Molecular
Cloning, Expression and Characterization of the Clones Encoding Soluble TF
Mutants
YU Min, ZHANG Yu-Gao, WANG Yu-Xiong, SONG
Hou-Yan*
( Department of Molecular Genetics, Medical School, Fudan University,
Shanghai 200032, China )
Abstract
Tissue factor (TF) plays an important role in the pathogenesis of
atherosclerotic,sepsis and disseminated intravascular coagulation (DIC).TF
pathway is therefore an attractive therapeutic target in a number of disease
states.Here two TF mutants were developed and named MCsTF and MFsTF, in which
the amino acids of active sites were mutated. Both of them were expressed in E.coli
and used to inhibit TF pathway through competitive FVII/VIIa binding with
TF.The results indicated that rMCsTF almost lost all activities of FX
activation and procoagulation, and rMFsTF lost 90% activity.The specific
catalytic constant (kcat/Km) of FX activation
by the complex formed by FVIIa with rMCsTF or rMFsTF were 2.0% and 3.7%,
respectively, compared to that of rsTF.The inhibition effects of the mutants
were studied in vitro,and it appeared that the prothrombin time were
prolonged in a dose-dependent manner.Therefore,these mutants of TF may become
new kind of specific inhibitors of TF pathway,as a promising drug for the
treatment of patients with over-expression of TF.
Key words soluble tissue factor mutant; tissue factor
pathway; inhibition of clot
*Corresponding author: Tel,
86-21-64033738; e-mail, [email protected]