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:3.4.21.7 (plasmin)
9,023 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We studied the synthesis and secretion of alpha-2-macroglobulin by cultures of human adherent cells. Much more alpha-2-macroglobulin (measured by radioimmunoassay) accumulated in media of established strains of adherent cells derived from embryonic lung than in media of established strains derived from adult skin or rheumatoid synovium. Alpha-2-macroglobulin accumulated in media of primary cultures of adherent cells from a variety of embryonic tissues. However, the amount of alpha-2-macroglobulin accumulating in media of subsequent passage of these cells declined for all strains except those derived from lung. Immunodiffusion and double-antibody immunoprecipitation studies of cell extracts and media after incubation of cells with l-[(35)S]methionine supported the radioimmunoassay finding that adherent cells from lung synthesized and secreted more alpha-2-macroglobulin than adherent cells from skin. Intracellular alpha-2-macroglobulin could not be detected by radio-immunoassay or visualized by immunofluorescent microscopy, suggesting that synthesized alpha-2-macroglobulin is rapidly secreted. Plasminogen-rich fibrin clots were lysed in culture media of adherent cells from embryonic lung and, to a lesser extent, heart. Adherent cells from other tissues, which produced less alpha-2-macroglobulin, did not lyse fibrin clots. However, all cultures of adherent cells contained pericellular fibronectin, a large, external, transformation-sensitive glycoprotein known to be cleaved by plasmin. We speculate that production of alpha-2-macroglobulin may be a means for protease-secreting normal cells to preserve cell surface integrity and that alpha-2-macroglobulin synthesized locally in lung may protect lung tissues from a variety of proteases.
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PMID:Synthesis and secretion of alpha-2-macroglobulin by cultured adherent lung cells. Comparison with cell strains derived from other tissues. 7 7

We have observed that treatment of rabbit synovial fibroblasts with proteolytic enzymes can induce secretion of collagenase (EC 3.4.24.7) and plasminogen activator (EC 3.4.21.-). Cells treated for 2-24 hr with plasmin, trypsin, chymotrypsin, pancreatic elastase, papain, bromelain, thermolysin, or alpha-protease but not with thrombin or neuraminidase secreted detectable amounts of collagenase within 16-48 hr. Treatment of fibroblasts with trypsin also induced secretion of plasminogen activator. Proteases initiated secretion of collagenase (up to 20 units per 10(6) cells per 24 hr) only when treatment produced decreased cell adhesion. Collagenase production did not depend on continued presence of proteolytic activity or on subsequent cell adhesion, spreading, or proliferation. Routine subculturing with crude trypsin also induced collagenase secretion by cells. Secretion of collagenase was prevented and normal spreading was obtained if the trypsinized cells were placed into medium containing fetal calf serum. Soybean trypsin inhibitor, alpha(1)-antitrypsin, bovine serum albumin, collagen, and fibronectin did not inhibit collagenase production. Although proteases that induced collagenase secretion also removed surface glycoprotein, the kinetics of induction of cell protease secretion were different from those for removal of fibronectin. Physiological inducers of secretion of collagenase and plasminogen activator by cells have not been identified. These results suggest that extracellular proteases in conjunction with plasma proteins may govern protease secretion by cells.
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PMID:Proteases induce secretion of collagenase and plasminogen activator by fibroblasts. 20 72

Limited proteolytic cleavage of fibronectin and plasma cold-insoluble globulin with cathepsin D produced two major fragments. The smaller, Mr = 72,000 fragment bound to collagen and contained most of the cysteine in the molecule. This region contains intrachain disulfide bonds which maintain a conformation that is necessary for interaction with collagen. Cleavage of the intact protein and the 72,000-dalton fragment with plasmin localized the collagen-binding region in cold-insoluble globulin to a sequence of about 42,000 daltons. This region is located approximately two-thirds of the linear distance from the NH2 terminus of each chain in the dimeric molecule.
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PMID:Isolation of a collagen-binding fragment from fibronectin and cold-insoluble globulin. 76 39

