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Thrombin Stimulates Tissue Plasminogen Activator Release from Cultured Human Endothelial Cells

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Thrombin Stimulates Tissue Plasminogen Activator Release from Cultured Human Endothelial Cells Thrombin stimulates tissue plasminogen activator release from cultured human endothelial cells. E G Levin, … , J Anderson, L A Harker J Clin Invest. 1984;74(6):1988-1995. https://doi.org/10.1172/JCI111620. Research Article The effect of thrombin on the release of tissue plasminogen activator from endothelial cells was studied in primary cultures of human umbilical vein endothelial cells. Tissue plasminogen activator concentration in conditioned medium was measured by a two-site radioimmunometric assay. The addition of increasing concentrations (0.01 to 10 U/ml) of thrombin to confluent cultures produced a saturable, dose-dependent increase in the rate of release of tissue plasminogen activator. A sixfold increase in tissue plasminogen activator concentration (from 2 to 12 ng/ml) occurred after the addition of 1 U/ml thrombin (8 X 10(-9) M) to cultures containing 5 X 10(4) cells/cm2. Enhanced release was not observed until 6 h after thrombin addition, reached a maximum rate of 1.3 ng/ml per h between 8 and 16 h, and then declined to 0.52 ng/ml per h after 16 h. The 6-h lag period before increased tPA release was reproducible and independent of thrombin concentration. Thrombin inactivated with diisopropylfluorophosphate or hirudin did not induce an increase in tissue plasminogen activator levels. A 50-fold excess of diisopropylfluorophosphate-treated thrombin, which inhibits binding of active thrombin to endothelial cell high affinity binding sites, did not inhibit the thrombin-induced increase. It is concluded that proteolitically active thrombin causes an increase in the rate of release of tissue plasminogen activator from cultured human endothelial cells. The 6-h interval between thrombin […] Find the latest version: https://jci.me/111620/pdf Thrombin Stimulates Tissue Plasminogen Activator Release from Cultured Human Endothelial Cells Eugene G. Levin, Ulla Marzec, Johanna Anderson, and Laurence A. Harker Roon Research Center for Arteriosclerosis and Thrombosis, Department of Basic and Clinical Research, Scripps Clinic and Research Foundation, La Jolla, California 92037 Abstract. The effect of thrombin on the release tissue plasminogen activator from cultured human en- of tissue plasminogen activator from endothelial cells dothelial cells. The 6-h interval between thrombin treat- was studied in primary cultures of human umbilical ment and enhanced tissue plasminogen activator release vein endothelial cells. Tissue plasminogen activator con- may reflect a delaying mechanism that transiently pro- centration in conditioned medium was measured by a tects hemostatic plugs from the sudden increase in the two-site radioimmunometric assay. The addition of in- local concentration of this fibrinolytic enzyme. creasing concentrations (0.01 to 10 U/ml) of thrombin to confluent cultures produced a saturable, dose-depen- Introduction dent increase in the rate of release of tissue plasminogen activator. A sixfold increase in tissue plasminogen acti- Thrombin has been shown to affect the synthesis, release, or vator concentration (from 2 to 12 ng/ml) occurred after activation of a variety of plasma components through its of these X interaction with the vascular endothelium ( 1-6). Two the addition of 1 U/ml thrombin (8 l0-9 M) to cultures demonstrated effects could play a role in the regulation of the containing 5 X 104 cells/cm2. Enhanced release was not fibrinolytic pathway: the activation of protein C (5), which observed until 6 h after thrombin addition, reached a generates an increase in plasma fibrinolytic activity in vivo maximum rate of 1.3 ng/ml per h between 8 and 16 h, (7), and the thrombin-induced decline of intracellular plasmin- and then declined to 0.52 ng/ml per h after 16 h. The ogen activator activity and plasminogen activator release from 6-h lag period before increased tPA release was repro- bovine aortic endothelial cells in culture (4). This latter effect ducible and independent of thrombin concentration. is quite dramatic, as shown by the suppression of plasminogen Thrombin inactivated with diisopropylfluorophosphate activator activity occurring within minutes after the addition or hirudin did not induce an increase in tissue plasmin- of thrombin. Studies performed to determine which of the two ogen activator levels. A 50-fold excess of diisopropyl- immunochemically distinct plasminogen activators released by which inhibits binding the bovine endothelial cells (urokinase or tissue plasminogen fluorophosphate-treated thrombin, activator [8]) was affected by the addition of thrombin dem- of active thrombin to endothelial cell high affinity onstrated a decline in the level of urokinase with no significant binding sites, did not inhibit the thrombin-induced effect upon tissue plasminogen activator (tPA)' activity (9). increase. It is concluded that proteolitically active Primary cultures of human umbilical vein endothelial cells thrombin causes an increase in the rate of release of release a single plasminogen activator, which is immunochem- ically related to tPA (10). Unlike in the supernatant of bovine This work was presented in part at the Annual Meeting of the endothelial cells, no urokinase is detected in the supernatant American Society of Hematology, San Francisco, California, 3-6 of cultured human endothelial cells. The tPA has a molecular December 1983, and published in abstract (1983. Blood. 