Characterization of domain deletion and/or duplication mutants of a recombinant chimera of tissue-type plasminogen activator and urokinase-type plasminogen activator (rt-PA/u-PA)
Nelles, L.; Lijnen, H.R.; Van Nuffelen, A.; Demarsin, E.; Collen, D.
Thrombosis and Haemostasis 64(1): 53-60
1990
ISSN/ISBN: 0340-6245 PMID: 2148847 Document Number: 357509
Chimeric molecules comprising the A-chain of tissue-type plasminogen activator (t-PA) and the catalytic domain of urokinase-type plasminogen activator (u-PA) have intact enzymatic characteristics of u-PA, but only partial fibrin-binding properties of t-PA (Nelles et al., J Biol Chem 1987; 262: 10855-62). The following domain deletion and/or duplication mutants of such a t-PA/u-PA chimera were constructed, purified and characterized: rt-PA-delta FE/u-PA, with deletion of the finger-like (F) and epidermal growth factor-like (E) domains, rt-PA-delta K1 delta K2/u-PA, with kringle 1 (K1) replaced by a second copy of kringle 2 (K2), and rt-PA-delta FEK1 delta K2/u-PA, with F and E domain deletions in rt-PA-delta K1 delta K2/u-PA. The specific activities on fibrin plates of the single-chain (sc) chimeras ranged between 68,000 IU/mg for rt-PA-delta K1 delta K2/scu-PA and 200,000 IU/mg for rt-PA-delta FEK1 delta K2/scu-PA, as compared to 120,000 IU/mg for rscu-PA. The specific activities of their plasmin-generated two-chain (tc) derivatives ranged between 120,000 IU/mg for rt-PA-delta K1 delta K2/tcu-PA and 240,000 IU/mg for rt-PA-delta FEK1 delta K2/tcu-PA, as compared to 100,000 IU/mg for rtcu-PA. All two-chain chimeras activated plasminogen following Michaelis-Menten kinetics, with catalytic efficiencies between 0.072 microM-1s-1 for rt-PA-delta K1 delta K2/tcu-PA and 0.081 microM-1 s-1 for rt-PA-delta FEK1 delta K2/tcu-PA, as compared to 0.088 microM-1 s-1 for rtcu-PA.