Three novel mutations of antithrombin inducing high-molecular-mass compounds.

Abstract

We have identified three novel mutations of the antithrombin (AT) gene in patients with thrombotic complications: a Cys 128 -» Tyr mutation, a G -» A mutation in the intervening sequence 4 (FVS4) 14 nucleotide 5' to exon 5, and a 9 bp deletion in the 3' end of exon 6 resulting in a short aberrant sequence after Arg 425. The latter mutation was associated with an Arg 47 -• His mutation in two compound heterozygous brothers. These three mutations led to the expression in the circulation of small amounts of inactive molecules with a high molecular mass in immunoblot analysis. In reducing conditions, these variant molecules had a normal molecular mass, which led us to postulate that these mutations prevent the formation of one intramolecular disulfidc bond and allow the formation of intermolecular disulfide bonds. Plasma from a heterozygous patient bearing the Cys 128 -• A ntithrombin (AT) is the main natural inhibitor of /\ thrombin and other coagulation proteases, and X A. its physiological importance was first shown in 1965 by the description of a hereditary AT deficiency in a family with unexplained thrombosis.1 The inhibitory effect of AT, due to the almost irreversible trapping of target proteases, is strongly enhanced by natural glycosaminoglycans such as heparin.The AT gene, the nucleotide sequence of which has recently been elucidated, 2 maps more than 13 480 bp, comprises 7 exons (1, 2, 3a, 3b, 4, 5, and 6), and encodes a polypeptide of 464 amino acids. Several intracellular modifications occur before the secretion of the folded protein, such as the formation of three intramolecular disulfide bonds in positions 8-128, 21-95, and 247-430 of the amino-acid sequence, glycosylation at four sites, and cleavage of a 32-amino-acid signal peptide (for a review see Reference 3). The structure of the mature protein presents large homologies with other serine protease inhibitors, forming a superfamily called serpins. Crystallographic study of cleaved a-1-antitrypsin provided a working model of three-dimensional serpin structure, 4 the validity of which was recently confirmed by crystal

Mutations in the shutter region of antithrombin result in formation of disulfide‐linked dimers and severe venous thrombosis

“…Unlike the classical type I variants that result from major deletions or the presence of premature stop codons, the conformational variants are secreted into the plasma, albeit as here, the variants form only 5–15% of the circulating antithrombin. One recognized disadvantageous consequence is the conversion of such unstable antithrombins to the latent conformation [9,20,24], with an accompanying inactivation of the functionally normal component [25]. However, the variants reported here do not appreciably undergo the latent transition, but rather the prime finding is the presence in the plasma of dimers of the variant antithrombin.…”

Intracellular Accumulation of Antithrombin Morioka(C95R), a Novel Mutation Causing Type I Antithrombin Deficiency

“…In the case of type I deficiency, AT (E⌬313) and AT (P429Stop) are degraded rapidly by proteasomes when these cDNAs are expressed in baby hamster kidney cells (40). The mutations AT (C128Y) and AT (C430F) were identified recently (41,42), but the intracellular fate of these ATs has not yet been elucidated. From our investigation, these ATs are likely to have a similar intracellular fate as AT (C95R).…”

Genetic Analysis of Mutations in Seven Japanese Families with Type I Antithrombin Deficiency

“…Emmerich et al reported that loss of an intramol ecular disulfide bond of AT could induce high-molecu lar-weight compounds and the pleiotropic phenotypes by partial misfolding and the formation of intermolecular disulfide bonds with other components in plasma. 9 How ever, we could not detect a variant molecule or a highmolecular-mass compound in the affected plasmas by immunoblotting.…”