62:287a). weight of 100,000, does not bind to fibrin, and is inactive. It Address reprint request to Dr. Levin, Department of Basic and has been suggested that these unusual characteristics result 10666 Clinical Research, Scripps Clinic and Research Foundation, from an association between the tPA and an endothelial cell North Torrey Pines Road, La Jolla, CA 92037. June synthesized inhibitor through a sodium dodecyl sulfate (SDS)- Receivedfor publication 5 March 1984 and in revisedform 19 found in the 1984. stable bond (10). This fibrinolytic inhibitor, culture medium of human endothelial cells, is present in J. Clin. Invest. © The American Society for Clinical Investigation, Inc. 1. Abbreviations used in this paper: DFP, diisopropylfluorophosphate; 0021-9738/84/12/1988/08 $ 1 .00 DMEM, Dulbecco's modified Eagle's medium; PAGE, polyacrylamide Volume 74, December 1984, 1988-1995 gel electrophoresis; tPA, tissue plasminogen activator. 1988 E. G. Levin, U. Marzec, J. Anderson, and L. A. Harker excess (10-12). The addition of purified human tPA to the RPMI- 1640. To determine the effect of transient exposure of thrombin endothelial cell conditioned medium resulted in an increase on endothelial cells, cultures were treated with thrombin for the in the molecular weight of this tPA by 40,000 and a disap- prescribed time, washed three times with RPMI-1640 containing 5% pearance of tPA activity (10). NuSerum prewarmed to 370C, and then incubated in I ml of RPMI- 1640-5% NuSerum. Because primary cultures of human endothelial cells do not release urokinase-like plasminogen activators it was of [355]methionine labeling of cultures was performed by washing confluent cultures twice with methionine-free DMEM interest to determine whether thrombin would affect plasmin- and adding 1 ml of the same medium containing 10 MCi/ml [35S]methionine for 15 ogen activator release in human endothelial cells in culture. min. The amount of radioactivity incorporated into cellular and Using a solid-phase radioimmunometric assay (13) we inves- excreted proteins was determined by trichloracetic acid precipitation tigated the effect of thrombin on the release of tPA from of cell extracts and conditioned medium, filtration, and counting of primary cultures of human endothelial cells. We show that precipitable radioactivity. thrombin induces an increase in the release of tPA in a dose Preparation of '25"-labeled affinity purified anti-tPA IgG. Affinity and time dependent manner. purified anti-tPA was prepared by passing 2 ml of the IgG fraction of rabbit antiserum (6 mg IgG/ml in 0.15 M sodium chloride-0.01 M phosphate, pH 7.2 [phosphate-buffered saline, PBS]) through a 2-ml Methods Sepharose 4B column containing -600 ;ig tPA. The column was washed with 0.5 M sodium chloride-0.0l M sodium phosphate, pH Materials. Human alpha-thrombin was a gift from Dr. J. W. Fenton 7.2, and IgG was eluted with 0.1 M glycine HCl, pH 3.0. To each (New York Department of Health, Albany, New York) and had a eluate fraction was added 50 Il of 1 M sodium phosphate pH 7.2 and specific activity of 3,642 U/mg thrombin. Diisopropylfluorophosphate a final concentration of 1% bovine serum albumin. The fractions (DFP) treated thrombin was prepared by reacting thrombin with 10 containing the IgG were pooled and dialyzed against PBS. The mM DFP at pH 7.2 for 1 h at 370C. antibodies were iodinated by the lactoperoxidase method (16). The Human tPA was a gift from Dr. D. Collen (University of Leuven, radioiodinated antibody was then added to 200 Ml of tPA-Sepharose Belgium). tPA-Sepharose was prepared with CNBr-activated Sepharose 4B containing 60 Ag tPA and mixed for 2 h at room temperature. The 4B (Pharmacia Fine Chemicals, Piscataway, NJ) and 100 ,g of tPA/ Sepharose was washed with 0.5 M sodium chloride-0.01 M sodium ml of gel. Antisera against tPA were raised in rabbits as described phosphate, pH 7.2 to remove unbound material. The igG was eluted previously (14). The IgG fractions were isolated by ammonium sulfate from the Sepharose with 0.1 M glycine-HCI, pH 3.0, and the eluate precipitation and DEAE-cellulose chromatography (8). was dialyzed against PBS. Plasminogen was prepared from human plasma by affinity chro- Radioimmunometric assay. The assay used was modified from matography on lysine-Sepharose (Pharmacia Fine Chemicals) as de- Rijken et al. (13). Rabbit anti-tPA IgG was diluted to a final concen- scribed (15). Plasminogen-free bovine fibrinogen and lactoperoxidase tration of 10 ,g/ml in 50 mM sodium borate buffer, pH 9.0, and 150 were obtained from Calbiochem-Behring Corp.
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