[Study on the molecular mechanism of antithrombin gene C2759T (Leu99Phe) mutation causing antithrombin deficiency].
OBJECTIVE
To study the molecular mechanism of antithrombin (AT) gene C2759T (Leu99Phe) mutation causing AT deficiency.


METHODS
A mutated AT cDNA expression plasmid ATM2759 was constructed by mega-primer method. ATM2759 and wild type AT cDNA expression plasmid ATN were transfected into COS7 cells or CHO cells by using Superfect reagent respectively for in vitro expression study and immunofluorescence assay.


RESULTS
The antigen levels of AT (AT:Ag) in the cell lysate of ATM2759 transfected COS7 cells and the cell culture supernatant were 174.97% and 35.63% of that of ATN transfected COS7 cells respectively, whereas the AT activity in the cell culture supernatant was 47.73% of the control's. Immunofluorescence analysis showed that the fluorescence intensity was significantly higher in ATM2759 transfected CHO cells than in those transfected with ATN.


CONCLUSIONS
Leu99Phe substitution may not affect the binding capacity of AT with heparin. Secretion defect and intracellular accumulation of the mutated AT protein might be the mechanisms of this mutation causing AT deficiency.
Fu
Qi-hua
QH
Institute of Transfusion Medicine, Blood Centre of Zhejiang Province, Hangzhou 310006, China.
Wang
Wen-bin
WB
Ding
Qiu-lan
QL
Zhou
Rong-fu
RF
Wu
Wen-man
WM
Hu
Yi-qun
YQ
Wang
Xue-feng
XF
Yan
Li-xing
LX
Wang
Zhen-yi
ZY
Wang
Hong-li
HL
D000818:Animals; D000990:Antithrombin III / Q000235:genetics* / Q000378:metabolism; D020152:Antithrombin III Deficiency / Q000235:genetics; D016466:CHO Cells; D019556:COS Cells; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003412:Cricetulus; D005455:Fluorescent Antibody Technique; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D014162:Transfection
D000990:Antithrombin III