Monoclonal Antibodies to Human 54,000 Molecular Weight Plasminogen Activator Inhibitor from Fibrosarcoma Cells - Inhibitor Neutralization and One-Step Affinity Purification

Summary

Mouse monoclonal antibodies were derived against a plasminogen activator inhibitor with a mol.wt. of ∼54,000 (54 K) from the human fibrosarcoma cell line HT-1080. Screening for hybrids producing antibodies directed against the inhibitor was performed with enzyme-linked immunosorbent assay and SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting. Four clones of hybridomas producing IgG₁ antibodies were further characterized. The inhibitor was purified ∼50-fold to homogeneity from conditioned cell culture fluid with a yield of ∼85% by a one-step procedure using Sepharose-conjugated monoclonal antibody. In the ¹²⁵I-fibrin plate assay one of the antibodies neutralized the effect of the inhibitor on urokinase-type plasminogen activator. Two of the antibodies bound complexes between urokinase-type plasminogen activator and inhibitor while the remaining two antibodies did not. The antibodies could be used for immunocytochemical localization of the inhibitor in HT-1080 cells. All four antibodies cross-reacted with a plasminogen activator inhibitor derived from cultured human umbilical cord endothelial cells.

Key words

Plasminogen activator inhibitor - Monoclonal antibody - Fibrosarcoma cells - Inhibitor neutralization - Purification

