UNIPROT:P00750 - FACTA Search

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

The binding, internalization, and degradation of tissue-type plasminogen activator (t-PA) were studied in a rat hepatoma (Novikoff) cell line. Binding of t-PA to specific saturable high affinity binding sites (Kd = 12 nM, 54,000 sites/cell) was followed by internalization and degradation and did not require a functional active site. The catabolism of t-PA was not inhibited by an excess of urokinase-type plasminogen activator (u-PA), and t-PA bound to Novikoff membranes was not complexed to PAI-1, suggesting a mechanism independent of PAI-1. Additionally, a mannose receptor is not involved since t-PA binding was not influenced by an excess of mannose, galactose, ovalbumin, or EDTA. Furthermore, the degradation of t-PA was not influenced by 10 mM 6-aminohexanoic acid, a lysine analogue. The t-PA receptor binds to and can be eluted from wheat germ agglutinin-Sepharose. Cross-linking of t-PA with partially purified receptor and ligand blot analysis, suggest that t-PA binds to two proteins, a principal one of 55 kDa and a minor one of 43 kDa. Novikoff cells are able also to bind (Kd = 1.4 nM, 25,000 sites/cell) and degrade u-PA. The binding was inhibited by pro-u-PA and the amino-terminal fragment of u-PA, but not by an excess of t-PA. The u-PA receptor, but not the t-PA receptor, was removed by treatment with phosphatidylinositol-specific phospholipase C. Our results show that the clearance receptor for t-PA on Novikoff cells is different from the mannose receptor and the PAI-1-dependent receptor described in other cells. The rat hepatoma cells are thus a good model to study the PAI-1 independent hepatocyte-specific clearance of t-PA.
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PMID:Demonstration of a specific clearance receptor for tissue-type plasminogen activator on rat Novikoff hepatoma cells. 131 32

The interaction of 125I-labelled tissue-type plasminogen activator (125I-t-PA) with freshly isolated rat parenchymal and endothelial liver cells was studied. Binding experiments at 4 degrees C with parenchymal cells and endothelial liver cells indicated the presence of 68,000 and 44,000 high-affinity t-PA-binding sites, with an apparent Kd of 3.5 and 4 nM respectively. Association of 125I-t-PA with parenchymal cells was Ca(2+)-dependent and was not influenced by asialofetuin, a known ligand for the galactose receptor. Association of 125I-t-PA with liver endothelial cells was Ca(2+)-dependent and mannose-specific, since ovalbumin (a mannose-terminated glycoprotein) inhibited the cell association of t-PA. Association of 125I-t-PA with liver endothelial cells was inhibited by anti-(human mannose receptor) antiserum. Anti-(galactose receptor) IgG had no effect on 125I-t-PA association with either cell type. Degradation of 125I-t-PA at 37 degrees C by both cell types was inhibited by chloroquine or NH4Cl, indicating that t-PA is degraded lysosomally. in vitro experiments with three monoclonal antibodies (MAbs) demonstrated that anti-t-PA MAb 1-3-1 specifically decreased association of 125I-t-PA with the endothelial cells, and anti-t-PA Mab 7-8-4 inhibited association with the parenchymal cells. Results of competition experiments in rats in vivo with these antibodies were in agreement with findings in vitro. Both antibodies decreased the liver uptake of 125I-t-PA, while a combination of the two antibodies was even more effective in reducing the liver association of 125I-t-PA and increasing its plasma half-life. We conclude from these data that clearance of t-PA by the liver is regulated by at least two pathways, one on parenchymal cells (not galactose/mannose-mediated) and another on liver endothelial cells (mediated by a mannose receptor). Results with the MAbs imply that two distinct sites on the t-PA molecule are involved in binding to parenchymal cells and liver endothelial cells.
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PMID:Characterization of the interaction both in vitro and in vivo of tissue-type plasminogen activator (t-PA) with rat liver cells. Effects of monoclonal antibodies to t-PA. 131 35

In the liver, tissue-type plasminogen activator (t-PA) is endocytosed by hepatic parenchymal (PC), endothelial (EC) and Kupffer (KC) cells. Although the endocytosis is receptor-mediated, it remains a matter of discussion which receptors are involved in this catabolic process. To evaluate the role of a protein-specific receptor, as well as the possible involvement of the galactose receptor on PC and the mannose receptor on EC, we have employed different glycosylation variants of t-PA in biochemical and immunocytochemical studies. Partial or total removal of carbohydrate side-chains by endoglycosidases did not prevent clearance and hepatic endocytosis of t-PA by either of the liver cell types. Blockade of the galactose and mannose receptors by co-application of a large excess of the glycoprotein ovalbumin remained without effect on the binding and uptake of t-PA by hepatic cells. However, the contribution of different liver cell types to the hepatic clearance of t-PA was to a certain extent dependent on the type of oligosaccharide chains removed. The mannose receptor on EC is partially responsible for the clearance of t-PA by this cell type, whereas the galactose receptor does not seem to be involved in this process. The results obtained in this study further demonstrate that the major portion of the hepatic catabolism of t-PA is independent of its carbohydrate side-chains.
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PMID:Evidence for carbohydrate-independent endocytosis of tissue-type plasminogen activator by liver cells. 132 74