The human NSCLC cell lines HS-24 (squamous cell carcinoma) and SB-3 (metastasis derived from an adenocarcinoma) were investigated in respect to cell interactions, motility and invasive properties. HS-24 revealed high self adhesion capacity. Testing the interactions with collagens type I/III or IV, laminin and fibronectin by adhesion, non directional motility and haptotaxis assays, tight interactions and stimulation, particularly with collagen type I/III, was detected. Proteinase inhibitors (E64, Stefin A or leupeptin) revealed a slightly negative influence. Invasion in vitro of lung explants was reduced by leupeptin in a dose dependent manner and slightly increased by plasmin. SB-3 cells revealed low self adhesion. As judged from interaction with fibronectin, these cells have low integrin receptor concentrations and thus reduced adhesiveness to extracellular matrix. Collagen type I/III was inhibitory for undirectional motility and not permissive for haptotaxis. Therefore, it may play a restrictive role during the spread in vivo of these cells. Colonization of lung explants was low and was not influenced by cathepsin B proteinase inhibitors. The results emphasize a particular role of collagens for primary site tumor and metastasis development.
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PMID:Cell interactions and motility in human lung tumor cell lines HS-24 and SB-3 under the influence of extracellular matrix components and proteinase inhibitors. 131 92

In this article we review a novel type of plasminogen activation on staphylococcal and streptococcal cells. The activation mechanism implies a specific binding of glu-plasminogen to bacterial surface via the lysine-binding sites of plasminogen. Association of plasminogen with bacterial surfaces greatly enhances the t-PA mediated activation which takes place only poorly in solution. The end product, surface-associated plasmin, is enzymatically active, protected against high molecular weight plasmin inhibitors and capable of converting itself from glu-plasmin to the lys-form. The modification is associated with an increased affinity of the bound lys-plasmin towards the binding molecules on bacterial surface. This novel way of retaining plasmin on the surface may be important for the bacteria to invade and penetrate surrounding tissues. Our data on the effect of plasmin on staphylococcal adherence indicate that plasmin is not very effective in cleaning bacteria from surfaces coated with extracellular matrix components, fibronectin and fibrinogen.
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PMID:Surface-associated activation of plasminogen on gram-positive bacteria. Effect of plasmin on the adherence of Staphylococcus aureus. 132 10

We have screened six human squamous carcinoma cell lines for their ability to invade connective tissue by using the experimentally modified chorioallantoic membrane of a chick embryo as an in vivo model of invasion. In confirmation of our earlier studies, all the invasive cell lines expressed high levels of surface-bound urokinase type plasminogen activator (uPA). However, some cell lines expressing this activity were not invasive, suggesting that surface uPA, although necessary, was not sufficient. Since in addition to fibronectin, that can be degraded by uPA or plasmin, chorioallantoic membrane connective tissue contains collagen, we examined the profile of collagenases secreted by the various cell lines in search for an activity that would coincide with the invasive phenotype. We found, using gelatin substrate gels, that type IV gelatinase was produced by all six cell types tested, three cell types produced the M(r) 92,000 gelatinase, and three a lower-molecular-weight activity, which we identified by immunoprecipitation with specific antibodies, and by a direct assay of activity, as interstitial collagenase. Only the latter cells were found to be highly invasive. We showed previously that continuous culture in vitro of one of the carcinoma cell lines, HEp3, led to a gradual extinction of their malignant phenotype. To confirm the correlation between invasion and the production of interstitial collagenase, we examined these two functions in cells freshly isolated from a HEp3 tumor and intermittently during passage in vitro. We found that, although the surface uPA activity was slightly diminished in the in vitro grown cultures, it was still within the range of values found in highly malignant cells, suggesting that it is not the reason for the decrease in invasiveness. In contrast, the reduction in interstitial collagenase closely followed the loss of the invasive phenotype; after 30 in vitro passages the cells were almost completely devoid of interstitial collagenase and unable to invade. The decrease in collagenase activity was not the result of an increased tissue inhibitor of metalloproteinases production.
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PMID:Invasion of connective tissue by human carcinoma cell lines: requirement for urokinase, urokinase receptor, and interstitial collagenase. 133 82

