Investigation by HPLC of the Catabolism of Recombinant Tissue Plasminogen Activator in the Rat

Roy Harris; Louis Garcia Frade; Lesley J Creighton; Paul S Gascoine; Maher M Alexandroni; Stephen Poole; Patrick J Gaffney

doi:10.1055/s-0038-1647645

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 1988; 60(01): 107-112
DOI: 10.1055/s-0038-1647645

Original Article

Schattauer GmbH Stuttgart

Investigation by HPLC of the Catabolism of Recombinant Tissue Plasminogen Activator in the Rat

Authors

Roy Harris

^*The National Institute for Biological Standards and Control, Madrid, Spain, South Mimms, Potters Bar, Herts UK
Louis Garcia Frade

^**The Ramon y Cajal Hospital, Madrid, Spain
Lesley J Creighton

^*The National Institute for Biological Standards and Control, Madrid, Spain, South Mimms, Potters Bar, Herts UK
Paul S Gascoine

^*The National Institute for Biological Standards and Control, Madrid, Spain, South Mimms, Potters Bar, Herts UK
Maher M Alexandroni

^*The National Institute for Biological Standards and Control, Madrid, Spain, South Mimms, Potters Bar, Herts UK
Stephen Poole

^*The National Institute for Biological Standards and Control, Madrid, Spain, South Mimms, Potters Bar, Herts UK
Patrick J Gaffney

^*The National Institute for Biological Standards and Control, Madrid, Spain, South Mimms, Potters Bar, Herts UK

Abstract

PDF Download

Summary

The catabolism of recombinant tissue plasminogen activator (rt-PA) was investigated after injection of radiolabelled material into rats. Both Iodogen and Chloramine T iodination procedures yielded similar biological activity loss in the resultant labelled rt-PA and had half lives in the rat circulation of 1 and 3 min respectively. Complex formation of rt-PA was investigated by HPLC gel exclusion (TSK G3000 SW) fractionation of rat plasma samples taken 1-2 min after ¹²⁵I-rt-PA injection. A series of radiolabelled complexes of varying molecular weights were found. However, 60% of the counts were associated with a single large molecular weight complex (350–500 kDa) which was undetectable by immunologically based assays (ELISA and BIA) and showed only low activity with a functional promoter-type t-PA assay. Two major activity peaks in the HPLC fractions were associated with Tree t-PA and a complex having a molecular weight of ̴ 180 kDa. HPLC fractionation to produce these three peaks at various timed intervals after injection of ¹²⁵I-rt-PA showed each to have a similar initial rate half life in the rat circulation of 4-5 min. The function of these complexes as yet is unclear but since a high proportion of rt-PA is associated with a high molecular weight complex with a short half life in the rat, we suggest that the formation of this complex may be a mechanism by which t-PA activity is initially regulated and finally cleared from the rat circulation.

Key words

Recombinant tissue plasminogen activator - Rat - Catabolic behaviour - HPLC

PDF (1099 kb)

