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).
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.(ABSTRACT TRUNCATED AT 250 WORDS)
Nelles
L
L
Center for Thrombosis and Vascular Research, University of Leuven, Belgium.
Lijnen
H R
HR
Van Nuffelen
A
A
Demarsin
E
E
Collen
D
D
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D003470:Culture Media; D005337:Fibrin / Q000378:metabolism; D005340:Fibrinogen / Q000378:metabolism; D005341:Fibrinolysin / Q000494:pharmacology; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D010960:Plasminogen Activators / Q000235:genetics*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012685:Sepharose / Q000031:analogs & derivatives; D010959:Tissue Plasminogen Activator / Q000235:genetics*; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics*
D000345:Affinity Labels; D000596:Amino Acids; D003470:Culture Media; D011993:Recombinant Fusion Proteins; C030675:lysine-sepharose; D005337:Fibrin; D005340:Fibrinogen; D012685:Sepharose; D010960:Plasminogen Activators; D010959:Tissue Plasminogen Activator; D005341:Fibrinolysin; D014568:Urokinase-Type Plasminogen Activator