Characterization of molecular defect of 13387-9delG mutated antithrombin in inherited type I antithrombin deficiency

As a major physiological inhibitor of thrombin and other coagulation proteases, antithrombin (AT) plays an important role in the maintenance of normal hemostasis and its deficiency is associated with a predisposition for familial venous thromboembolic disease. Recently, we found a novel mutation (13387-9delG) in the antithrombin gene that is associated with type I AT deficiency. To examine the molecular pathologic mechanism of this mutation causing type I AT deficiency, the wild-type and the mutant AT constructs were expressed in COS-7 cells or Chinese Hamster Ovary cells. No AT antigen could be detected by enzyme-linked immunosorbent assay in the conditioned media of cells expressing the mutant protein, and the AT antigen level was reduced in cell lysates. The mutant AT-expressing cells did not have less intracellular mRNA levels than the wild-type transfectants as estimated by quantitative reverse transcriptase-polymerase chain reaction. Metabolic and pulse-chase experiments showed the newly synthesized wild-type AT protein was gradually secreted into the media, whereas no labeled mutant AT protein was detected in the media and the total amount of radioactivity was significantly reduced in the cells during the chase periods. By immunofluorescence analysis, the staining of the mutant AT was weaker than that of the wild type, and was predominantly diffuse without perinuclear enhancement. These results indicate that the 13387-9delG mutation, which disrupts the disulfide bridge Cys247-Cys430, impairs the secretion and stability of the truncated AT protein associated with intracellular degradation.