[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.