Binding of Tissue Plasminogen Activator to Cultured Human Fibroblasts

Summary

The binding of ¹²⁵I-labeled, one-chain tissue plasminogen activator (t-PA) by WI-38 cultured human lung fibroblasts was investigated. Binding of t-PA to WI-38 monolayers was specific, saturable and temperature dependent. One and two-chain t-PAs were comparable in their ability to compete with ₁₂₅I-labeled, one- chain t-PA for binding to fibroblasts, while no inhibition of binding was observed with a 500-fold molar excess of urokinase. Studies with various compounds suggest that neither the catalytic site, the fibrin binding site, nor the carbohydrate moieties on t-PA are involved in its binding to WI-38 cells. At higher temperatures, the amount of cell-bound ₁₂₅I-t-PA that was removed by either incubation in binding buffer containing an excess of unlabeled t-PA, or by brief treatment with acidic buffer, was small (approximately 20%) suggesting that much of the t-PA is internalized. Electrophoretic analysis of extracts prepared from cells that had been incubated with ¹²⁵I-t-PA revealed the presence of a major band of 70,000 M_r, which corresponds to intact t-PA. Our results suggest that WI-38 fibroblasts are capable of binding and internalizing t-PA, and that these processes involve a receptor site specific for t-PA.

Keywords

Plasminogen - Type plasminogen activator - WI - 38 human fibroblasts - Binding

• References

• 1 Collen D. On the regulation and control of fibrinolysis. Thromb Haemostas 1980; 43: 77-82
• 2 Rijken DC, Collen D. Purification and characterization of the plasminogen activator secreted by human melanoma cells in culture. J Biol Chem 1981; 256: 7035-7041
• 3 Hoylaerts M, Rijken DC, Lijnen HR, Collen D. Kinetics of the activation of plasminogen by human tissue plasminogen activator. J Biol Chem 1982; 257: 2912-2919
• 4 Ranby D. Studies on kinetics of plasminogen activation by tissue plasminogen activator. Biochim Biophys Acta 1982; 704: 461-469
• 5 Van de Werf F, Bergman SR, Fox KA A, DeGeest H, Hoyng CF, Sobel BE, Collen D. Coronary thrombolysis with intravenously administered human tissue-type plasminogen activator produced by recombinant DNA technology. Circulation 1984; 69: 605-610
• 6 Van Mourik JA, Lawrence DA, Loskutoff DJ. Purification of an inhibitor of plasminogen activator (antiactivator) synthesized by endothelial cells. J Biol Chem 1984; 259: 14914-14921
• 7 Colucci M, Paramo JA, Collen D. Generation in plasma of a fastacting inhibitor of plasminogen activator in response to an endotoxin stimulation. J Clin Invest 1985; 75: 818-824
• 8 Korninger C, Stassen JM, Collen D. Turnover of human extrinsic (tissue type) plasminogen activator in rabbits. Thromb Haemostas 1981; 46: 658-661
• 9 Verstraete M, Bounameaux H, de Cock F, Van de Werf F, Collen D. Pharmacokinetics and systemic fibrinogenolytic effects of recombinant human tissue-type plasminogen activator (rt-PA) in humans. J Pharmacol Exp Ther 1985; 235: 506-512
• 10 Fuchs H, Berger H, Pizzo SV. Catabolism of human tissue plasminogen activator in mice. Blood 1985; 65: 539-544
• 11 Emeis JJ, van den Hoogen CM, Jense D. Hepatic clearance of tissue- type plasminogen activator in rats. Thromb Haemostas 1985; 54: 661-664
• 12 Ashwell G, Harford J. Carbohydrate specific receptors of the liver. Ann Rev Biochem 1982; 51: 531-554
• 13 Bakhit C, Lewis D, Bilings R, Malfroy B. Cellular catabolism of recombinant tissue-type plasminogen activator. J Biol Chem 1987; 262: 8716-8720
• 14 Rijken DC, Wijngaards G, Welbergen J. Immunological characterization of plasminogen activator activities in human tissues and body fluids. J Lab Clin Med 1981; 97: 477-486
• 15 Hajjar KA, Hamel NH, Harpel PC, Nachman RL. Binding of tissue plasminogen activator to cultured human endothelial cells. J Clin Invest 1987; 80: 1712-1719
• 16 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-685
• 17 Urden G, Johansson U, Chmielewska J, Brandt J, Wiman B. Monoclonal antibodies towards the fast inhibitor of tissue plasminogen activator from human plasma and serum. Thromb Haemostas 1986; 55: 383-387
• 18 Degen JL, Estensen RD, Nagamine Y, Reich E. Induction and desensitization of plasminogen activator gene expression by tumor promoters. J Biol Chem 1985; 266: 12426-12433
• 19 Olefsky JM, Kao M. Surface binding and rates of internalization of¹²⁵I-insulin in adipocytes and IM-9 lymphocytes. J Biol Chem 1982; 257: 8667-8671
• 20 Anderson P, Yip YK, Vilcek J. Human interferon is internalized and degraded by cultured fibroblasts. J Biol Chem 1983; 258: 6497-6501
• 21 Bird TA, Saklatvala J. Studies on the fate of receptor-bound¹²⁵I- interleukin 1B in porcine synovial fibroblasts. J Immunol 1987; 139: 92-97
• 22 Ichinose A, Takio K, Fujikawa K. Localization of the binding site of tissue-type plasminogen activator to fibrin. J Clin Invest 1986; 78: 163-169
• 23 Van Zonneveld A, Veerman H, Pannekoek H. On the interaction of the finger and the kringle-2 domain of tissue-type plasminogen activator with fibrin. J Biol Chem 1986; 261: 14214-14218
• 24 Lijnen HR, Uytterhoeven M, Collen D. Inhibition of trypsin-like serine proteinases by tripeptide arginyl and lysyl chloremethylketones. Thromb Res 1984; 34: 431-437
• 25 Rijken DC, Emeis JJ, Gerwig GJ. On the composition and function of the carbohydrate moiety of tissue-type plasminogen activator from human melanoma cells. Thromb Haemostas 1985; 54: 788-791
• 26 Dini G, Fibi G, Pasquali F, Del Rosso M. Plasminogen activator: morphological evidence of binding internalization and delivery to lysosomes in 3T3 mouse fibroblasts. Histochem J 1985; 17: 333-341
• 27 Ny T, Elgh F, Lund B. The structure of the human tissue-type plasminogen activator gene: Correlation of intron and exon structures to functional and structural domains. Proc Natl Acad Sci USA 1984; 81: 5355-5359
• 28 Hekman CM, Loskutoff DJ. Kinetic analysis of the interactions between plasminogen activator inhibitor 1 and both urokinase and tissue plasminogen. activator Arch Biochem Biophys 1988; 262: 199-210