Subscribe to RSS
DOI: 10.1055/s-0038-1646397
Mediators of Induction of Augmented Expression of Plasminogen Activator Inhibitor Type-1 in Hep G2 Cells by Platelets
Publication History
Received 24 August 1990
Accepted 14 February 1991
Publication Date:
25 July 2018 (online)
![](https://www.thieme-connect.de/media/10.1055-s-00035024/199102/lookinside/thumbnails/10-1055-s-0038-1646397-1.jpg)
Summary
Plasminogen activator inhibitor type-1 (PAI-1) is a physiologic modulator of the fibrinolytic system. We have shown previously that PAI-1 biosynthesis in cultured cells depends on several factors in serum. Because platelets are richly endowed with specific growth factors and because the release reaction is an integral part of thrombosis, the present study was performed to determine whether platelets augment PAI-1 production and if so, to define mediators responsible. Hep G2 cells were used to determine whether platelet lysates increased PAI-1 synthesis in a dose and time-dependent manner. In cells labeled metabolically with 35S-methionine for 6 h, an increase in labeled PAI-1 was elicited indicative of de novo synthesis as well as increased secretion of PAI-1 mediated by platelet lysates. Steady state levels of both the 3.2 and 2.2 kb forms of PAI-1 mRNA increased after 2 h and peaked in 3-5 h in a dose-dependent fashion as well. Incubation of Hep G2 cells with collagen activated platelets resulted in a similar induction of PAI-1 mRNA. The increase in PAI-1 mRNA occurred with exposure of the cells to platelet lysates for intervals as brief as 15 min and was not inhibited by cycloheximide indicating its independence of new protein synthesis. In order to identify the factors in platelets responsible for the induction of PAI-1 synthesis in the Hep G2 cell model system, neutralizing antibodies were used to inhibit specific platelet associated growth factors. Antibodies to transforming growth factor-β (TGF-β) and to the epidermal growth factor (EGF)/transforming growth factor alpha (TGF-α) receptor inhibited the platelet lysate-mediated increase in PAI-1 protein by 77%. The results indicate that physiologic concentrations of platelet derived TGF-β and EGF/TGF-α increase PAI-1 expression in Hep G2 cells and suggest that the augmentation of PAI-1 synthesis induced by platelets is mediated by specific platelet associated growth factors that may contribute to observed increases in circulating PAI-1 levels in patients with disorders manifested by thrombosis.
-
References
- 1 Saksela O, Rifkin DB. Cell-associated plasminogen activation: Regulation and physiological functions. Annu Rev Cell Biol 1988; 4: 93-126
- 2 Laiho M, Keski-Oja J. Growth factors in the regulation of pericellular proteolysis: A review. Cancer Res 1989; 49: 2533-53
- 3 Loskutoff DJ. Type 1 plasminogen activator inhibitor and its potential influence on thrombolytic therapy. Semin Thromb Hemostas 1988; 14: 100-9
- 4 Kruithof EKO, Tran-Thang C, Bachmann F. The fast-acting inhibitor of tissue-type plasminogen activator in plasma is also the primary plasma inhibitor of urokinase. Thromb Haemostas 1986; 55: 65-9
- 5 Kruithof EKO, Gudinchet A, Bachmann F. Plasminogen activator inhibitor 1 and plasminogen activator inhibitor 2 in various disease states. Thromb Haemostas 1988; 59: 7-12
- 6 Kluft C, Verheijen JH, Jie AFH, Rijken DC, Preston FE, Sue-Ling HM, Jespersen J, Aasen AO. The postoperative fibrinolytic shutdown: a rapidly reverting acute phase pattern for the fast-acting inhibitor of tissue-type plasminogen activator after trauma. Scand J Clin Lab Invest 1985; 45: 605-10
- 7 Colucci M, Paramo JA, Collen D. Generation in plasma of a fast-acting inhibitor of plasminogen activator in response to endotoxin stimulation. J Clin Invest 1985; 75: 818-24
- 8 Engebretsen LF, Kierulf P, Brandtzaeg P. Extreme plasminogen activator inhibitor and endotoxin values in patients with meningococcal disease. Thromb Res 1986; 42: 713-6
- 9 Lucore CL, Sobel BE. Interactions of tissue-type plasminogen activator with plasma inhibitors and their pharmacologic implications. Circulation 1988; 77: 660-9
- 10 Bertozzi P, Astedt B, Zenzius L, Lynch K, LeMaire F, Zapol W, Chapman Jr HA. Depressed bronchoalveolar urokinase activity in patients with adult respiratory distress syndrome. N Engl J Med 1990; 322: 890-7
- 11 Sprengers ED, Kluft C. Plasminogen activator inhibitors. Blood 1987; 69: 381-7
- 12 Lucore CL, Fujii S, Wun T-C, Sobel BE, Billadello JJ. Regulation of the expression of type 1 plasminogen activator inhibitor in Hep G2 cells by epidermal growth factor. J Biol Chem 1988; 263: 15845-8
- 13 Sawdey M, Podor TJ, Luskutoff DJ. Regulation of type 1 plasminogen activator inhibitor gene expression in cultured bovine aortic endothelial cells. J Biol Chem 1989; 264: 10396-401
- 14 Fujii S, Lucore CL, Hopkins WE, Billadello JJ, Sobel BE. Potential attenuation of fibrinolysis by growth factors released from platelets and their pharmacologic implications. Am J Cardiol 1989; 63: 1505-11
