymes. Mast cells produce two major proteinases, chymase and tryptase, whose physiological functions are poorly known. In the present study we have analyzed the ability of purified human mast cell tryptase to digest pericellular matrices of human fibroblasts. Isolated matrices of human fibroblasts and fibroblast conditioned medium were treated with tryptase, and alterations in the radiolabeled polypeptides were observed in autoradiograms of sodium dodecyl sulphate polyacrylamide gels. It was found that an M(r) 72,000 protein was digested to an M(r) 62,000 form by human mast cell tryptase while the plasminogen activator inhibitor, PAI-1, was not affected. Cleavage of the M(r) 72,000 protein could be partially inhibited by known inhibitors of tryptase but not by aprotinin, soybean trypsin inhibitor, or EDTA. Fibroblastic cells secreted the M(r) 72,000 protein into their medium and it bound to gelatin as shown by analysis of the medium by affinity chromatography over gelatin-Sepharose. The soluble form of the M(r) 72,000 protein was also susceptible to cleavage by tryptase. Analysis using gelatin containing polyacrylamide gels showed that both the intact M(r) 72,000 and the M(r) 62,000 degraded form of the protein possess gelatinolytic activity after activation by sodium dodecyl sulphate. Immunoblotting analysis of the matrices revealed the cleavage of an immunoreactive protein of M(r) 72,000 indicating that the protein is related to type IV collagenase. Further analysis of the pericellular matrices indicated that the protease sensitive extracellular matrix protein fibronectin was removed from the matrix by tryptase in a dose-dependent manner. Fibronectin was also susceptible to proteolytic degradation by tryptase. The data suggest a role for mast cell tryptase in the degradation of pericellular matrices.
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PMID:Pericellular substrates of human mast cell tryptase: 72,000 dalton gelatinase and fibronectin. 146 68

Widespread intravascular coagulation is common in patients with sepsis. Coagulation abnormalities may result from exposure to endotoxin, from tumor necrosis factor alpha or interleukin 1 release, or from the actions of a more specific mediator, such as vascular permeability factor. The result is marked activation of the contact and coagulation systems; simultaneously, there is decreased fibrinolysis and depressed levels of the inhibitors of the contact and coagulation systems. Multiple agents are being studied to correct these abnormalities. Antithrombin III holds promise because it inhibits a number of factors important in contact and coagulation activation, not just thrombin. Plasminogen activators may prove helpful in increasing fibrinolysis during sepsis; because they have been associated with rebound thrombin generation, however, plasminogen activators may be most effective if used in conjunction with hirudin or a synthetic hirudin analogue. Bradykinin may offset hypotension in sepsis. Protein C may inhibit thrombin formation and also complex with plasminogen activator inhibitor 1, thereby promoting fibrinolysis. Other agents that may prove effective include alpha 1-antitrypsin Pittsburgh, C1-esterase inhibitor, monoclonal antibodies to contact factors, soybean trypsin inhibitors, thrombomodulin, prostaglandin I2, and aprotinin. There are no data to support the use of heparin or fibronectin, except in limited circumstances.
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PMID:Modulators of coagulation. A critical appraisal of their role in sepsis. 162 18

Chromosome studies were carried out after a 24-hour harvest of unstimulated bone marrow aspirate cell cultures from a 75-year-old male with a clinical diagnosis of acute myelomonocytic leukemia (FAB M4). Analysis of nine cells after trypsin-Giemsa banding (GTG) revealed two cell lines with a mosaic chromosome pattern, 46,XY/46,XY,t(7;19)(q22;p13.3). A review of the recent literature reveals one case of childhood ALL with a 46,XY/46,XY,t(7;19)(q11;q13) chromosome pattern [1] and a 46,XY,t(3q;11q),t(7q;19p),t(15;17)(q26;q22) in one patient with ANLL (FAB M3) [2]. The t(7;19)(q22;p13.3) seen in our case has not been reported as the sole specific clonal chromosome rearrangement in myeloid neoplasia. Interestingly, the plasminogen activator inhibitor type I, multi-drug resistance, and erythropoietin genes are located at band 7q22 and the insulin receptor gene is located at band 19p13.3. Both sites contain fragile site loci. The possible role of these fragile sites, genes, or other genes in the rearrangement can only be surmised.
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PMID:Atypical (7;19) translocation in acute myelomonocytic leukemia. 175 94

