A Slow Clearing, Fibrin-Specific, PAI-1 Resistant Variant of t-PA (T103N, KHRR 296-299 AAAA)

Nicholas F Paoni; Bruce A Keyt; Canio J Refino; Alice M Chow; Hung V Nguyen; Lea T Berleau; Julie Badillo; Luis C Peña; Kevin Brady; Florian M Wurm; John Ogez; William F Bennett

doi:10.1055/s-0038-1649571

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 1993; 70(02): 307-312
DOI: 10.1055/s-0038-1649571

Original Articles

Fibrinolysis

Schattauer GmbH Stuttgart

A Slow Clearing, Fibrin-Specific, PAI-1 Resistant Variant of t-PA (T103N, KHRR 296-299 AAAA)

Authors

Nicholas F Paoni

¹The Department of Cardiovascular Research, Genentech., Inc., South San Francisco, CA, USA
Bruce A Keyt

¹The Department of Cardiovascular Research, Genentech., Inc., South San Francisco, CA, USA
Canio J Refino

¹The Department of Cardiovascular Research, Genentech., Inc., South San Francisco, CA, USA
Alice M Chow

¹The Department of Cardiovascular Research, Genentech., Inc., South San Francisco, CA, USA
Hung V Nguyen

¹The Department of Cardiovascular Research, Genentech., Inc., South San Francisco, CA, USA
Lea T Berleau

¹The Department of Cardiovascular Research, Genentech., Inc., South San Francisco, CA, USA
Julie Badillo

¹The Department of Cardiovascular Research, Genentech., Inc., South San Francisco, CA, USA
Luis C Peña

¹The Department of Cardiovascular Research, Genentech., Inc., South San Francisco, CA, USA
Kevin Brady

¹The Department of Cardiovascular Research, Genentech., Inc., South San Francisco, CA, USA
Florian M Wurm

²The Department of Cell Culture R & D, Genentech., Inc., South San Francisco, CA, USA
John Ogez

³The Department of Recovery Process R & D, Genentech., Inc., South San Francisco, CA, USA
William F Bennett

¹The Department of Cardiovascular Research, Genentech., Inc., South San Francisco, CA, USA

Abstract

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Summary

Site directed mutagenesis was used to construct a t-PA variant that contains an additional glycosylation site in the first kringle domain (T103N) combined with a tetra-alanine substitution in the protease domain (KHRR 296-299 AAAA). This combination variant has a plasma clearance rate that is 4.5-fold slower in rats and 5.4-fold slower in rabbits than t-PA. It is also less than one tenth as active as t-PA towards plasminogen in the presence of fibrinogen, and has approximately twice the normal activity in the presence of fibrin. It shows substantial resistance to the fast acting inhibitor, plasminogen activator inhibitor-1 (PAI-1), requiring a 10-fold greater molar excess of PAI-1 to reduce its activity by 50%, compared to t-PA. This is the result of a reduction of nearly 100-fold in the second order rate constant for PAI-1 inactivation. These results show that it is possible to combine mutations in different domains of t-PA to construct a variant which is simultaneously slower clearing, less reactive towards plasminogen in the absence of a fibrin clot, and resistant to inactivation by PAI-1.

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References

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