- 15 Lund LR, Riccio A, Andreasen PA, Nielsen LS, Kristensen P, Laiho M, Saksela O, Blasi F, Dano K. Transforming growth factor-P is a strong and fast acting positive regulator of the level of type-1 plasminogen activator inhibitor mRNA in WI-38 human lung fibroblasts. EMBO J 1987; 6: 1281-6
- 16 Emeis JJ, Kooistra T. Interleukin 1 and lipopolysaccharide induce an inhibitor of tissue-type plasminogen activator in vivo and in cultured endothelial cells. J Exp Med 1986; 163: 1260-6
- 17 Schleef RR, Bevilacqua MP, Sawdey M, Gimbrone Jr MA, Loskutoff DJ. Cytokine activation of vascular endothelium. J Biol Chem 1988; 263: 5797-803
- 18 Ross R, Raines EW, Bowen-Pope DF. The biology of platelet-derived growth factor. Cell 1986; 46: 155-69
- 19 Assoian R, Komoriya KA, Meyers CA, Miller DM, Sporn MB. Transforming growth factor-β in human platelets. J Biol Chem 1983; 258: 7155-60
- 20 Derynck R. Transforming growth factor a. Cell 1988; 54: 593-5
- 21 Oka Y, Orth DN. Human plasma epidermal growth factor/p-urogastrone is associated with blood platelets. J Clin Invest 1983; 72: 249-59
- 22 Fitzgerald DJ, Catella F, Roy L, FitzGerald GA. Marked platelet activation in vivo after intravenous streptokinase in patients with acute myocardial infarction. Circulation 1988; 77: 142-50
- 23 Kerins DM, Roy L, FitzGerald GA, Fitzgerald DJ. Platelet and vascular function during coronary thrombolysis with tissue-type plasminogen activator. Circulation 1989; 80: 1718-25
- 24 Nieuwenhuis HK, van Oosterhout JJG, Rozemuller E, van Iwaarden F, Sixma JJ. Studies with a monoclonal antibody against activated platelets: Evidence that a secreted 53,000-molecular weight lysosomelike granule protein is exposed on the surface of activated platelets in the circulation. Blood 1987; 70: 838-45
- 25 George JN, Pickett EB, Saucerman S, McEver RP, Kunicki TJ, Kieffer N, Newman PJ. Platelet surface glycoproteins. Studies on resting and activated platelets and platelet membrane microparticles in normal subjects, and observations in patients during adult respiratory distress syndrome and cardiac surgery. J Clin Invest 1986; 78: 340-8
- 26 Knowles BB, Hoew CC, Aden DP. Human hepatocellular carcinoma cell lines secrete the major plasma proteins and hepatitis B surface antigen. Science 1980; 209: 497-9
- 27 Declerck PJ, Alessi M, Verstreken M, Kruithof EKO, Juhan-Vague I, Collen D. Measurement of plasminogen activator inhibitor 1 in biologic fluids with a murine monoclonal antibody-based enzyme-linked immunosorbent assay. Blood 1988; 71: 220-5
- 28 Feinberg A, Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 1983; 132: 6-13
- 29 Dillmann WH, Barrieux A, Neeley WE, Conteras P. Influence of thyroid hormone on the in vitro translational activity of specific mRNAs in the rat heart. J Biol Chem 1983; 258: 7738-45
- 30 Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987; 162: 156-9
- 31 Kessler SW. Rapid isolation of antigens from cells with a staphylococcal protein A-antibody absorbent: parameters of the interactions of antibody antigen complexes with protein A. J Immunol 1975; 115: 1617-24
- 32 Laemmli UK. Cleavage of structural protein during assembly of the head of bacteriophage T4. Nature 1970; 227: 680-5
- 33 Bostrom K, Boren J, Wettesten M, Sjoberg A, Bondjers G, Wiklund O, Carlsson P, Olofsson S-O. Studies on the assembly of apo B-100-containing lipoproteins in HepG2 cells. J Biol Chem 1988; 263: 4434-42
- 34 Westerhausen DR, Hopkins WE, Billadello JJ. Multiple transforming growth factor-β inducible elements regulate expression of the plasminogen activator inhibitor type-1 gene in Hep G2 cells. J Biol Chem 1991; 266: 1092-1100
- 35 Transcriptional regulation of plasminogen activator inhibitor type-1 mRNA in Hep G2 cells by epidermal growth factor. Nucl Acids Res 1991; 19: 163-8
- 36 Dinarello CA. Interleukin-1 and the pathogenesis of the acute-phase response. N Engl J Med 1984; 311: 1413-8
- 37 Perlmutter DH, Dinarello CA, Punsal PI, Colten HR. Cachectin/tumor necrosis factor regulates hepatic acute-phase gene expression. J Clin Invest 1986; 78: 1349-54
- 38 Poli V, Cortese R. Interleukin 6 induces a liver-specific nuclear protein that binds to the promoter of acute-phase genes. Proc Natl Acad Sci USA 1989; 86: 8202-6
- 39 Gauldie J, Richards C, Harnish D, Lansdorp P, Baumann H. Interferon β2/B-cell stimulatory factor type 2 shares identity with monocyte-derived hepatocyte-stimulating factor and regulates the major acute phase protein response in liver cells. Proc Natl Acad Sci USA 1987; 84: 7251-5
- 40 Mackiewicz A, Ganapathi MK, Schultz D, Brabenec A, Weinstein J, Kelley MF, Kushner I. Transforming growth factor β1 regulates production of acute-phase proteins. Proc Natl Acad Sci USA 1990; 87: 1491-5
- 41 Fujii S, Lucore CL, Sobel BE. Induction of plasminogen activator inhibitor by products released from platelets. Circulation 1990; 82: 1485-93