A Monoclonal Antibody that Does Not Recognize Tissue-Type Plasminogen Activator Bound to Its Naturally Occurring Inhibitor

Summary

Hybridoma clones specific for tissue-type plasminogen activator (tPA) were screened in solid-phase radioimmunoassays for their reactivity toward free tPA and tPA complexed to the endothelial cell-derived, (3-migrating plasminogen activator inhibitor (β-PAI). Two monoclonal antibodies (MABs) were identified with quite distinct properties. The first, MAB LI72D₁, bound to free tPA but did not recognize tPA complexed to the β-PAI, whereas the second, MAB HI72C₁, bound both to free tPA and to tPA in complex with β-PAI. These properties were maintained when the MABs were immobilized to plastic micro- titer wells, thus permitting the development of immunoradiometric assays (IRMAs) to quantitate free tPA and total tPA antigen in various samples. The IRMAs were employed to analyze the tPA in media conditioned by several human cell types. The results indicate that in some cases, tPA may be present entirely as a free and active enzyme, while in others it apparently exists entirely in complex with β-PAI. Interestingly, some cells appear to contain both forms of tPA.

Key words

Plasminogen activator - Inhibitor - Immunoradiometric assay - Monoclonal antibody - Immobilized enzyme

• References

• 1 Rijken DC, Hoylaerts M, Collen D. Fibrinolytic properties of one-chain and two-chain human extrinsic (tissue-type) plasminogen activator. J Biol Chem 1982; 257: 2920-2925
• 2 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
• 3 Erickson LA, Schleef RR, Ny T, Loskutoff DJ. The fibrinolytic system of the vessel wall. Clin Haematol 1985; 14: 513-530
• 4 Holmberg L, Lecander I, Persson B, Astedt 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
• 5 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
• 6 Loskutoff DJ, Ny T, Sawdey M, Lawrence D. The fibrinolytic system of cultured endothelial cells: Modulation by plasminogen activator inhibitor. In: Proteases in Biological Control and Biotechnology Cunningham D, Long G. (eds) Alan R Liss, Inc; New York: 1986. (in press)
• 7 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
• 8 Hekman CM, Loskutoff DJ. Endothelial cells produce a latent inhibitor of plasminogen activators that can be activated by denatur-ants. J Biol Chem 1985; 260: 11581-11587
• 9 Philips M, Juul A, Thorsen S. Human endothelial cells produce a plasminogen activator inhibitor and a tissue-type plasminogen activator-inhibitor complex. Biochim Biophys Acta 1984; 802: 99-110
• 10 Erickson LA, Ginsberg MH, Loskutoff DJ. Detection and partial characterization of an inhibitor of plasminogen activator in human platelets. J Clin Invest 1984; 74: 1465-1473
• 11 Booth NA, Anderson JA, Bennett B. Platelet release protein which inhibits plasminogen activators. J Clin Pathol 1985; 38: 825-830
• 12 Erickson LA, Hekman CM, Loskutoff DJ. The primary plasminogen activator inhibitors in endothelial cells, platelets, serum, and plasma are immunologically related. Proc Natl Acad Sci USA 1985; 82: 8710-8714
• 13 Hedner U. Inhibitors of the plasminogen activation by urokinase in human serum. In: Thrombosis and Urokinase Paoletti R, Sherry S. (eds) Academic Press; London: 1977: 119-128
• 14 Chmielewska J, Ranby M, Wiman B. Evidence for a rapid inhibitor to tissue plasminogen activator in plasma. Thromb Res 1983; 31: 427-436
• 15 Walker JE, Gow L, Campbell DM, Ogston D. The inhibition by plasma of urokinase and tissue activator-induced fibrinolysis in pregnancy and the puerperium. Thromb Haemostas 1983; 49: 21-23
• 16 Juhan-Vague I, Moerman B, de Cock F, Aillaud MF, Collen D. Plasma levels of a specific inhibitor of tissue-type plasminogen activator (and urokinase) in normal and pathological conditions. Thromb Res 1984; 33: 523-530
• 17 Erickson LA, Ginsberg MH, Loskutoff DJ. An inhibitor of tissue-type plasminogen activator in human platelets, serum, and plasma. In: Progress in Fibrinolysis Davidson JF, Donati MB, Coccheri S. (eds) hurchill Livingstone; Edinburgh: 1985. Vol. VII: pp. 137-140
• 18 Erickson LA, Hekman CM, Loskutoff DJ. Denaturant-induced stimulation of the p-migrating plasminogen activator inhibitor in endothelial cells and serum. Blood (in press)
• 19 Kawano T, Marimoto K, Uemura Y. Partial purification and properties of urokinase inhibitor from human placenta. J Biochem 1970; 67: 333-342
• 20 Mullertz S, Clemmensen I. The primary inhibitor of plasmin in human plasma. Biochem J 1976; 159: 545-553
• 21 Kruithof E KO, Tran-Thang C, Ransijn A, Backmann F. Demonstration of a fast-acting inhibitor of plasminogen activators in human plasma. Blood 1984; 64: 907-913
• 22 Schleef RR, Sinha M, Loskutoff DJ. Characterization of two monoclonal antibodies against human tissue-type plasminogen activator. Thromb Haemostas 1985; 53: 170-175
