Characterization of thrombin- and plasmin-resistant mutants of recombinant human single chain urokinase-type plasminogen activator.
Recombinant human single-chain urokinase-type plasminogen activator (suc-PA) (SM0: wild type) and its variants resistant to plasmin and/or thrombin (SM1: Lys135 to Gln; SM3: Phe157 to Asp; and SM4: Lys135 to Gln and Phe157 to Asp) have been constructed by site-directed mutagenesis with the aim of producing more efficient thrombolytic agents [Miyake, T. et al. (1988) J. Biochem. 104, 643-647]. In the present study, we characterized the recombinant variant scu-PAs expressed in Escherichia coli. They appeared to have structural integrity because their heat-stabilities, immunological reactivities, and circular dichroism spectra were essentially identical to those of each other and of native scu-PA (nscu-PA). In the presence of thrombin, SM3 and SM4 showed efficient clot lysis by all of the assays used, compared with SM0, SM1, and nscu-PA. While in the absence of thrombin, when measured by a fibrin plate method in a purified system, SM3 and SM4 had lower specific activities than SM0, SM1, and nscu-PA, because of their catalytic constants for conversion to the two-chain form (tcu-PA) by plasmin are lower. However, SM4 lysed clots as efficiently as SM0 in plasma by retaining the single-chain form, whereas SM0 was partly converted to the two-chain form.
Eguchi
Y
Y
Division of Thrombosis and Hemostasis, Jichi Medical School, Tochigi.
Sakata
Y
Y
Matsuda
M
M
Osada
H
H
Numao
N
N
Ohmori
M
M
Kondo
K
K
D001345:Autoradiography; D002384:Catalysis; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000187:drug effects / Q000378:metabolism; D005341:Fibrinolysin / Q000494:pharmacology*; D005342:Fibrinolysis / Q000187:drug effects; D006801:Humans; D005779:Immunodiffusion; D007457:Iodine Radioisotopes; D007700:Kinetics; D009154:Mutation; D010960:Plasminogen Activators / Q000032:analysis / Q000235:genetics*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000032:analysis; D013917:Thrombin / Q000494:pharmacology*; D014568:Urokinase-Type Plasminogen Activator / Q000032:analysis / Q000235:genetics*
D007457:Iodine Radioisotopes; D011994:Recombinant Proteins; D010960:Plasminogen Activators; D013917:Thrombin; D005341:Fibrinolysin; D014568:Urokinase-Type Plasminogen Activator