Antithrombin-III-Hamilton: a gene with a point mutation (guanine to adenine) in codon 382 causing impaired serine protease reactivity

Abstract

Antithrombin-III-Hamilton is a structural mutant of antithrombin III with defective serine protease reactivity, demonstrable in three members of a French Canadian family. The propositus, a 54-year-old man with a history of recurrent thromboembolic events, and his two asymptomatic grown children are heterozygous for the mutant antithrombin III gene. In all three individuals, the immunoreactive antithrombin III level is normal, while the antithrombin and antifactor Xa activity is approximately 50% of the control value. Two dimensional immunoelectrophoresis of antithrombin-III-Hamilton in the presence of heparin is normal. Purified antithrombin-III-Hamilton did not form thrombin-antithrombin III complex when incubated with thrombin for up to 30 minutes. The normal and mutant antithrombin III alleles of the propositus could be distinguished by linkage to Pstl restriction fragment length polymorphisms (RFLP). Genomic DNA from the propositus was cloned into EMBL 3 phage vectors and two clones containing nearly complete copies of the antithrombin-III-Hamilton allele were identified. Exon 6 of both clones was subcloned into M13 phage vector and sequenced, revealing a G----A point mutation in the first base of codon 382. Codon 382 codes for alanine in the normal allele and for threonine in the antithrombin-III-Hamilton allele. Alanine-382, 12 residues from the reactive center, is a highly conserved amino acid in the family of serine protease inhibitors known as the serpins. We postulate that, as a result of the substitution of threonine for alanine in antithrombin-III-Hamilton, either the tertiary structure or the hydrophobicity of the thrombin-binding region is altered, causing aberrant conformation of the Arg-393-Ser-394 bond at the reactive center impairing the interaction between antithrombin-III-Hamilton and the activated serine proteases.

Regulation of Thrombin Activity by Antithrombin and Heparin

“…3 and 4), is substituted by Thr thus are poor inhibitors of thrombin and instead behave predominantly as substrates of the enzyme. [133][134][135][136][137] In agreement with these findings, analogous congenital mutants of C1 esterase inhibitor 129 and a congenital mu tant of α 2 -antiplasmin in which an Ala is inserted be tween the P8 and P9 residues 138 behave in a similar manner. However, an antithrombin variant with Ala 384 substituted by Ser was shown to retain appreciable activ ity, particularly against Factor Xa.…”

Role of the C‐sheet in the maturation of N‐glycans on antithrombin: functional relevance of pleiotropic mutations

“…The characterization of natural mutations identified in patients with antithrombin deficiency has helped us to discover and characterize both functional domains of this key anticoagulant, such as the HBS, as well as to define new mechanisms involved in the deficiency of this serine protease inhibitor . Moreover, these studies might also help to identify the clinical prognosis associated with each mutation .…”

Antithrombin: The Principal Inhibitor of Thrombin

“…This amino acid is conserved in all serpins that retain proteinase inhibitory activity. Initial studies suggested that substitution of Ala 382 in antithrombin Hamilton 130 and Ala 384 in antithrombin Charleville 131 caused functionally inactive mutants. Closer examination, however, showed that these and similar variants (antithrombins Glasgow II and Vicenza) 132,133 are cleaved at their reactive site bonds following their interactions with thrombin but do not form inhibitory complexes with them.…”