• 23 Holmberg L, Bladh B, Astedt B. Purification of urokinase by affinity chromatography. Biochem Biophys Acta 1976; 445: 215-222
• 24 Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72: 248-254
• 25 David GS, Reisfeld RA. Protein iodination with solid state lactoperoxidase. Biochemistry 1974; 13: 1014-1021
• 26 Levin E, Loskutoff DJ. Cultured bovine endothelial cells produce both urokinase and tissue-type plasminogen activators. J Cell Biol 1982; 94: 631-636
• 27 Thornton SC, Mueller SN, Levine EM. Human endothelial cells: use of heparin in cloning and long-term serial cultivation. Science 1983; 282: 623-625
• 28 Schleef RR, Sinha M, Loskutoff DJ. Immunoradiometric assay to measure the binding of a specific inhibitor to tissue-type plasminogen activator. J Lab Clin Med 1985; 106: 408-415
• 29 Levin EG. Latent tissue plasminogen activator produced by human endothelial cells in culture: evidence for an enzyme-inhibitor complex. Proc Natl Acad Sci USA 1983; 80: 6804-6808
• 30 Weber K, Osborn M. The reliability of molecular weight determination by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem 1969; 244: 4406-4412
• 31 Granelli-Piperno A, Reich E. A study of pro tease and protease-inhibitor complexes in biological fluids. J Exp Med 1978; 148: 223-234
• 32 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
• 33 Loskutoff DJ. The fibrinolytic system of cultured endothelial cells: deciphering the balance between plasminogen activation and inhibition. In: Progress in Fibrinolysis Davidson JF, Donati MB, Coccheri S. (eds) Churchill Livingstone; Edinburgh: 1985. Vol. VII: pp 15-22
• 34 Rijken DC, Juhan-Vague I, de Cock F, Collen D. Measurement of human tissue-type plasminogen activator by a two-site immunoradiometric assay. J Lab Clin Med 1983; 101: 274-284
• 35 MacGregor IR, Prowse CV. Tissue plasminogen activator in human plasma measured by radioimmunoassay. Thromb Res 1983; 31: 461-474
• 36 Bergsdorf N, Nilsson T, Wallen P. An enzyme linked immunosorbent assay for determination of tissue plasminogen activator applied to patients with thromboembolic disease. Thromb Haemostas 1983; 50: 740-744
• 37 Matsuo O, Kato K, Matsuo C, Matsuo T. Determination of tissue plasminogen activator by an enzyme-immunoassay method. Anal Biochem 1983; 135: 58-63
• 38 Holvoet P, Cleemput H, Collen D. Assay of human tissue-type plasminogen activator (tPA) with an enzyme-linked immunosorbent assay (ELISA) based on three murine monoclonal antibodies to tPA. Thromb Haemostas 1985; 54: 684-687
• 39 Rijken DC, Juhan-Vague I, Collen D. Complexes between tissue-type plasminogen activators and proteinase inhibitors in human plasma, identified with an immunoradiometric assay. J Lab Clin Med 1983; 101: 285-294
• 40 Rijken DC, van Hinsbergh V WM, Sens E HC. Quantitation of tissue-type plasminogen activator in human endothelial cell cultures by use of an enzyme immunoassay. Thromb Res 1984; 33: 145-153
• 41 Roblin RO, Young PL, Bell TE. Concomitant secretion by transformed SVWI38-VA13-2RA cells of plasminogen activator(s) and substance(s) which prevent their detection. Biochem Biophys Res Commun 1978; 82: 165-172
• 42 Hauert J, Tissot JD, Bachmann F. Demonstration of two forms of tissue-type-related and of prekallikrein-related plasminogen activators in human plasma. In: Progress in Fibrinolysis Davidson JF, Bachmann F, Bouvier CA, Kruithof E KO. (eds) Churchill Livingstone; Edinburgh: 1983. Vol. VI. pp. 62-64
• 43 Stalder M, Hauert J, Kruithof E KO, Bachmann F. Release of vascular plasminogen activator (v-PA) after venous stasis: elec-trophoretic-zymographic analysis of free and complexed v-PA. Brit J Haem 1985; 61: 169-176
• 44 Isacson S, Nilsson IM. Defective fibrinolysis in blood and vein walls in recurrent idiopathic venous thrombosis. Acta Chir Scand 1972; 138: 313-319
• 45 Pettersson G, Brandt J, Wallen P. Monoclonal antibodies to plasminogen activator from human uterine tissue. In: Progress in Fibrinolysis Davidson JF, Bachmann F, Bouvier CA, Kruithof E KO. (eds) Churchill Livingstone; Edinburgh: 1983. Vol. VI. pp. 191-194
• 46 Angles-Cano E, Sultan Y. A solid-phase fibrin immunoassay for the specific detection of monoclonal antibodies against different epitopic determinants of tissue-plasminogen activators. J Immunol Methods 1984; 69: 115-127
• 47 Nielson LS, Hansen JG, Andreasen PA, Skriver L, Dan K, Zeuthen J. Monoclonal antibody to human 66,000 molecular weight plasminogen activator from melanoma cells. Specific enzyme inhibition and one-step affinity purification. EMBO J 1983; 2: 115-119
• 48 MacGregor IR, Micklem LR, James K, Pepper DS. Characterization of epitopes on human tissue plasminogen activator recognized by a group of monoclonal antibodies. Thromb Haemostas 1985; 53: 45-50