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J. Biochem, 1990, Vol. 108, No. 1 72-79
© 1990 Japanese Biochemical Society

research-article

Characterization of Thrombin- and Plasmin-Resistant Mutants of Recombinant Human Single Chain Urokinase–Type Plasminogen Activator1

Yutaka Eguchi*, Yoichi Sakata*,2, Michio Matsuda*, Hiroshi Osada**, Naganori Numao**, Muneki Ohmori** and Kiyoshi Kondo**

*Division of Thrombosis and Hemostasis, Institute of Hematology, Jichi Medical School Minamikawachi-machi, Tochigi 329-04
**Sagami Chemical Research Center Sagamihara, Kanagawa 229

2To whom all correspondence should be addressed

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: Lys136 to Gln and Phe167 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.

1This work was supported In part by a Research Grant for Cardiovascular Disease (62C-1) from the Ministry of Health and Welfare of Japan and Grant-in-Aid for Scientific Research on Priority Areas No. 63616512 from the Ministry of Education, Science and Culture of Japan.


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