• References

• 1 Collen D. On the regulation and control of fibrinolysis. Thromb Haemostas 1980; 43: 77-89
• 2 Gelehrter TD, Barouski-Miller PA, Coleman PL, Cwikel BJ. Hormonal regulation of plasminogen activator in rat hepatoma cells. Mol Cell Biochem 1983; 53/54: 11-21
• 3 Danø K, Andreasen PA, Grøndahl-Hansen J, Kristensen P, Nielsen LS, Skriver L. Plasminogen activators, tissue degradation and cancer. Adv Cancer Res 1985; 44: 139-266
• 4 Kawano T, Morimoto K, Uemura Y. Urokinase inhibitor in human placenta. Nature 1968; 217: 253-254
• 5 Holmberg L, Lecander I, Persson B, Åstedt B. An inhibitor from placenta specifically binds urokinase and inhibits plasminogen activator released from ovarian carcinoma in tissue culture. Biochim Biophys Acta 1978; 544: 128-137
• 6 Seifert SC, Gelehrter TD. Mechanism of dexamethasone inhibition of plasminogen activator in rat hepatoma cells. Proc Natl Acad Sci USA 1978; 75: 6130-6133
• 7 Baker JB, Low DA, Simmer RL, Cunningham DD. Protease-nexin: a cellular component that links thrombin and plasminogen activator and mediates their binding to cells. Cell 1980; 21: 37-45
• 8 Loskutoff DJ, Edgington TS. An inhibitor of plasminogen activator in rabbit endothelial cells. J Biol Chem 1981; 256: 4142-4145
• 9 Coleman PL, Barouski-Miller PA, Gelehrter TD. Dexamethasone-induced inhibitor of fibrinolytic activity in hepatoma cells. J Biol Chem 1982; 257: 4260-4264
• 10 Chmielewska J, Ranby M, Wiman B. Evidence for a rapid inhibitor to tissue plasminogen activator in plasma. Thromb Res 1983; 31: 427-436
• 11 Emeis JJ, van Hinsbergh VW M, Verheijen JH, Wijngaards G. Inhibition of tissue-type plasminogen activator by conditioned medium from cultured human and porcine vascular endothelial cells. Biochem Biophys Res Commun 1983; 110: 392-398
• 12 Golder JP, Stephens RW. Minactivin: A human monocyte product which specifically inactivates urokinase-type plasminogen activator. Eur J Biochem 1983; 136: 517-522
• 13 Loskutoff DJ, van Mourik JA, Erickson LA, Lawrence D. Detection of an unusually stable fibrinolytic inhibitor produced by bovine endothelial cells. Proc Natl Acad Sci USA 1983; 80: 2956-2960
• 14 Cwikel BJ, Barouski-Miller PA, Coleman PL, Gelehrter TD. Dexamethasone induction of an inhibitor of plasminogen activator in HTC hepatoma cells. J Biol Chem 1984; 259: 6847-6851
• 15 Kruithof EK O, Tran-Thang C, Rasijn A, Bachmann F. Demonstration of a fast-acting inhibitor of plasminogen activators in human plasma. Blood 1984; 64: 907-913
• 16 Philips M, Juul AG, Thorsen S. Human endothelial cells produce a plasminogen activator inhibitor and a tissue-type plasminogen activator-inhibitor complex. Biochim Biophys Acta 1984; 802: 099-110
• 17 Vassalli JD, Dayer JM, Wohlvend A, Belin D. Concomitant secretion of prourokinase and of a plasminogen activator-specific inhibitor by cultured human monocytes-macrophages. J Exp Med 1984; 159: 1653-1668
• 18 Thorsen S, Philips M. Isolation of tissue-type plasminogen activator-inhibitor complexes from human plasma. Evidence for a rapid plasminogen activator inhibitor. Biochim Biophys Acta 1984; 802: 111-118
• 19 Loskutoff DJ. The fibrinolytic system of cultured endothelial cells: Deciphering the balance between plasminogen activation and inhibition. In: Progress in Fibrinolysis 1985; 7: 15-22
• 20 Saksela O, Hovi T, Vaheri A. Urokinase-type plasminogen activator and its inhibitor secreted by cultured human monocyte-macrophages. J Cell Physiol 1985; 122: 125-132
• 21 Stephens RW, Golder JP, Fayle DR H, Hume DA, Hapel AJ, Allan W, Ferdham CJ, Doe WF. Minactivin expression in human monocyte and macrophage populations. Blood 1985 66. in press
• 22 Åstedt B, Lecander I, Brodin T, Lundblad A, Löw K. Purification of a specific placental plasminogen activator inhibitor by monoclonal antibody and its complex formation with plasminogen activator. Thromb Haemostas 1985; 53: 122-125
• 23 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
• 24 Andreasen PA, Nielsen LS, Kristensen P, Grøndahl-Hansen J, Skriver L, Danø K. Plasminogen activator inhibitor from human fibrosarcoma cells binds urokinase-type plasminogen activator, but not its proenzyme. J Biol Chem 1986 (in press)
• 25 Nielsen LS, Andreasen PA, Grøndahl-Hansen J, Skriver L, Danø K. Plasminogen activators catalyse conversion of inhibitor from fibrosarcoma cells to an inactive form with an apparently lower molecular weight. FEBS Lett 1986 in press
• 26 Köhler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 1975; 256: 495-497
• 27 Danø K, Møller V, Ossowski L, Nielsen LS. Purification and characterization of a plasminogen activator from mouse cells transformed by an oncogenic virus. Biochim Biophys Acta 1980; 613: 542-555
• 28 Nielsen LS, Hansen JG, Skriver L, Wilson EL, Kaltoft K, Zeuthen J, Danø K. Purification of zymogen to plasminogen activator from human glioblastoma cells by affinity chromatography with monoclonal antibody. Biochemistry 1982; 24: 6410-6415
• 29 Nielsen LS, Hansen JG, Andreasen PA, Skriver L, Danø K, Zeuthen J. Monoclonal antibody to human 66000 molecular weight plasminogen activator from melanoma cells. Specific enzyme inhibition and one-step affinity purification. EMBO J 1983; 2: 115-119
• 30 Köhler G, Milstein C. Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion. Eur J Immunol 1976; 6: 511-519
• 31 Jaffe EA, Hoyer LW, Nachman RL. Synthesis of antihemophilic factor antigen by cultured human endothelial cells. J Clin Invest 1973; 52: 2757-2764
• 32 Kaltoft K, Nielsen LS, Zeuthen J, Danø K. Monoclonal antibody that specifically inhibits a human Mr 52,000 plasminogen-activating enzyme. Proc Natl Acad Sci USA 1982; 79: 3720-3723
• 33 Engvall E, Perlmann P. Enzyme-linked immunosorbent assay, ELISA. III. Quantitation of specific antibodies by enzyme-labeled anti-immunoglobulin in antigen-coated tubes. J Immunol 1972; 109: 129-135
• 34 Shulman M, Wilde CD, Köhler G. A better cell line for making hybridomas secreting specific antibodies. Nature 1978; 276: 269-270
• 35 Grøndahl-Hansen J, Nielsen LS, Kristensen P, Grøndahl-Hansen V, Andreasen PA, Danø K. Plasminogen activator in psoriatic scales is of the tissue-type PA as identified by monoclonal antibodies. Brit J Dermatol 1985; 113: 257-263
• 36 Danø K, Reich E. Plasminogen activator from cells transformed by an oncogenic virus. Inhibitors of the activation reaction. Biochim Biophys Acta 1979; 566: 138-151
• 37 Deutsch DG, Mertz ET. Plasminogen: Purification from human plasma by affinity chromatography. Science 1970; 170: 1095-1096
• 38 Danø K, Reich E. Serine enzymes released by cultured neoplastic cells. J Exp Med 1978; 147: 745-757
• 39 Granelli-Piperno A, Reich E. A study of proteases and protease> inhibitor complexes in biological fluids. J Exp Med 1978; 148: 223-234
• 40 Erickson LA, Lawrence DA, Loskutoff DJ. Reverse fibrin autography: A method to detect and partially characterize protease inhibitors after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Anal Biochem 1984; 137: 454-463
• 41 Graham RC, Karnovsky MJ. The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultra-structural cytochemistry by a new technique. J Histochem Cytochem 1966; 14: 291-302
• 42 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951; 193: 265-275
• 43 Sprengers ED, Verheijen JH, van Hinsbergh VW M, Emeis JJ. Evidence for the presence of two different fibrinolytic inhibitors in human endothelial cell conditioned medium. Biochim Biophys Acta 1984; 801: 163-170
• 44 Erickson LA, Ginsberg MH, Loskutoff DJ. Detection and partial characterization of an inhibitor of plasminogen activator in human platelets. J Clin Invest 1984; 74: 1465-1472
• 45 Loskutoff DJ, Roegner K, Erickson LA, Schleef RR, Huttenlocher A, Coleman PL, Gelehrter TD. The dexamethasone-induced inhibitor of plasminogen activator in hepatoma cells is antigenically related to an inhibitor produced by bovine aortic endothelial cells. Thromb Haemostas 1986; 55: 08-11
• 46 Andreasen PA, Nielsen LS, Grøndahl-Hansen J, Skriver L, Zeuthen J, Stephens RW, Danø K. Inactive proenzyme to tissue-type plasminogen activator from human melanoma cells, identified after affinity purification with a monoclonal antibody. EMBO J 1984; 3: 51-56