References

References
1 Hedner O, Nilsson IM. The role of fibrinolysis. Clin Haematol 1981; 10: 327-342
2 Hoylaerts M, Rijken DC, Lijnen HR, Collen D. Kinetics of the activation of plasminogen by human tissue plasminogen activator. Role of fibrin J Biol Chem 1982; 257: 2912-2919
3 Gaffney PJ. Fibrinolysis in “Thrombosis & Haemostasis”. Bloom AL, Thomas DP. (eds) Churchill-Livingstone; Edinburgh: 1987. pp 223-244
4 Collen D. On the regulation and control of fibrinolysis. Thromb Haemostas 1980; 43: 77-89
5 Verstraete M, Bournameaux M, De Cock F, Van der Werf F, Collen D. Pharmacokinetics and systemic fibrinogenolytic effects of recombinant tissue-type plasminogen activator in humans;. J Pharm Exp Ther 1985; 235: 506-512
6 Korninger C, Stassen JM, Collen D. Turnover of human extrinsic (tissue-type) plasminogen activator in rabbits. Thromb Haemostas 1981; 46: 658-661
7 Beebe DP, Aronson DL. Turnover of human tissue plasminogen activator in rabbits. Thromb Res 1986; 43: 663-674
8 Nilsson S, Einarsson M, Ekvam S, Haggroth L, Mattsson Ch. Turnover of tissue plasminogen activator in normal and hepatectomized rabbits. Thromb Res 1985; 39: 511-521
9 Fuchs HE, Berger H, Pizzo SV. Catabolism of human tissue plasminogen activator in mice. Blood 1985; 65: 539-544
10 Rijken DC, Juhan-Vague I, Collen D. Complexes between tissue type plasminogen activator and proteinase inhibitors in human plasma, identified with an immunoradiometric assay. J Lab Clin Med 1983; 101: 285-294
11 Thorsen S, Phillips M. Isolation of tissue type plasminogen activator-inhibitor complexes from human plasma. Evidence for a rapid plasminogen activator inhibitor Biochim Biophys Acta 1984; 802: 111-118
12 TIMI Study Group. The thrombolysis in myocardial infarction trial. N Engl J Med 1985; 312: 932-936
13 Pennica D, Holmes WE, Kohr WJ, Harkins RN, Vehar GA, Ward CA, Bennett WF, Yelverton E, Seeburg PH, Heyneker HL, Goeddel DV, Collen D. Cloning and expression of human tissue type plasminogen activator. Nature 1983; 301: 214-221
14 Garcia FradeL J, Poole S, Hanley S, Creighton LJ, Curtis AD, Gaffney PJ. Bioavailability in rats of human recombinant tissue plasminogen activator after subcutaneous and intramuscular injection. Thromb Haemostas 1986; 56: 299-301
15 Campbell MN, Johnson G. Chloramine T-review. Chem Rev 1978; 78: 65
16 Fraker PJ, Speck JC. Protein and cell membrane iodinations with sparingly soluble chloramide. Biochem Biophys Res Commun 1978; 80: 849-857
17 Verheijen JH, Mullart E, Chang GT G, Kluft C, Wijngaards G. A simple, sensitive spectrophotometric assay for extrinsic (tissue-type) plasminogen activator applicable to measurements in plasma. Thromb Haemostas 1982; 48: 266-269
18 Mahmoud M, Gaffney PJ. Bioimmunoassay (BIA) of tissue plasminogen activator (t-PA) and its specific inhibitor (t-PA/INH). Thromb Haemostas 1985; 53: 356-359
19 Rijken DC, Van Hinsberg VW M, Sens EM C. Quantitation of tissue type plasminogen activator in human endothelial cultures by use of an enzyme immunoassay. Thromb Res 1984; 33: 145-153
20 Gaffney PJ, Curtis AD. A collaborative study of a proposed international standard for tissue plasminogen activator (t-PA). Thromb Haemostas 1985; 53: 134-136
21 Bergmann SR, Fox KA A, Ter-Pogossian MM, Sobel BE, Collen D. Clot-selective coronary thrombolysis with tissue type plasminogen activator. Science 1983; 220: 1181-1183
22 Collen D, Lijnen HR. New approaches in thrombolytic therapy. Arteriosclerosis 1984; 4: 579-585
23 Verstraete M, Bory M, Collen D, Erbel R, Lennane RJ. et al Randomised trial of intravenous recombinant tissue-type plasminogen activator versus intravenous streptokinase in acute myocardial infarction. Lancet 1985; 1: 842-847
24 Rijken DC, Emeis JJ. Clearance of the heavy and light polypeptide chains of human tissue-type plasminogen activator in rats. Biochem J 1986; 238: 643-646
25 Emeis JJ, Van de Hoogen CM, Jense D. Hepatic clearance of tissue-type plasminogen activators in rats. Thromb Haemostas 1985; 54: 661-664
26 Korninger C, Collen D. Neutralisation of human extrinsic (tissue type) plasminogen activator in human plasma: no evidence for a specific inhibitor. Thromb Haemostas 1981; 46: 662-665
27 Kruithof EK O, Tran-Thang C, Ransijn A, Bachmann F. Demonstration of a fast acting inhibitor of plasminogen activator in human plasma. Blood 1984; 64: 907-913
28 Verheijen JH, Chang GT G, Kluft C. Evidence for the occurrence of a fast acting inhibitor for tissue-type plasminogen activator in human plasma. Thromb Haemostas 1984; 51: 392-395
29 Wiman B, Chmielewska J, Ranby M. Inactivation of tissue plasminogen activator in plasma. Demonstration of a complex with a new rapid inhibitor J Biol Chem 1984; 259: 3644-3647
30 Nilsson IM, Tengborn L. A family with thrombosis associated with high level of tissue plasminogen activator inhibitor. Haemostasis 1984; 14: 24
31 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