We have undertaken a structural and functional analysis of recombinant plasminogen activator inhibitor type 1 (PAI-1) produced in Escherichia coli using site-directed mutagenesis and immunochemistry. Expression of recombinant PAI-1 yielded an inhibitor that was functionally indistinguishable from PAI-1 made in human endothelial cells. Mutations in both the reactive center P1 and P1' residues (Arg-Met) and a putative secondary binding site for plasminogen activators on PAI-1 have been engineered to assess their functional effects. The inhibition of a panel of serine proteases, including plasminogen activators, trypsin, elastase, and thrombin, has been studied. Substitution of the P1 arginine residue with lysine or the P1' residue with either valine or serine had no detectable effect on the rate of inhibition of plasminogen activators. However, replacement of both P1 and P1' by Met-Ser produced a variant with no detectable plasminogen activator inhibitor activity. Mutations introduced into either Asp102 or Lys104 in the second site did not affect the rate of inhibition of plasminogen activators. Complementary immunochemical experiments using antibodies directed against the same two regions of the PAI-1 protein confirm that the reactive center is the primary determinant of inhibitory activity and that the putative second site is not a necessary functional region.
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PMID:Mutational and immunochemical analysis of plasminogen activator inhibitor 1. 212 Feb 33

Cultured human endothelial cells synthesize and secrete two types of plasminogen activator, tissue plasminogen activator (t-PA) and urokinase (u-PA). Previous work from this laboratory (Hajjar, K.A., Hamel, N. M., Harpel, P. C., and Nachman, R. L. (1987) J. Clin. Invest. 80, 1712-1719) has demonstrated dose-dependent, saturable, and high affinity binding of t-PA to two sites associated with cultural endothelial cell monolayers. We now report that an isolated plasma membrane-enriched endothelial cell fraction specifically binds 125I-t-PA at a single saturable site (Kd 9.1 nM; Bmax 3.1 pmol/mg membrane protein). Ligand blotting experiments demonstrated that both single and double-chain t-PA specifically bound to a Mr 40,000 membrane protein present in detergent extracts of isolated membranes, while high molecular weight, low molecular weight, and single-chain u-PA associated with a Mr 48,000 protein. Both binding interactions were reversible and cell-specific and were inhibitable by pretreatment of intact cells with nanomolar concentrations of trypsin. The relevant binding proteins were not found in subendothelial cell matrix, failed to react with antibodies to plasminogen activator inhibitor type 1 and interacted with their respective ligands in an active site-independent manner. The isolated t-PA binding site was resistant to reduction and preserved the capacity for plasmin generation. In contrast, the isolated u-PA binding protein was sensitive to reduction, and did not maintain the catalytic activity of the ligand on the blot. The results suggest that in addition to sharing a matrix-associated binding site (plasminogen activator inhibitor type 1), both t-PA and u-PA have unique membrane binding sites which may regulate their function. The results also provide further support for the hypothesis that plasminogen and t-PA can assemble on the endothelial cell surface in a manner which enhances cell surface generation of plasmin.
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PMID:Identification and characterization of human endothelial cell membrane binding sites for tissue plasminogen activator and urokinase. 215 65

The binding of type 1 plasminogen activator inhibitor (PAI-1) to the extracellular matrix (ECM) of cultured bovine aortic endothelial cells was investigated using purified 125I-labeled or L-[35S]methionine-labeled PAI-1 as probes. Little specific binding of latent PAI-1 to ECM previously depleted of endogenous PAI-1 could be demonstrated. In contrast, the guanidine-activated form of PAI-1 bound to ECM in a dose- and time-dependent manner, and binding was saturable. The dissociation constant (Kd) for this interaction was estimated to be 60 nM by Scatchard analysis, and approximately 6 pmol of activated PAI-1 was bound per cm2 of ECM. Binding was relatively specific since unlabeled, activated PAI-1 competed with 35S-labeled PAI-1 for binding to ECM, but latent PAI-1 did not. Moreover, PAI-2, protein C inhibitor (i.e. PAI-3), protease nexin-1, and alpha 2-antiplasmin were not able to compete. Tissue-type plasminogen activator (tPA) also inhibited binding, but diisopropyl fluorophosphate-inactivated tPA did not. Pretreatment of ECM with tPA, urokinase-type PA, or thrombin had no effect on its ability to subsequently bind PAI-1, whereas trypsin, plasmin, and elastase pretreatment greatly reduced its ability to bind PAI-1. Guanidine-activated, radiolabeled PAI-1 resembled active endogenous PAI-1 since it was unstable in solution but stable when bound to ECM. In addition, it formed complexes with tPA that had a relatively low affinity for ECM. These data suggest that ECM of bovine aortic endothelial cells contains a protease-sensitive structure that binds active PAI-1 tightly and relatively selectively and that this association stabilizes PAI-1 against the spontaneous loss of activity that occurs in solution.
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PMID:Binding of type 1 plasminogen activator inhibitor to the extracellular matrix of cultured bovine endothelial cells. 249 80

