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 no significant difference. Oligomerization (not aggregation) of serpin is a characteristic feature due to insertion of one reactive loop into another serpin molecule, and molecular flexibility and/or instability of the mutant antithrombin seems to inhibit this phenomenon. Examination of three-dimensional structure of antithrombin based on the spatial coordinates obtained from crystallographic studies showed hydrophobic interactions including Phe 368 on the surface region of antithrombin. To further substantiate the role of this amino acid residue in the inhibitory activity, site-directed mutagenesis was performed and resultant mutant antithrombin cDNA clones were expressed in a cell-free system. Only Leu variant can bind weakly to the target enzyme thrombin, suggesting a bulky hydrophobic amino acid may play an important role in this position.
The second patient was a 47-year-old male with right popliteal arterial embolism. The plasma heparin cofactor activity and the immunologic antithrombin level in the plasma were 46% and 64%, respectively. No other members of the family showed deficient symptoms. DNA sequence analysis of the amplified antithrombin exons revealed a TTCTG-insertional repeat in codon 214, which results in premature termination of antithrombin polypeptide. With SSCP analysis, only the patient's sample showed polymorphic pattern due to the presence of the mutant allele, which is in accordance with the fact that none of the other tested individuals showed antithrombin deficiency. No detectable amount of prematurely terminated antithrombin polypeptide was identified when immunobloting analysis was performed. These results suggested that only the functional antithrombin expressed from the normal allele is being secreted into the bloodstream, whereas truncated antithrombin without secretion-competent status in the endoplasmic reticulum is retained and subsequently degraded.
