Biochemical and thrombolytic properties of a low molecular weight form (comprising Leu144 through Leu411) of recombinant single-chain urokinase-type plasminogen activator.
The cDNA encoding a low Mr derivative (residues 144-411) of human single-chain urokinase-type plasminogen activator was cloned, the recombinant low Mr single-chain urokinase-type plasminogen activator (rscu-PA-32k) was expressed in Chinese hamster ovary cells, and the translation product was purified to homogeneity from conditioned cell culture medium. rscu-PA-32k is very similar to intact recombinant single-chain urokinase-type plasminogen activator in terms of its very low activity (120 IU/mg) on a chromogenic substrate for urokinase (pyroglutamylglycylarginine p-nitroanilide), its plasminogen-dependent fibrinolytic activity on fibrin plates (specific activity = 170,000 IU/mg), its plasminogen activating potential, and the lack of specific binding to fibrin. In a rabbit jugular vein thrombosis model, comparable thrombolysis was obtained with rscu-PA-32k as compared to low molecular weight two-chain urokinase (50% lysis at 2.1 and 1.6 mg/kg infused over 4 h). Thrombolysis was associated with much less extensive systemic fibrinogen breakdown with rscu-PA-32k than with two-chain urokinase (residual fibrinogen at 50% lysis of 71 and 10%, respectively). It is concluded that the functional properties of rscu-PA-32k, expressed with a high efficiency, are similar to those of its previously characterized natural counterpart.
Lijnen
H R
HR
Center for Thrombosis and Vascular Research, University of Leuven, Belgium.
Nelles
L
L
Holmes
W E
WE
Collen
D
D
D000818:Animals; D002460:Cell Line; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D006224:Cricetinae; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004792:Enzyme Precursors; D005337:Fibrin / Q000378:metabolism; D005341:Fibrinolysin / Q000378:metabolism; D005342:Fibrinolysis; D005343:Fibrinolytic Agents; D007601:Jugular Veins; D007700:Kinetics; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism; D010960:Plasminogen Activators / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011817:Rabbits; D011994:Recombinant Proteins / Q000378:metabolism*; D013927:Thrombosis / Q000378:metabolism; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D004792:Enzyme Precursors; D005343:Fibrinolytic Agents; D010446:Peptide Fragments; D011994:Recombinant Proteins; D005337:Fibrin; D004247:DNA; D010960:Plasminogen Activators; D005341:Fibrinolysin; D014568:Urokinase-Type Plasminogen Activator