Thrombospondin (TSP), an adhesive integrin-binding protein of plasma and platelets, was detected in preretinal traction membranes from patients with idiopathic (8/8) and traumatic (7/8) proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy (PDR) (6/8). TSP immunoreactivity was compared to the pattern of von Willebrand factor, plasma transglutaminase (blood coagulation factor XIII), fibronectin, and mononuclear phagocytes, using double-label immunofluorescence microscopy. TSP was partially colocalised with the endothelial cell marker, von Willebrand factor, in PDR. The codistribution of catalytic factor XIII and two cross-linking substrates, fibronectin and TSP, suggests a functional role of the enzyme in the extracellular matrix build-up in PVR and PDR. No significant TSP synthesis by mononuclear phagocytes was observed. Western blotting indicated a plasmin-mediated intravitreal breakdown of presumably plasmatic TSP in PVR and PDR.
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PMID:Thrombospondin: a new attachment protein in preretinal traction membranes. 135 87

Binding of the adhesive ligand fibrinogen and the monoclonal antibody PAC1 to platelet glycoprotein (GP) IIb-IIIa is dependent on cell activation and inhibited by Arg-Gly-Asp (RGD)-containing peptides. Previously, we identified a sequence in a hypervariable region of PAC1 (mu-CDR3) that mimics the activity of the antibody. Here we examine whether monoclonal antibodies to this idiotypic determinant in PAC1 can mimic GP IIb-IIIa by binding to fibrinogen. Mice were immunized with a peptide derived from the mu-CDR3 of PAC1. Four antibodies were obtained that recognized fibrinogen as well as a recombinant form of the variable region of PAC1. However, they did not bind to other RGD-containing proteins, including von Willebrand factor, fibronectin, and vitronectin. Several studies suggested that these anti-PAC1 peptide antibodies were specific for GP IIb-IIIa recognition sites in fibrinogen. Three such sites have been proposed: two RGD-containing regions in the A alpha chain, and the COOH terminus of the gamma chain (gamma 400-411). Two of the antibodies inhibited fibrinogen binding to activated platelets, and all four antibodies bound to the fibrinogen A alpha chain on immunoblots. Antibody binding to immobilized fibrinogen was partially inhibited by monoclonal antibodies specific for the two A alpha chain RGD regions. However, the anti-PAC1 peptide antibodies also bound to plasmin-derived fibrinogen fragments X and D100, which contain gamma 400-411 but lack one or both A alpha RGD regions. This binding was inhibited by an antibody specific for gamma 400-411. When fragment D100 was converted to D80, which lacks gamma 400-411, antibody binding was reduced significantly (p less than 0.01). Electron microscopy of fibrinogen-antibody complexes confirmed that each antibody could bind to sites on the A alpha and gamma chains. These studies demonstrate that certain anti-PAC1 peptide antibodies mimic GP IIb-IIIa by binding to platelet recognition sites in fibrinogen. Furthermore, they suggest that the gamma 400-411 region of fibrinogen may exist in a conformation similar to that of an A alpha RGD region of the molecule.
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PMID:Anti-idiotypic antibodies against an antibody to the platelet glycoprotein (GP) IIb-IIIa complex mimic GP IIb-IIIa by recognizing fibrinogen. 137 Aug 32