The relationship between plasminogen activator (PA)/plasminogen activator inhibitor (PAI) activity and morphological differentiation was investigated in human neuroblastoma (NB) cells treated with retinoic acid (RA). Conditioned medium from nine NB cell lines and one closely related neuroepithelioma line was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and zymography. All NB cell lines were shown to secrete urokinase (UK)-type PA (mol. wt., 52 kDa), and all except two produced tissue PA (mol. wt., 65 kDa). Identification of the PAs was made based on molecular weight and sensitivity to inhibition by anti-UK and anti-tPA antibodies. Several cell lines expressed PA inhibitory molecules; two molecular-weight forms were observed (35 and 40 kDa) in different cell lines. Complex formation with [125]I-labelled proteases revealed specific binding with UK and trypsin but not thrombin, plasmin, or kallikrein. After treatment for 6 days with 1 microM RA, six of the cell lines exhibited an increase in cell-associated and/or secreted tPA activity, corresponding to morphological differentiation of the cells as manifested by extensive neurite outgrowth. A decrease in UK and UK-complex secretion was observed in several of these cell lines. Three cell lines exhibiting no detectable morphological alterations with RA treatment also showed no dramatic changes in PA/PAI activity. These results suggest that morphological differentiation of NB cells may be associated with alterations in the regulation of PA activity.
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PMID:Effect of retinoic acid on human neuroblastoma: correlation between morphological differentiation and changes in plasminogen activator and inhibitor activity. 259 Sep 98

We have isolated three cDNA clones for human alpha 2-plasmin inhibitor (alpha 2-PI). Two clones are from human hepatoma cell line, Hep G2, and cover the entire protein coding region plus the 3'-flanking region up to the poly(A) sequence, and the other clone is from human liver and contains the carboxyl-terminal half. The total length of the cDNAs is 2.29 kb, corresponding to more than 95% of the full-length mRNA. alpha 2-PI seems to consist of 452 amino acid residues plus 39 amino acid residues for the signal peptide. The amino acid sequence shows 23 to 28% homology to those of five other protease inhibitors, plasminogen activator inhibitor (PAI), protein C inhibitor (PCI), alpha 1-antitrypsin (alpha 1-AT), antithrombin III (AT III), and alpha 1-antichymotrypsin (alpha 1-AC). alpha 2-PI seems to be the most distantly related among these inhibitors. Comparison of the phylogenetic trees of proteases and their inhibitors indicates that four proteases, namely elastase (or trypsin), chymotrypsin, plasminogen activator, and thrombin, may have evolved concurrently with the corresponding inhibitors. However, alpha 2-PI and PCI seem to have evolved asynchronously from their substrates. The data suggest that alpha 2-PI may originally have inhibited some protease other than plasmin, and protein C may have had an inhibitor different from the present one early in its evolutionary history.
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PMID:Structure of human alpha 2-plasmin inhibitor deduced from the cDNA sequence. 283 Feb 48

To identify factors potentially influencing expression of type 1 plasminogen activator inhibitor (PAI-1), we characterized the human tissue-specific distribution of PAI-1 mRNA and the influence of epidermal growth factor (EGF) on expression of steady state levels of PAI-1 mRNA and secretion of PAI-1 by Hep G2 cells. Two species of PAI-1 mRNA (3.2 and 2.2 kilobases) were detected, and the ratio of the two varied among tissues (3 to 5:1) in contrast to the 1:1 ratio detected in Hep G2 cells. Expression of PAI-1 mRNA was inversely related to the distribution of tissue-type plasminogen activator mRNA (2.3 kilobases). Nu-Serum, a growth media supplement, increased steady state levels of PAI-1 mRNA 5-fold within 3 h. Factors responsible were found to be trypsin-sensitive and dialysis-resistant. Antisera to EGF attenuated Nu-Serum-induced increases of PAI-1 mRNA by 57%, suggesting that EGF or EGF homologous peptides contributed to the response. EGF elicited increases of PAI-1 mRNA levels in a dose-dependent manner. Induction was rapid (7-fold at 3 h with 5 ng/ml) and complete within 10 h. The response was not attenuated by cycloheximide (25 micrograms/ml). Factor X and glyceraldehyde-3-phosphate dehydrogenase mRNA did not increase. Increased levels of PAI-1 antigen were detected in conditioned media of Hep G2 cells by 4 h and were maximal at 8 h (6-fold). We conclude that the expression of PAI-1 mRNA is tissue-specific and regulated by epidermal growth factor in Hep G2 cells.
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PMID:Regulation of the expression of type 1 plasminogen activator inhibitor in Hep G2 cells by epidermal growth factor. 284 25


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