Biological and Thrombolytic Properties of Proenzyme and Active Forms of Human Urokinase – II. Turnover of Natural and Recombinant Urokinase in Rabbits and Squirrel Monkeys

Summary

The turnover of recombinant pro-urokinase (Rec-pro-UK), recombinant urokinase (Rec-UK) and natural urinary urokinase (Nat-UK) was studied in rabbits and in squirrel monkeys (Samiri sciureus). Following intravenous injection, urokinase activity disappeared rapidly from the blood. The initial disappearance rate could be described by a single exponential term with a t½ of 3 to 6 min for each molecular form of urokinase in both species. Urokinase related antigen, measured with a radioimmunoassay in the plasma of the squirrel monkeys disappeared with a t½ of 3.5 min for Rec-pro-UK, 6.0 min for Rec-UK and 8.0 min for Nat-UK.

The clearance and organ distribution of Rec-pro-UK, Rec-UK and Nat-UK was studied with the use of ¹²⁵I-labeled preparations. In each case the radioactivity initially disappeared rapidly from the plasma, also with a t½ of a few min, but then the disappearance rate slowed down. Labeled Rec-UK in which the active site histidine was irreversibly blocked by alkylation, disappeared equally rapidly from the plasma. Measurement of the organ distribution of ¹²⁵I at different time intervals revealed that all three types of urokinase were rapidly accumulated in the liver, which was followed by release of degradation products in the blood. Experimental hepatectomy prolonged the t½ of each type of urokinase very markedly (t½ >30 min).

These findings indicate that urokinase is rapidly removed from the blood by clearance and degradation in the liver. Recognition by the liver does not require a functional active site and is not mediated via carbohydrate side chains. Inactivation by plasma protease inhibitors does not represent a significant pathway of urokinase inhibition in vivo.

Keywords

Recombinant urokinase - Recombinant pro-urokinase - Turnover in rabbits - Turnover in squirrel monkeys - Clearance mechanism - Inactivation

• References

• 1 Heyneker HL, Holmes WE, Vehar GA. Preparation of functional human urokinase proteins. European Patent Application nr 83103629.8, Publication nr 0092182A2, European Patent Office. Munich, West-Germany:
• 2 Fraker PJ, Speck JC. Protein and cell membrane iodinations with a sparingly soluble chloroamide, 1, 3, 4, 6-tetrachloro-3a, 6a-diphenyl- glycoluril. Biochem Biophys Res Commun 1978; 80: 849-857
• 3 Lijnen HR, Uytterhoeven M, Collen D. Inhibition of trypsin-like serine proteinases by tripeptide arginyl and lysyl chloromethyl ketones. Thromb Res 1984; 34: 431-437
• 4 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
• 5 Plow EF, De Cock F, Collen D. Immunochemical characterization of the plasmin-antiplasmin system. Basis for the specific detection of the plasmin-antiplasmin complex by latex agglutination assays J Lab Clin Med 1979; 93: 199-209
• 6 Ashwell G, Morell AG. The role of surface carbohydrates in the hepatic recognition and transport of circulating glycoproteins. Adv Enzymol 1974; 41: 99-128
• 7 Prieels JP, Pizzo SV, Glasgow LR, Paulsen JC, Hill RL. Hepatic receptor that specifically binds oligosaccharides containing fusosyl alpha 1 leads to 3N-acetylglucosamine linkages. Proc Natl Acad Sci USA 1978; 75: 2215-2219
• 8 Stahl PD, Rodman JS, Miller MJ, Schlesinger PH. Evidence for receptor-mediated binding of glycoproteins, glycoconjugates, and lysosomal glycosidases by alveolar macrophages. Proc Natl Acad Sci USA 1978; 75: 1399-1403
• 9 Kawasaki T, Etoh R, Yamashina I. Isolation and characterization of a mannan-binding protein from rabbit liver. Biochem Biophys Res Commun 1978; 81: 1018-1024
• 10 Shifman MA, Pizzo SV. The in vivo metabolism of antithrombin III and antithrombin III complexes. J Biol Chem 1982; 275: 3243-3248
• 11 Gonias SL, Balber AE, Hubbard WJ, Pizzo SV. Ligand binding, conformational change and plasma elimination of human, mouse and rat alpha-macroglobulin proteinase inhibitors. Biochem J 1983; 209: 99-105
• 12 Collen D, Wiman B. Turnover of antiplasmin, the fast-acting plasmin inhibitor of plasma. Blood 1979; 53: 313-324
• 13 Tajima H, Ishiguro J, Nonaka R, Kurita M, Tanaka S, Ogawa N. Metabolic fate of urokinase. Chem Pharm Bull 1974; 22: 727-735
• 14 Matsuo O, Kosugi T, Mihara H. Urokinase inactivation rate in the rabbit: effect of circulatory isolation of the liver, spleen and kidneys. Haemostasis 1978; 7: 367-372
• 15 Fletcher AP, Alkjaersig N, Sherry S, Genton E, Hirsch J, Bachmann F. The development of urokinase as a thrombolytic agent. Maintenance of a sustained thrombolytic state in man by its intravenous infusion J Lab Clin Med 1965; 65: 713-731
• 16 Collen D. Mechanisms of inhibition of tissue-type plasminogen activator in blood. Thromb Haemostas 1983; 50: 678
• 17 Collen D, Stassen JM, Blaber M, Winkler M, Verstraete M. Biological and thrombolytic properties of proenzyme and active forms of human urokinase. III. Thrombolytic properties of natural and recombinant urokinase in rabbits with experimental jugular vein thrombosis. Thromb Haemostas 1984; 51: 27-30