The binding of tissue-type plasminogen activator (t-PA) to membranes prepared from human liver was investigated, and a specific, saturable, high-affinity binding site (Kd = 3.4 nM) was identified. The binding of t-PA to liver membranes was not affected by an excess of D-mannose or D-galactose, or by active urokinase (u-PA), whereas binding of t-PA to membranes prepared from human HepG2 hepatoma cells was inhibited by u-PA. HepG2-membrane-bound t-PA was fully complexed to PA inhibitor 1 (PAI-1), whereas liver-membrane-bound t-PA was not complexed. Gel filtration on Sephacryl S300 of membrane proteins solubilized in deoxycholate revealed that high-affinity t-PA binding activity elutes at an apparent molecular mass of 40 kDa. Monoclonal antibodies specific for the growth factor and the kringle 2 domains inhibited the binding of t-PA to liver membranes and the catabolism of t-PA by rat hepatoma cells. Human liver membranes also bound u-PA; binding was inhibited by pro-u-PA, the N-terminal fragment of u-PA, but not by the 33 kDa form of u-PA or by t-PA. Our results show that human liver membranes contain a specific 40 kDa binding protein for t-PA that is different from the PAI-1-dependent receptor described on HepG2 cells and the mannose receptor isolated from human liver.
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PMID:Characterization of the binding of plasminogen activators to plasma membranes from human liver. 144 49

Various studies have shown that mannose receptors rapidly eliminate glycoproteins and microorganisms bearing high mannose-type carbohydrate chains from the blood circulation. The purpose of this study was to characterize the mannose receptor in the liver, which in vivo is involved in the rapid clearance of tissue-type plasminogen activator from the circulation. Human liver membranes were solubilized in Triton X-100, and the solution was applied to a tissue-type plasminogen activator Sepharose column. Bound proteins were eluted with ethylenediaminetetraacetate (10 mmol/L). A second, similar purification step rendered a single liver protein of 175,000 daltons. A combination of ligand blotting and a chromogenic assay for tissue-type plasminogen activator demonstrated that the identified liver protein is a mannose receptor because it bound tissue-type plasminogen activator, this tissue-type plasminogen activator binding being fully inhibited by 0.2 mol/L D-mannose. Western-blot analysis revealed that the isolated liver protein is immunologically identical to the human mannose receptor from placenta. Treatment of the liver protein and the placenta mannose receptor with trypsin yielded the same pattern of proteolytic degradation products as identified on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We conclude that the physiologically relevant mannose receptor for tissue-type plasminogen activator clearance isolated from human liver is immunologically and structurally similar to or identical with the human mannose receptor isolated from placenta.
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PMID:Isolation and characterization of the mannose receptor from human liver potentially involved in the plasma clearance of tissue-type plasminogen activator. 161 83

The glycoprotein tissue-type plasminogen activator (t-PA, alteplase, CAS 105857-23-6) is a serine protease consisting of 527 amino acids and can activate plasminogen to plasmin, which subsequently dissolves the fibrin network of a thrombus. This activation occurs selectively on the thrombus, making recombinant t-PA a very effective agent in the treatment of thromboembolic disorders. t-PA has a short in vivo half-life and is rapidly removed from the circulation by the liver. The catabolism of t-PA involves receptor-mediated endocytosis and intracellular degradation in several cell types of the liver namely hepatic endothelial, parenchymal and Kupffer cells. Liver endothelial cells have been reported to possess a specific uptake system for t-PA based on the recognition of the high mannose carbohydrate structures on Asn117. To further elucidate the involvement of the mannose receptor on sinusoidal endothelial cells in the hepatic catabolism of t-PA and to identify the mechanisms involved, biochemical as well as electron microscopic studies were performed. The biochemical studies revealed that the removal of the mannose side chain in t-PA significantly reduced its clearance and degradation in isolated perfused livers. The binding of t-PA to preparations of primary hepatocytes and liver cell membranes could not be competed for by various sugars and glycoproteins, and was not dependent on the presence of carbohydrates on the molecule. This ruled out a major relevance of the sugar moieties of t-PA in its recognition by liver cells that were not of endothelial origin.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Endocytosis of the recombinant tissue plasminogen activator alteplase by hepatic endothelial cells. 190 31

Previous studies have shown that tissue-type plasminogen activator (t-PA) in blood is cleared by the liver partially through a mannose-specific uptake system. The present study was undertaken to investigate, in a purified system, whether t-PA is recognized by the mannose receptor which is expressed on macrophages and liver sinusoidal cells. The mannose receptor was isolated and purified from bovine alveolar macrophages and migrated as a single protein band at Mr 175,000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Ligand blotting revealed that this protein specifically bound t-PA. The t-PA-receptor interaction was further characterized in a binding assay, which showed saturable binding with an apparent dissociation constant of 1 nM. t-PA binding required calcium ions and was negligible in the presence of EDTA or at acid pH. Mannose-albumin was an effective inhibitor, whereas galactose-albumin did not have a significant effect. From a series of monosaccharides tested, D-mannose and L-fucose were the most potent inhibitors, N-acetyl-D-glucosamine was a moderate inhibitor, whereas D-galactose and N-acetyl-D-galactosamine were ineffective. t-PA, deglycosylated by endoglycosidase H, did not interact with the receptor. It is concluded that the mannose receptor specifically binds t-PA, probably through its high mannose-type oligosaccharide.
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PMID:Binding of tissue-type plasminogen activator by the mannose receptor. 190 88

