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DOI: 10.1055/s-0038-1648418
Determinants of Induction of Increased Synthesis of Plasminogen Activator Inhibitor Type-1 in Human Endothelial Cells by t-PA
Publication History
Received 09 April 1991
Accepted after revision 21 August 1991
Publication Date:
02 July 2018 (online)
![](https://www.thieme-connect.de/media/10.1055-s-00035024/199202/lookinside/thumbnails/10-1055-s-0038-1648418-1.jpg)
Summary
Tissue-type plasminogen activator (t-PA) induces synthesis of a rapidly acting inhibitor, plasminogen activator inhibitor type-1 (PAI-1) in human umbilical vein endothelial cells (HUVEC) in culture. In vivo, an analogous process may induce negative feedback on the fibrinolytic system. To define specific determinants in the t-PA molecule contributing to the induction, PAI-1 synthesis was characterized in 35S-methionine labeled HUVEC in response to several molecular variants of t-PA. Catalytically active variants devoid of several specific structural domains in the A-chain retained the capacity to form complexes with PAI-1 and to induce increased concentrations of total PAI-1 (free and complexed) in conditioned media without depleting PAI-1 from the extracellular matrix. Surprisingly, a mutant t-PA with markedly reduced catalytic activity reflecting replacement of the active site serine with threonine (S478T) formed complexes with PAI-1 and induced increased PAI-1 synthesis as well. However, in contrast to wild-type t-PA and A-chain variants, it did not release 35S-methionine labeled PAI-1 from the extracellular matrix. Thus, its effects appeared to reflect increased secretion exclusively. Our results suggest that induction of PAI-1 synthesis in HUVEC by t-PA depends on its protease domain but that an active site serine is not a requirement.
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References
- 1 Beebe DR. Binding of tissue plasminogen activator to human umbilical vein endothelial cells. Thromb Res 1987; 46: 241-254
- 2 Hajjar KA, Hamel NM, Harpel PC, Nachman RL. Binding of tissue plasminogen activator to cultured human endothelial cells. J Clin Invest 1987; 80: 1712-1719
- 3 Barnathan ES, Kuo A, Van der Keyl H, McCrae KR, Larsen GR, Cines DB. Tissue-type plasminogen activator binding to human endothelial cells. J Biol Chem 1988; 263: 7792-7799
- 4 Sakata Y, Okada M, Noro A, Mastuda M. Interaction of tissue-type plasminogen activator and plasminogen activator inhibitor 1 on the surface of endothelial cells. J Biol Chem 1988; 263: 1960-1969
- 5 Kruithof EKO. Plasminogen activator inhibitors - A review. Enzyme 1988; 40: 113-121
- 6 Hekman CM, Loskutoff DJ. Endothelial cells produce a latent inhibitor of plasminogen activators that can be activated by dénaturants. J Biol Chem 1985; 260: 11581-11587
- 7 Mimuro J, Schleef RR, Loskutoff DJ. Extracellular matrix of cultured bovine aortic endothelial cells contains functionally active type 1 plasminogen activator inhibitor. Blood 1987; 70: 721-728
- 8 Knudsen BS, Nachman RL. Matrix plasminogen activator inhibitor. Modulation of the extracellular proteolytic environment. J Biol Chem 1988; 263: 9476-9481
- 9 Fujii S, Lucore CL, Hopkins WE, Billadello JJ, Sobel BE. Induction of synthesis of plasminogen activator inhibitor type-1 by tissue-type plasminogen activator in human hepatic and endothelial cells. Thromb Haemostas 1990; 64: 412-419
- 10 Reilly TM, Greenplate G, Timmemans PBMWM. Tissue plasminogen activator-induced secretion of type-1 plasminogen activator inhibitor in cultured human fibroblasts. Thromb Res 1989; 55: 619-625
- 11 Jaffe EA, Nachman RL, Becker CK, Minick CR. Culture of human endothelial cells derived from umbilical veins. J Clin Invest 1973; 52: 2745-2756
- 12 Monge JC, Lucore CL, Fry ETA, Sobel BE, Billadello JJ. Characterization of interaction of active-site serine mutants of tissue-type plasminogen activator with plasminogen activator inhibitor-1. J Biol Chem 1989; 264: 10922-10925
- 13 Angles-Cano E. A spectrophotometric solid-phase fibrin-tissue plasminogen activator activity assay (SOFIA-t-PA) for high-fibrin-affinity tissue plasminogen activators. Anal Biochem 1986; 153: 201-210
- 14 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: 1617-1624
- 15 Laemmli UK. Cleavage of structural protein during assembly of the head of bacteriophage T4. Nature 1970; 227: 680-685
- 16 Van Hinsbergh VWM. Regulation of the synthesis and secretion of plasminogen activators by endothelial cells. Haemostasis 1988; 18: 307-327
- 17 Pannekoek H, de Vries C, van Zonneveld AJ. Mutants of human tissue-type plasminogen activator (t-PA): Structural aspects and functional properties. Fibrinolysis 1988; 2: 123-132
- 18 Beebe DP, Miles LA, Plow EF. A linear amino acid sequence involved in the interaction of t-PA with its endothelial cell receptor. Blood 1989; 74: 2034-2037
- 19 Wittwer AJ, Sanzo MA. Effect of peptides on the inactivation of tissue plasminogen activator by plasminogen activator inhibitor-1 and on the binding of tissue plasminogen activator to endothelial cells. Thromb Haemostas 1990; 64: 270-275
- 20 Russell ME, Quertermous T, Declerck PJ, Collen D, Haber E, Homey CJ. Binding of tissue-type plasminogen activator with human endothelial cell monolayers. J Biol Chem 1990; 265: 2569-2575
- 21 Ramakrishnan V, Sinicropi DV, Dere R, Darbonne WC, Bechtol KB, Baker JB. Interaction of wild-type and catalytically inactive mutant forms of tissue-type plasminogen activator with human umbilical vein endothelial cell monolayers. J Biol Chem 1990; 265: 2755-2762
- 22 Wiman B, Collen D. On the kinetics of the reaction between human antiplasmin and plasmin. Eur J Biochem 1978; 84: 573-578
- 23 Travis J, Salvesen GS. Human plasma proteinase inhibitors. Annu Rev Biochem 1983; 52: 655-709
- 24 Lijnen HR, Van Hoef B, Collen D. On the reversible interaction of plasminogen activator inhibitor-1 with tissue-type plasminogen activator and with urokinase-type plasminogen activator. J Biol Chem 1991; 266: 4041-4044
- 25 Lawrence DA, Strandberg L, Ericson J, Ny T. Structure-function studies of the SERPIN plasminogen activator inhibitor type 1. Analysis of chimeric strained loop mutants. J Biol Chem 1990; 265: 20293-20301