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Thromb Haemost 1986; 55(01): 094-097
DOI: 10.1055/s-0038-1661455
Original Article
Schattauer GmbH Stuttgart

Isolation and Preliminary Characterisation of Active B-Chain of Recombinant Tissue-Type Plasminogen Activator

I Dodd
The Beecham Pharmaceuticals Research Division, Biosciences Research Centre, Great Burgh, Epsom, Surrey, UK
,
R Fears
The Beecham Pharmaceuticals Research Division, Biosciences Research Centre, Great Burgh, Epsom, Surrey, UK
,
J H Robinson
The Beecham Pharmaceuticals Research Division, Biosciences Research Centre, Great Burgh, Epsom, Surrey, UK
› Author Affiliations
Further Information

Publication History

Received 05 August 1985

Accepted 14 November 1985

Publication Date:
19 July 2018 (online)

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Summary

Purified 2-chain recombinant tissue-type plasminogen activator (t-PA) was reduced under mild conditions - 10 mM dithiothreitol/ 5° C/1.5 h - and the two chains were separated by chromatography on lysine Sepharose. The t-PA B chain was fully active as determined by its activity towards the chromogenic substrate S-2288 (H-D-ile-pro-arg p-nitroanilide). Analysis by sodium dodecyl sulphate polyacrylamide gel electrophoresis under reducing or non-reducing conditions revealed a single polypeptide at Mr = 35,000 or 29,000 respectively. In addition, under non-reducing conditions a fibrinolytic band at apparent Mr = 29,000 was present after fibrin zymography. The N-terminal sequence was confirmed as ile-lys-gly. The t-PA B chain had a specific amidolytic activity, using S-2288, of 170,000 to 210,000 SU/mg protein. (This compares to a specific activity of the native 2-chain t-PA of 170,000 SU/mg). It resembles urokinase-type plasminogen activator in its inability to be stimulated by fibrin and its dose response on human fibrin plates. However, t-PA B-chain was stimulated to almost the same extent as t-PA by poly-D-lysine. The isoelectric points, at pH 5.6 and 5.7, fall outside the range generally quoted for t-PA preparations (pH 7.8-8.8).

Key words

Tissue-type plasminogen activator - Fibrinolysis - Urokinase-type plasminogen activator
 

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