Tissue-type plasminogen activator (t-PA) has a short half-life in the circulation because the enzyme is rapidly cleared by the liver. This short review summarizes recent literature concerning mechanisms of uptake and degradation of t-PA in the liver. In vivo studies in rats show that degradation takes place via a lysosomal pathway. Saturation of the uptake system at high t-PA concentrations suggests a receptor-mediated mechanism. Competition experiments with various glycoproteins indicate that the asialoglycoprotein receptor is not involved, but they point to a role for the mannose receptor, which recognizes t-PA via its high mannose-type oligosaccharide on the first kringle domain. Both in vivo and in vitro studies with isolated liver cells demonstrate that parenchymal cells, as well as liver endothelial cells, are involved in the clearance of t-PA. Parenchymal cells, as the hepatoma cell line Hep G2, endocytose t-PA via a still unknown, possibly t-PA specific receptor, while liver endothelial cells catabolize t-PA via the mannose receptor.
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PMID:Receptor-mediated endocytosis of tissue-type plasminogen activator (t-PA) by liver cells. 210 99

The mechanism of uptake of radio-iodinated tissue plasminogen activator (125I-t-PA) was studied in rats. When trace amounts of 125I-t-PA were injected alone, the clearance followed a biphasic pattern in which 65% and 35% were cleared with alpha- and beta-kinetics (t1/2 (alpha) = 0.6 min, and t1/2 (beta) = 6.4 min), respectively. Co-injection with excess unlabelled t-PA or mannan changed the uptake kinetics to the monophasic beta-elimination pattern. Mannosylated albumin and ovalbumin, both of which bind to the hepatic mannose receptor, reduced the proportion of t-PA cleared with t1/2 (alpha) to 48% and 21%, respectively. A corresponding increase in the beta-elimination of t-PA was observed. The t1/2 (alpha) and t1/2 (beta) were unchanged. Studies on the clearance of 125I-ovalbumin also showed a biphasic elimination with an initial rapid phase, t1/2 (alpha), accounting for only 39% of the clearance of ovalbumin, as compared to 65% in the case of t-PA. Macromolecules with affinity for the galactose-receptor only, such as asialofetuin, or galactosylated albumin, did not significantly affect the clearance kinetics at the concentrations used. Asialoorosomucoid, which also carries galactosyl residues in the terminal position, reduced somewhat (from 65% to 48%) the proportion cleared with alpha-kinetics. Very high concentrations of galactose and N-acetyl-galactosamine, which are also known to compete for binding to the galactose receptor, lowered the proportion of t-PA cleared in the late beta-phase (reduced from 35% to 26% with galactose and to 19% with N-acetyl-galactosamine).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Clearance of tissue plasminogen activator by mannose and galactose receptors in the liver. 216 Jan 32

The interaction in vivo of 125I-labeled tissue-type plasminogen activator (t-PA) with the rat liver and the various liver cell types was characterized. Intravenously injected 125I-t-PA was rapidly cleared from the plasma (t1/2 = 1 min), and 80% of the injected dose associated with the liver. After uptake, t-PA was rapidly degraded in the lysosomes. The interaction of 125I-t-PA with the liver could be inhibited by preinjection of the rats with ovalbumin or unlabeled t-PA. The intrahepatic recognition site(s) for t-PA were determined by subfractionation of the liver in parenchymal, endothelial, and Kupffer cells. It can be calculated that parenchymal cells are responsible for 54.5% of the interaction of t-PA with the liver, endothelial cells for 39.5%, and Kupffer cells for only 6%. The association of t-PA with parenchymal cells was not mediated by a carbohydrate-specific receptor and could only be inhibited by an excess of unlabeled t-PA, indicating involvement of a specific t-PA recognition site. The association of t-PA with endothelial cells could be inhibited 80% by the mannose-terminated glycoprotein ovalbumin, suggesting that the mannose receptor plays a major role in the recognition of t-PA by endothelial liver cells. An excess of unlabeled t-PA inhibited the association of 125I-t-PA to endothelial liver cells 95%, indicating that an additional specific t-PA recognition site may be responsible for 15% of the high affinity interaction of t-PA with this liver cell type. It is concluded that the uptake of t-PA by the liver is mainly mediated by two recognition systems: a specific t-PA site on parenchymal cells and the mannose receptor on endothelial liver cells. It is suggested that for the development of strategies to prolong the half-life of t-PA in the blood, the presence of both types of recognition systems has to be taken into account.
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PMID:Characterization of the interaction in vivo of tissue-type plasminogen activator with liver cells. 314 72

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