The effect of extracellular matrix composition on the location, amount, and activity of cell-associated urokinase-type plasminogen activator was tested using HT-1080 cells adherent to either fibronectin or vitronectin. Specific immunoprecipitation of newly synthesized urokinase indicated that cells adherent to fibronectin synthesized 2-3-fold more urokinase than cells adherent to vitronectin. Complexes of urokinase and plasminogen activator inhibitor type 1 (PAI-1) were detected in cell layers of vitronectin-adherent but not fibronectin-adherent cells. Inhibition of PAI-1 using a neutralizing monoclonal antibody resulted in a 3-fold increase in urokinase enzymatic activity on vitronectin adherent cells. Urokinase activity on fibronectin adherent cells was only slightly increased following PAI-1 neutralization. Examination of both HT-1080 and normal human fibroblast cells by immunofluorescent microscopy localized urokinase-type plasminogen activator to discrete, focal areas underneath cells adherent to vitronectin. Urokinase was not detectable by immunofluorescence on cells adherent to fibronectin. The addition of exogenous prourokinase to locate urokinase receptors on adherent HT-1080 cells indicated that the focal localization of cell-surface urokinase resulted from the clustering of urokinase receptors following adhesion to vitronectin but not fibronectin-coated substrates. These results suggest that vitronectin can contribute to the control of cell-surface plasmin activity by regulating the synthesis of urokinase and directing the localization of urokinase receptors.
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PMID:Vitronectin regulates the synthesis and localization of urokinase-type plasminogen activator in HT-1080 cells. 137 87

The precursor of matrix metalloproteinase 9 (proMMP-9), also known as '92 kDa progelatinase/type IV procollagenase', was purified from the conditioned medium of U937 monocytic leukaemia and HT1080 fibrosarcoma cell lines stimulated with phorbol 12-myristate 13-acetate. ProMMP-9 in these culture media is non-covalently complexed with the 29 kDa tissue inhibitor of metalloproteinases (TIMP), but free proMMP-9 was separated from the TIMP-proMMP-9 complex by chromatography on Green A Dyematrex gel. The final product was homogeneous on SDS/PAGE, with a molecular mass of 88 kDa without reduction and 92 kDa with reduction. Treatment of proMMP-9 with 4-aminophenylmercuric acetate converted the 88 kDa precursor into 80 kDa and 68 kDa forms. Gelatin-containing zymographic analysis showed zones of lysis associated with all three species. However, only the 68 kDa species was shown to be catalytically active by its ability to bind to alpha 2-macroglobulin. In the presence of an equimolar amount of TIMP, only the 80 kDa species was generated by treatment with 4-aminophenylmercuric acetate, but no enzyme activity was detected. This indicates that TIMP binds to the 80 kDa intermediate and inhibits the generation of the active 68 kDa species. Eight endopeptidases (trypsin, chymotrypsin, plasmin, plasma kallikrein, thrombin, cathepsin G, neutrophil elastase and thermolysin) were tested for their ability to activate proMMP-9. Of them, trypsin was the most effective activator of proMMP-9. Only partial activation (10-30%) was observed with plasmin, cathepsin G and chymotrypsin. The active forms generated by trypsin were identified as 80 kDa, 74 kDa and 66 kDa by their abilities to bind to alpha 2-macroglobulin. In the presence of an equimolar amount of TIMP, proMMP-9 was also converted into the same molecular-mass species by trypsin, but they were not proteolytically active. This suggests activated MMP-9 is inhibited by TIMP. Activated MMP-9 digested gelatin, type-V collagen, reduced carboxymethylated transferrin and, to a lesser extent, type-IV collagen and laminin A chain. The specific activity against gelatin was estimated to be 15,000 units/mg (1 unit = 1 microgram of gelatin degraded/min at 37 degrees C) by titration with alpha 2-macroglobulin. Comparative studies on digestion of gelatin and collagen types IV and V by MMP-9 and MMP-2 indicated that both enzymes degrade these substrates into similar fragments. However, the susceptibilities of laminin, fibronectin and reduced carboxymethylated transferrin to these two MMPs were sufficiently different to indicate differences in substrate specificities between these two closely related proteinases.
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PMID:Purification and characterization of matrix metalloproteinase 9 from U937 monocytic leukaemia and HT1080 fibrosarcoma cells. 137 48


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