Molecular pathology of the hereditary dysfunctional antithrombins

Abstract

Two cases of hereditary antithrombin deficiency were investigated on the molecular level. The first propositus was a 25-year-old male patient with recurrent deep vein thrombosis and pulmonary enbolism. The heparin cofactor activity of the patient was about half of the normal level, but the antigenic level in plasma was normal. Heparin affinity of the antithrombin in the patient``s plasma was also normal. Familial studies revealed reduced antithrombin activity in his mother and the maternal aunt. Seven exons of antithrombin gene were amplified from the patient and from his parents and were sequenced. A novel T to C point mutation in exon 5 converting Phe 368 (T$\underline{T}$C) to Ser (T$\underline{C}$C) was identified in the patient and his mother. Interestingly, enzymatically amplified exon 5 from the patient and his mother had only the mutant sequence as confirmed by SSCP (sigle-stranded DNA conformational polymorphism) anaylsis and direct sequencing, etc. Since molecular analyses of the amplified exon 5 from the maternal aunt who had antithrombin deficiency revealed the presence of normal allele, the mother and the patient was regarded as heterozygous; the mechanism by which the normal allele was not amplified is yet unknown. As this is a unique variant which has not been reported, a name ``Antithrombin Seoul`` was proposed. Antithrombin proteins were purified from the citrated plasma by heparin-agarose affinity chromatography and Superose HR 6 gel filtration. Apparent second order rate constant of the variant antithrombin was about two-fold lower than that of normal. Conventional biochemical technique did not differentiated the variant protein from the normal form. When tryptophan fluorescence was monitored upon equilibrium thermal unfolding, however, antithrombin Seoul unfolded at a lower temperature, suggesting decreased thermal stability. In the normal sample, oligomerization product was appeared in the prolonged incubation, but antithrombin Seoul showed...