The Plasmin Heavy Chain - Urokinase Conjugate: A Specific Thrombolytic Agent

Summary

Low molecular weight urokinase (LMW-UK) was coupled to the heavy chain of plasmin to make it able to bind to fibrin. The purified conjugate (PHC-UK conjugate), which consisted of equimolar concentrations of each starting material had a molecular weight of 93,600, bound tightly to fibrin-monomer-Sepharose and was not washed off with 1 M NaCl, but was eluted specifically with s-amino caproic acid. The conjugate showed higher fibrinolytic activity than HMW-UK. A control conjugate prepared by coupling human serum albumin to LMW-UK (HSA-UK conjugate) showed the same fibrinolytic activity as HMW-UK. The half-lives of these two conjugates in rabbits were about 3 times that of HMW-UK. In an experimental pulmonary embolism model in rabbits, the PHC-UK conjugate showed about 10 times higher thrombolytic activity than HMW-UK, while the HSA-UK conjugate showed similar thrombolytic activity as HMW-UK, and moreover caused severe systemic fibrinogen breakdown. Thus the significant increase in thrombolytic activity after injection of PHC-UK conjugate into rabbits may be due to its newly acquired fibrin binding activity, and not to increase in its half-life. It is concluded that the PHC-UK conjugate may be useful in treatment of thrombosis.

Keywords

Plasmin heavy chain-urokinase conjugate - Fibrin binding activity - Thrombolytic activity in rabbits

• References

• 1 Sherry S. Therapeutic fibrinolysis: past, present and future. In: Thrombosis and urokinase Paoletti R, Sherry S. (eds) Academic Press; London: 1977: 1-9
• 2 Porter JM, Goodnight Jr SH. The clinical use of fibrinolytic agents. Am J Surg 1977; 134: 217-221
• 3 Duckert F. Urokinase. In: Fibrinolytics and Antifibrinolytics Mark-wardt F. (ed) Springer; New York: 1978: 209-237
• 4 Cole ER, Bachmann FW. Purification and properties of a plasminogen activator from pig heart. J Biol Chem 1977; 252: 3729-3737
• 5 Allen RA, Pepper DS. Isolation and properties of human vascular plasminogen activator. Thromb Haemostas 1981; 45: 43-50
• 6 Husain SS, Lipinski B, Gurewich V. Rapid purification of a high-affinity plasminogen activator from human blood plasma by specific adsorption on fibrin/Celite. Proc Natl Acad Sci USA 1981; 78: 4265-4269
• 7 Rijken DC, Collen D. Purification and characterization of plasminogen activator secreted by human melanoma cells in culture. J Biol Chem 1981; 256: 7035-7041
• 8 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
• 9 Wun TC, Ossowski L, Reich E. A proenzyme form of human urokinase. J Biol Chem 1982; 257: 7262-7268
• 10 Husain SS, Gurewich V, Lipinski B. Purification and partial characterization of single-chain high-molecular-weight form of urokinase from human urine. Arch Biochem Biophys 1983; 220: 31-38
• 11 Kasai S, Arimura H, Nishida M, Suyama T. Thrombolytic properties of an inactivate proenzyme form of human urokinase secreted from human kidney cells. Cell Struct Funct 1985; 10: 151-159
• 12 Sottrup-Jansen L, Claeys H, Zajdel M, Petersen TE, Magnusson S. The primary structure of human plasminogen: Isolation of two lysine-binding fragments and one "mini-"plasminogen (MW, 38,000) by elastase-catalyzed-specific limited proteolysis. In: Progress in Chemical Fibrinolysis and Thrombolysis Davidson JF, Rowan RM, Desnoyers PC. (ed) Raven Press; New York: 1978. 3 191-209
• 13 Wiman B, Wallen P. The specific interaction between plasminogen and fibrin. A physiological role of the lysine binding site in plasminogen. Thromb Res 1977; 1: 213-222
• 14 Liu FT, Zinnecker M, Hamaoka T, Katz DH. New procedures for preparation and isolation of conjugates of proteins and a synthetic copolymer of d-amino acids and immunochemical characterization of such conjugates. Biochemistry 1979; 18: 690-697
• 15 Holmberg L, Bladh B, Astedt B. Purification of urokinase by affinity chromatography. Biochim Biophys Acta 1976; 445: 215-222
• 16 Travis J, Pannell R. Selective removal of albumin from plasma by affinity chromatography. Clin Chim Acta 1973; 49: 49-57
• 17 Heene DL, Matthias FR. Adsorption of fibrinogen derivatives on insolubilized fibrinogen and fibrinmonomer. Thromb Res 1973; 2: 137-154
• 18 Rickli EE, Otavsky WI. A new method of isolation and some properties of the heavy chain of human plasmin. Eur J Biochem 1975; 59: 441-447
• 19 Claesson G, Friberger P, Knos M, Eriksson E. Methods for determination of prekallikrein in plasma, glandular kallikrein and urokinase. Haemostasis 1978; 7: 76-78
• 20 Matsuo O, Collen D, Verstrate M. On the fibrinolytic and thrombolytic properties of active-site p-anisoylated streptokinase-plasminogen complex (BRL 26921). Thromb Res 1981; 24: 347-358
• 21 Edy J, De Cock F, Collen D. Inhibition of plasmin by normal and antiplasmin-depleted human plasma. Thromb Res 1976; 8: 513-518
• 22 Vermylen C, De Vreker R, Verstrate M. A rapid enzymatic method for assay of fibrinogen. Fibrin polymerization time (FPT-test). Clin Chim Acta 1963; 8: 418-424
• 23 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193: 265-275
• 24 Maezawa S, Takagi T. Monitoring of the elution from a high-performance gel chromatography column by a spectrophotometer, a low-angle laser light scattering photometer and a precision differential refractometer as a versatile way to determine protein molecular weight. J Chromatogr 1983; 280: 124-130
• 25 Wallen P, Wiman B. Characterization of human plasminogen II. Separation and partial characterization of different molecular forms of human plasminogen. Biochem Biophys Acta 1972; 257: 122-134
• 26 Kim HP, Byun SM, Yeom YI, Kim SW. Immobilization of urokinase on agarose matrices. J Pharm Sci 1983; 72: 225-228
• 27 Sugitachi A, Takagi K, Imaoka S, Kosaki G. Immobilization of plasminogen activator urokinase on nylon. Thromb Haemostas 1978; 39: 426-436
• 28 Maksimenko AV, Torchilin VP. Water-soluble urokinase derivatives of combined action. Thromb Res 1985; 38: 277-288
• 29 Shimizu K, Takatsuka J, Kaneko H, Sakurai M, Takeuchi S, Igarashi M, Asada T. Characteristics of urokinase modified with methoxypoly-ethylene glycol. Thromb Haemostas 1983; 50: 388 (Abstr)
• 30 Maksimenko AV, Torchilin VP. Water-soluble urokinase derivatives with increased affinity to the fibrin clot. Thromb Res 1985; 38: 289-295
• 31 Varadi A, Patthy L. Kringle 5 of human plasminogen carries a benzamidine-binding site. Biochem Biophys Res Commun 1981; 103: 97-102
• 32 Samama M, Cazenave B, Otero AM. Urokinase I and II activity. Thromb Haemostas 1978; 40: 578-580
• 33 Bode C, Matsueda GR, Hui KY, Haber E. Antibody-directed urokinase; A specific fibrinolytic agent. Science 1985; 229: 765-767