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

We have identified three novel mutations of the antithrombin (AT) gene in patients with thrombotic complications: a Cys 128 --> Tyr mutations, a G --> A mutation in the intervening sequence 4 (IVS4) 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 disulfide bond and allow the formation of intermolecular disulfide bonds. Plasma from a heterozygous patients bearing the Cys 128 --> Tyr mutation and from a compound heterozygote bearing the Arg 47 --> His mutation and the 9 bp deletion in exon 6 were passed through a heparin-sepharose column. In both cases a population of high-molecular-weight AT molecules with no binding affinity and no AT activity was separated from a population of normal molecules in the first patient, together with a population of molecules with a reduced binding affinity for heparin due to the substitution of Arg 47, in the compound heterozygote. The common feature of these three mutations is that they lead to partial misfolding and to the formation of intermolecular disulfide bonds with other plasma components, inducing the pleiotropic phenotypes observed.