Uptake and Degradation of Tissue Plasminogen Activator in Rat Liver

1988 ◽  
Vol 59 (03) ◽  
pp. 474-479 ◽  
Author(s):  
Monica Einarsson ◽  
Bård Smedsrød ◽  
Håkan Pertoft
Keyword(s):  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Rat Liver ◽  
Liver Cells ◽  
Terminal Phase ◽  
Tissue Plasminogen ◽  
Liver Endothelial Cells ◽  
Parenchymal Cells

SummaryThe mechanism of uptake of tissue plasminogen activator (tPA) in rat liver was studied. Radio-iodinated tPA was removed from the circulation after intravenous administration in a biphasic mode. The initial half life, t1/2(α), and the terminal phase, t1/2(β), were determined to be 0.5 min and 7.5 min, resp. Separation of the liver cells by collagenase perfusion and density centrifugation, revealed that the uptake per cell was two to three times higher in the non-parenchymal cells than in the parenchymal cells.Endocytosis of fluorescein isothiocyanate-labelled or 125I-labelled tPA was studied in pure cultures of liver cells in vitro. Liver endothelial cells and parenchymal cells took up and degraded tPA. Endocytosis was more efficient in liver endothelial cells than in parenchymal cells, and was almost absent in Kupffer cells.Competitivb inhibition experiments showing that excess unlabelled tPA could compete with the uptake and degradation of 125I-tPA, suggested that liver endothelial cells and parenchymal cells interact with the activator in a specific manner. Endocytosis of trace amounts of 125I-tPA in cultures of liver endothelial cells and parenchymal cells was inhibited by 50% in the presence of 19 nM unlabelled tPA. Agents that interfere with one or several steps of the endocytic machinery inhibited uptake and degradation of 125I-tPA in both cell types.These findings suggest that 1) liver endothelial cells and parenchymal cells are responsible for the rapid hepatic clearance of intravenously administered tPA; 2) the activator is taken up in these cells by specific endocytosis, and 3) endocytosed tPA is transported to the lysosomes where it is degraded.

Tissue Plasminogen Activator is Endocytosed by Mannose and Galactose Receptors of Rat Liver Cells

1988 ◽  
Vol 59 (03) ◽  
pp. 480-484 ◽  
Author(s):  
Bård Smedsrød ◽  
Monica Einarsson ◽  
Håkan Pertoft
Keyword(s):  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Rat Liver ◽  
Side Chains ◽  
Diisopropyl Fluorophosphate ◽  
Tissue Plasminogen ◽  
Rat Liver Cells ◽  
Liver Endothelial Cells ◽  
Parenchymal Cells

SummaryExperiments were carried out to charact erize the specificity of uptake of tPA in rat liver cells. Endocytosis in liver endothelial cells of the native carbohydrate variants of tissue plasminogen activator (tPA), and tPA inactivated by diisopropyl fluorophosphate was found to be competitive, suggesting that the determinant being recognized by these cells is different from the active site. Fibronectin and urokinase, which show partial homology with tPA, did not compete with tPA for uptake in liver endothelial cells. Hyaluronic acid, collagen, or IgG, which are endocytosed by specific receptors in liver endothelial cells, did not interfere with the uptake.Reduced endocytosis by liver endothelial cells was observed with tPA modified in the carbohydrate side chains, suggesting that these structures are important for uptake. Ovalbumin, mannan, mannose, fructose, and EDTA, but not galactose, effectively inhibited uptake in liver endothelial cells of both native and diisopropyl fluorophosphate-inhibited tPA, but had very little effect on the uptake of tPA modified in the carbohydrate side chains.Endocytosis of native tPA by parenchymal cells could be inhibited by galactose, ovalbumin, and EDTA, but not by mannose.These results suggest that endocytosis of tPA by liver endothelial cells and parenchymal cells is mediated by the mannose and galactose receptors, respectively.

scholarly journals 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

1992 ◽  
Vol 284 (2) ◽  
pp. 545-550 ◽  
Author(s):  
M Otter ◽  
J Kuiper ◽  
R Bos ◽  
D C Rijken ◽  
T J van Berkel
Keyword(s):  
Endothelial Cells ◽  
Mannose Receptor ◽  
Liver Cells ◽  
Tissue Type ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Liver Endothelial Cells ◽  
Parenchymal Cells

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.

Tissue Plasminogen Activator Expression in Endothelial Cells Exposed to Cyclic Strain in Vitro

1992 ◽  
Vol 1 (1) ◽  
pp. 43-50 ◽  
Author(s):  
Toshiaki Iba ◽  
Bauer E. Sumpio
Keyword(s):  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Cyclic Strain ◽  
Neutral Position ◽  
Messenger Ribonucleic Acid ◽  
Tissue Plasminogen ◽  
Pai 1

