Isolation and Characterization of Plasminogen Activator from Pig Leucocytes

Summary

Plasminogen activator was isolated from disrupted pig leucocytes by the aid of DEAE chromatography, gel filtration on Sephadex G-100 and final purification on CM cellulose, or by preparative gel electrophoresis.

Isolated plasminogen activator corresponds No. 3 band of the starting sample of leucocyte cells (that is composed from 10 gel electrophoretic bands).

pH optimum was found to be in pH range 8.0–8.5 and the highest pH stability is between pH range 5.0–8.0.

Inhibition studies of isolated plasminogen activator were performed with EACA, AMCHA, PAMBA and Trasylol, using Anson and Astrup method. By Astrup method 100% inhibition was found with EACA and Trasylol and 30% with AMCHA. PAMBA gave 60% inhibition already at concentration 10^–3 M/ml. Molecular weight of plasminogen activator was determined by gel filtration on Sephadex G-100. The value obtained from 4 different samples was found to be 28000–30500.

• References

• 1 Abiko Y, Iwamota M, Tomikawa M. Plasminogen - plasmin system. V. A stochiometric equilibrium complex of plasminogen and a synthetic inhibitor. Biochimica Biophysica Acta 1969; 185: 424
• 2 Alkjaersig N, Fletcher A. P, Sherry S. Epsilon-aminocaproic acid, an inhibitor of plasminogen activation. Journal of Biological Chemistry 1959; 234: 832
• 3 Anson M. L. Estimation of pepsin, trypsin, papain and cathepsin with hemoglobin. Journal of General Physiology 1939; 22: 79
• 4 Anderson L. Fibrinolysis and its control. South American Medical Journal 1972; 46: 182
• 5 Astrup T, Thorsen S. The physiology of fibrinolysis. Medical Clinic of North America 1972; 56: 153
• 6 Bachmann F, Fletcher A. P, Alkjaersig N, Sherry S. Partial purification and properties of the plasminogen activator from pig heart. Biochemistry 1964; 3: 1578
• 7 Beard E. L, Busuttil R. W, Gottshalk S. K. Stress induced release of plasminogen activator from lysosomes. Thrombosis et Diathesis Haemorrhagica 1969; 21: 20
• 8 Bleyl V. Fibrinolyse durch Leukozyten. In: Hiemeyer V. (ed.) Therapeutische und experimentelle Fibrinolyse F. K. Schattauer Verlag; 59 1969
• 9 Brockway W. J, Castellino F. J. Measurement of the binding of anti-fibrinolytic amino acid to various plasminogens. Archives of Biochemistry and Biophysics 1972; 151: 194
• 10 Burchardt P, Marscek M. Fibrinolyse als diagnostischer Test beim Prostata Carcinom. Der Urologe 1971; 10: 3
• 11 Dumazert C, Marcelet Y. Sur un nouveau catalyseur de minéralisation en vue du dosage de l’azote par la methode de Kjeldahl. I. Application on dosage de l’azote dans les protéines, les poudres d’organes, les fèces. Bulletin de la Société de Chimie Biologique 1938; 20: 212
• 12 Easa-Helmy M, Easa M. D, Casarett G. W. Effect of EACA on radiation-induced increase in capillary permeability. Radiology 1973; 106: 679
• 13 Farnely Q. R, Charkrabarti R, Evans J. F. Mode of action of phen-formin plus ethyloestrenol on fibrinolysis. Lancet 1971; 723 April 10
• 14 Glas P, Astrup T. Thromboplastin and plasminogen activator in tissue of the rabbit. American Journal of Physiology 1970; 219: 1140
• 15 Halawa B, Frydecka I, Wawrzkiewicz M. Proteolytic activity of leucocytes in patients treated by Curietherapie. Radiobiología Radiotherapia 1971; 5: 591
• 16 Johnson S, Skoza L, Tse O. A. Studies of plasmin inhibition and plas-minogen activation and its inhibition. In: Hiemeyer V. (ed.) Therapeutische und experimentelle Fibrinolyse F. K. Schattauer Verlag; 1969: 105
• 17 Kok P, Astrup T. Isolation and purification of a tissue plasminogen activator and its comparison with urokinase. Biochemistry 1969; 8: 79
• 18 Kopitar M, Kregar I, Lebez D. Leucocyte proteinases II. Partial purification of proteinases present in cathepsin D preparation. Enzymologica 1971; 41: 129
• 19 Kopitar M, Turk V, Coti Č. V, Stegnar M. Leucocyte proteinases and protein digestion. Symposium of Intracellular Gatabolism at Schloss Reinhardsbrunn. Friedrichroda, GDR, 7-11 May, 1973 1973. in the press.
• 20 Kwaan H. C, Astrup T. Tissue repair in presence of locally applied inhibitors of fibrinolysis. Experimental Molecular Pathology 1969; 11: 82
• 21 Kwaan H. C. Disorders of fibrinolysis. Medical Clinic of North America 1972; 56: 163
• 22 Lebez D, Kopitar M. Leucocyte proteinases. Low molecular weight cathepsins of F and G type. Enzymologia 1970; 39: 271
• 23 Lassen M. Heat denaturation of plasminogen in the fibrin plate method. Acta Physiologica Scandinavica 1952; 27: 371
• 24 Lesuk A, Torminiello L, Traven J. H, Groff J. L. Biochemical and biophysical studies of human urokinase. Thrombosis et Diathesis Haemorrhagica 1967; 18: 293
• 25 Matsuoka M, Saktjragawa N, Shimaoka M. Studies on fibrinolytic activities in normal human leucocytes. Acta Medica Biologica 1969; 16: 91
• 26 Prokopowicz J. Distribution of fibrinolytic and proteolytic enzymes in subcellular fractions of human granulocytes. Thrombosis et Diathesis Haemorrhagica 1968; 19: 84
• 27 Riddle J. M, Baenhart M. I. Ultrastructural study of fibrin dissolution via emigrated polymorphonuclear neutrophils. American Journal of Pathology 1964; 45: 805
• 28 Semar M, Skoza L, Johnson A. J. Partial purification and properties of a plasminogen activator from human erythrocytes. Journal of Clinical Investigation 1969; 48: 1777
• 29 Sherman L. A. Fibrinogen turnover: demonstration of multiple pathway of catabolism. Journal of Laboratory and Clinical Medicine 1972; 79: 710
• 30 Stegnar M, Kopitar M, Accetto B, Lebez D. Fibrinolytic and plasminogen activator activity in proteinase fractions from pig leucocytes. Acta Medica Iugoslavica 1973. in the press.
• 31 Tatarsky I, Sinakos Z, Lrrien M. J, Bernhart J. Leucocytes et fibrinolyse II. Etude des leucocytes pathologiques. Nouvelle Revue Francaise d’Hematologie 1967; 7: 95
• 32 Whitaker J. B. Determination of molecular weights of proteins by gel filtration on Sephadex. Analytical Chemistry 1963; 35: 1950
• 33 Wunschmann-Henderson B, Goldstein I, Henderson E. S, Astrup T. Variations in fibrinolytic activity of leucemic cell lines. Federation Proceedings 1971; 30: 338
• 34 Wunschmann-Henderson B, Horwitz D. L, Astrup T. Release of plasminogen activator from viable leucocytes of man, babboon , dog and rabbit. Proceedings of the Society for Experimental Biology and Medicine 1972; 141: 634