Purification of a Specific Placental Plasminogen Activator Inhibitor by Monoclonal Antibody and Its Complex Formation with Plasminogen Activator

Summary

A monoclonal antibody of IgG_2a-type was obtained against a specific fast acting plasminogen activator inhibitor found in placenta. The placental inhibitor was purified by affinity chromatography using the monoclonal antibody and additionally in a FPLC-system. A strong complex formation was found between the inhibitor and urokinase and also with the two-chain form of plasminogen activator of the tissue-type. A weaker complex was found between the placental inhibitor and the one- chain form of the tissue-type activator.

Key words

Monoclonal antibody - Plasminogen activator - Coagulation tests - Urokinase - Placental inhibitor

• References

• 1 Kawano T, Morimoto K, Uemura Y. Urokinase inhibitor in human placenta. Nature 1968; 217: 253-254
• 2 Aoki N, Kawano T. Inhibition of plasminogen activators by naturally occurring inhibitors in man. Am J Physiol 1972; 223: 1334-1337
• 3 Åstedt B, Pandolfi M. Nilsson Inga Marie. Inhibitory effect of placenta on plasminogen activation in human organ culture Proc Soc Exp Biol Med 1972; 139: 1421-1424
• 4 Holmberg L, Lecander Ingegerd, 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
• 5 Lecander Ingegerd, Roblin R, Åstedt B. Differential inhibition of two molecular forms of melanoma cell plasminogen activator by a placental inhibitor. Br J Haematol 1984; 57: 407-412
• 6 Engwall E, Perlman P. Enzyme-linked immunosorbent assayELISA. III. Quantitation of specific antibodies by enzyme-labelled antiimmunoglobulin antigen-coated tubes. J Immunol 1972; 109: 129-135
• 7 Nowinski RC, Lostrom ME, Tam MR, Stone MR, Burnette WM. The isolation of hybrid cell lines producing monoclonal antibodies against the p15 (E) protein of ecotropic murine leukemia. Virology 1979; 93: 111-126
• 8 Fazekas deSt, Groth S, Scheidegger D. Production of monoclonal antibodies: strategy and tactics. J Immunol Methods 1980; 35: 1-21
• 9 Kennett RH. Fusion protocols. In Monoclonal antibodies. Kennett, McKeam, Bechtol. (Eds.) Plenum Press; New York: 1980. pp. 365-367
• 10 Ouchterlony Ö, Nilsson L-Å. Immunodiffusion and immunoelectrophoresis. In: Handbook of Experimental Immunology. Weir Blackwell DM. (Ed) 2. Ed Oxford: 1973. pp 19-39
• 11 Ey PL, Prowse SJ, Jenkin CR. Isolation of pure IgG₁, IgG_2a and IgG_2b immunoglobulins from mouse serum using protein A Sepharose. Immunochemistry 1978; 15: 429-436
• 12 Astup T, Müllertz S. The fibrin plate method for estimating fibrinolytic activity. Arch Biochem Biophys 1952; 40: 346-351
• 13 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
• 14 Wallén P, RÅnby M, Bergsdorf N, Kok P. Purification and characterization of tissue plasminogen activator: on the occurrence of two different forms and their enzymatic properties. In Progress in Fibrinolysis Davidson JF, Nilsson IM, Åstedt B. (Eds.) Churchill Livingstone; 1981. 5 16-23
• 15 Holmberg L, Bladh B, Åstedt B. Purification of Leo urokinase by affinity chromatography. Biochim Biophys Acta 1976; 445: 215-222
• 16 Weber K, Osborn M. The reliability of molecular weight determinations by dodcyl sulphate-polyacrylamide gel electrophoresis. J Biol Chem 1969; 244: 4406-4412
• 17 Laurell CB. Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies. Anal Biochem 1966; 15: 45-52
• 18 Hedner U, Åstedt B. Studies on fibrinolytic inhibitors during pregnancy. Acta Obstet Gynecol Scand 1971; 50: 99-103
• 19 Unkeless I, Danø K, Kellerman GM, Reich E. Fibrinolysis associated with oncogenic transformation. Partial purification and characterization of the cell factor, a plasminogen acitivator J Biol Chem 1974; 249: 4295-4305
• 20 Christensen U, Holmberg L, Bladh B, Åstedt B. Kinetics of the reaction between urokinase and an inhibitor of fibrinolysis from placental tissue. Thromb Haemostas 1982; 48: 24-26
• 21 Noordhoek Hegt V, Brakman P. Histochemical study of an inhibitor of fibrinolysis in the human arterial wall. Nature 1974; 248: 75-76
• 22 Fomasari PM, Pandolfi M, Åstedt B. On the proteolytic inhibitory effect of human foetal and neoplastic tissues. Thromb Res 1976; 8: 829-836
• 23 Holmberg L, Kristoffersson Ann-Charlotte, Lecander Ingegerd, Wallen P, Åstedt B. Immunoradiometric quantification of tissue plasminogen activator secreted by fetal organs. Comparison with urokinase Scand J Clin Lab Invest 1982; 42: 347-354
• 24 Åstedt B, Holmberg L, Lecander Ingegerd, Thorell J. Radioimmunoassay of urokinase for quantification of plasminogen activators released in ovarian tumour cultures. Eur J Cancer 1981; 17: 239-244