The effects of cyclic strain on the production of tissue plasminogen activator (tPA) and type 1 plasminogen activator inhibitor (PAI-1) by cultured endothelial cells (EC) were examined. Human saphenous vein EC were seeded in selective areas of culture plates with flexible membrane bottoms (corresponding to specific strain regions) and grown to confluence. Membranes were deformed by vacuum (-20 kPa) at 60 cycles/min (0.5 s strain alternating with 0.5 s relaxation in the neutral position) for 5 days. EC grown in the periphery were subjected to 7-24% strain, while cells grown in the center experienced less than 7% strain. The results show a significant increase in immunoreactive tPA production on days 1, 3 and 5 compared to day 0 in EC subjected to more than 7% cyclic strain. There was no significant elevation of tPA in the medium of EC subjected to less than 7% strain. tPA activity could only be detected in the medium of EC subjected to more than 7% cyclic strain. PAI-1 levels in the medium were not significantly different in either group. In addition, immunocytochemical detection of intracellular tPA and messenger ribonucleic acid (mRNA) expression of tPA (assessed by the reverse transcriptase polymerase chain reaction utilizing tPA specific sense and antisense primers) was significantly increased in EC subjected to more than 7% cyclic strain. We conclude that a 60 cycles/min regimen of strain that is greater than 7% can selectively stimulate tPA production by EC in vitro and may contribute to the relative nonthrombogenicity of the endothelium in vivo.

Plasma from patients with cirrhosis increases tissue plasminogen activator release from vascular endothelial cells in vitro

2008 ◽  
Vol 18 (3) ◽  
pp. 186-190 ◽  
Author(s):  
Takeshi Hayashi ◽  
Asahi Kamogawa ◽  
Shinsei Ro ◽  
Kai Yamaguchi ◽  
Yutaro Kobayashi ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Vascular Endothelial Cells ◽  
Vascular Endothelial ◽  
Tissue Plasminogen

Binding of Tissue Plasminogen Activator to Vascular Grafts

1989 ◽  
Vol 61 (01) ◽  
pp. 131-136 ◽  
Author(s):  
Richard A Harvey ◽  
Hugh C Kim ◽  
Jonathan Pincus ◽  
Stanley Z Trooskin ◽  
Josiah N Wilcox ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Fibrinolytic Activity ◽  
Polymer Surface ◽  
Enzymic Activity ◽  
Vascular Prostheses ◽  
Chemically Modified ◽  
Insoluble Surfactant ◽  
Tissue Plasminogen

SummaryTissue plasminogen activator labeled with radioactive iodine (125I-tPA) was immobilized on vascular prostheses chemically modified with a thin coating of water-insoluble surfactant, tridodecylmethylammonium chloride (TDM AC). Surfactant- treated Dacron, polytetrafluoroethylene (PTFE), silastic, polyethylene and polyurethane bound appreciable amounts of 125I- tPA (5-30 μg 125I-tPA/cm2). Upon exposure to human plasma, the amount of 125I-tPA bound to the surface shows an initial drop during the first hour of incubation, followed by a slower, roughly exponential release with a t½ of appoximately 75 hours. Prostheses containing bound tPA show fibrinolytic activity as measured both by lysis of clots formed in vitro, and by hydrolysis of a synthetic polypeptide substrate. Prior to incubation in plasma, tPA bound to a polymer surface has an enzymic activity similar, if not identical to that of the native enzyme in buffered solution. However, exposure to plasma causes a decrease in the fibrinolytic activity of both bound tPA and enzyme released from the surface of the polymer. These data demonstrate that surfactant-treated prostheses can bind tPA, and that these chemically modified devices can act as a slow-release drug delivery system with the potential for reducing prosthesis-induced thromboembolism.

Specificity of the Thrombin-Induced Release of Tissue Plasminogen Activator from Cultured Human Endothelial Cells

1986 ◽  
Vol 56 (02) ◽  
pp. 115-119 ◽  
Author(s):  
Eugene G Levin ◽  
David M Stern ◽  
Peter P Nawroth ◽  
Richard A Marlar ◽  
Daryl S Fair ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Protein C ◽  
Primary Cultures ◽  
Activated Protein C ◽  
Specific Inhibitor ◽  
Factor Xa ◽  
Human Endothelial Cells ◽  
Tissue Plasminogen

SummaryThe addition of thrombin (9 nM) to primary cultures of human endothelial cells induces a 6- to 7-fold increase in the rate of release of tissue plasminogen activator (tPA). Several other serine proteases which specifically interact with endothelial cells were also analyzed for their effect on tPA release. Gamma-thrombin, an autocatalytic product of α-thrombin, promoted tPA release but was less effective than α-thrombin. A maximum increase of 5.5-fold was observed, although a concentration of γ-thrombin 20 times greater than α-thrombin was required. The response to Factor Xa was similar to α-thrombin, although the stimulation was significantly reduced by the addition of hirudin or DAPA suggesting that prothrombin activation was occurring. The simultaneous addition of prothrombin with Factor Xa resulted in enhanced tPA release equal to that observed with an equimolar concentration of active α-thrombin. Thus, under these conditions, Factor Xa-cell surface mediated activation of prothrombin can lead to a secondary effect resulting from cell-thrombin interaction. Activated protein C, which has been implicated as a profibrinolytic agent, was also tested. No change in tPA release occurred after the addition of up to 325 nM activated protein C in the presence or absence of proteins. Factor IXa and plasmin were also ineffective. The effect of thrombin on the endothelial cell derived plasminogen activator specific inhibitor was also studied. Thrombin produced a small but variable release of the inhibitor with an increase of less than twice that of non-thrombin treated controls.

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