Mechanisms of tissue-type plasminogen activator (tPA) clearance by the liver.
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Animals↗
Biomedical subjects
M Otter
Publications and source records attributed to M Otter.
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.
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.
Animals↗
Isolation and characterization of the mannose receptor from human liver potentially involved in the plasma clearance of tissue-type plasminogen activator.
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.
Blotting, Western↗
Binding of tissue-type plasminogen activator by the mannose receptor.
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.
Animals↗
Receptor-mediated endocytosis of tissue-type plasminogen activator (t-PA) by liver cells.
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.
Amino Acid Sequence↗
Binding and degradation of tissue-type plasminogen activator by the human hepatoma cell line Hep G2.
In this study, binding and degradation of tissue-type plasminogen activator (t-PA) by the human hepatoma cell line Hep G2 was investigated. Binding at 4 degrees C was time-dependent and reached a maximum after ca. 2 hours. Scatchard analysis of saturation experiments showed about 170,000 high affinity binding sites for t-PA per cell with an apparent Kd of 90 nM. These binding sites were calcium-dependent. Part of the binding to the hepatoma cells was non-saturable, owing to a large amount of low affinity binding sites which are at least partially located on the extracellular matrix of the cells. Competition with mannose- and galactose-terminated glycoproteins had no effect on total binding of 125I-t-PA. Degradation products of 125I-t-PA were found in the supernatant after a short lag phase and then increased linearly for at least 5 hours at 37 degrees C. Degradation could be inhibited by chloroquine, NH4Cl and NaN3. We conclude that the human hepatoma cell line Hep G2 has a specific binding mechanism for t-PA which is not mediated by known carbohydrate receptor systems. Binding is followed by cellular uptake and degradation in the lysosomes.
Binding, Competitive↗
Characterization of the interaction in vivo of tissue-type plasminogen activator with liver cells.
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.
Animals↗
Streaming potentials in chemically modified bone.
Direct streaming experiments with no significant mechanical deformation have been performed to determine the constituent or constituents of bovine tibia mainly responsible for the production of streaming potentials. Following the approach of Frank and Grodzinsky (5), selective removal of either main constituent--collagen or hydroxyapatite--of the tissue was performed by chemical means, and the streaming potential was remeasured. Demineralized samples were prepared by soaking in formic acid/sodium citrate, and anorganic samples were prepared by boiling and also by sodium hypochlorite treatment. Demineralized samples demonstrated zeta potentials close to those of whole bone samples, whereas anorganic samples had much smaller zeta potentials. Collagen rather than hydroxyapatite mineral is therefore implicated as the constituent of whole bone dominating the streaming potential, in agreement with the observation that collagenous tissues containing no mineral phase, i.e., tendon and cartilage, also exhibit streaming potentials. No sign change was observed in whole bone at high NaCl concentration in solutions containing calcium or at low pH. Although the sign of the signal for anorganic bone could be reversed by the addition of Ca2+ or PO4(3+) to the test solutions, the sign of the signal in neither whole bone nor demineralized bone could be so affected. Sign changes in whole bone were observed only with solutions containing basic organic molecules, e.g., protamine sulphates.
Animals↗
Acute myeloblastic leukemia two years after diagnosis of non-Hodgkin lymphoma.
The 18-year-old white male developed acute myeloblastic leukemia (AML) 25 months after diagnosis of poorly differentiated lymphocytic lymphoma, diffuse pattern (PDLL-D), involving cervical, supraclavicular, and mediastinal lymph nodes as well as bone marrow. Treatment of the lymphoma consisted of 2,000 rads to the mantel area and 18 months of chemotherapy with intravenous (IV) methotrexate (400 mg/m2), vincristine, and prednisone, alternating every two weeks with IV cyclophosphamide (1,000 mg/m2), vincristine, and prednisone plus monthly intrathecal methotrexate. Thereafter, a complete remission was maintained without therapy until the onset of AML. Several pseudodiploid clones containing multiple structural rearrangements and a hypodiploid clone were identified in the circulating blood at the time of diagnosis of AML. Induction therapy consisting of cytosine arabinoside, 5-azacytidine, vincristine, and prednisone was unsuccessful, and the patient died of sepsis two months after diagnosis. This case calls attention to the increased risk for subsequent acute nonlymphocytic leukemia in patients previously treated for nonhodgkin lymphoma.
Adolescent↗
Sister chromatid exchange in lymphocytes from patients with acute lymphoblastic leukemia.
Sister chromatid exchange (SCE) frequencies were studied in peripheral lymphocytes from 16 patients with newly diagnosed acute lymphoblastic leukemia (ALL) prior to the initiation of chemotherapy. The mean SCE frequency (mean +/- SE) for these patients was 12.2 +/- 0.2 per metaphase, which was significantly higher (P less than 0.001) than the mean SCE score for 14 age-matched controls, 7.6 +/- 0.2. Five of these patients were studied again while they were receiving maintenance therapy consisting primarily of daily 6-mercaptopurine and weekly methotrexate. Their remission SCE levels remained significantly higher than controls (P less than 0.005). In addition, SCE levels were studied in 7 long-term survivors of ALL. Three of these patients had been receiving continuous maintenance therapy for at least 3 years. Their mean SCE scores were significantly greater than controls (P less than 0.005). The other 4 patients had finished their final course of chemotherapy at least 8 months prior to the time of sampling, and their mean SCE scores were not significantly different from controls (P greater than 0.10). These data indicate that untreated patients with ALL have increased SCE levels which remain elevated during periods of remission maintained with chemotherapy. However, long-term survivors of ALL who are in remission and off chemotherapy do not demonstrate significantly increased SCE frequencies.
Adolescent↗