Familial Variant of Antithrombin III (AT III Bligny, 47Arg to His) Associated with Protein C Deficiency

Abstract

SummaryThe association of a variant of antithrombin III (AT III Bligny) and protein C deficiency is described in a 36-year-old patient having suffered from severe thrombotic episodes. His mother has protein C deficiency and showed a single episode of thrombophlebitis following surgery. His father, sister and daughter have the variant AT III and are asymptomatic. The abnormal AT III was characterized in plasma by the discrepancy between a normal progressive activity and a reduced heparin cofactor activity. This variant AT III was purified, separated from the normal protein by heparin-Sepharose chromatography and was eluted with increased NaCI concentrations. At pH 7.4, the variant AT III eluted at lower (0.3 to 0.5 M) NaCI concentrations than normal (1 to 1.5 M) AT III, thus demonstrating a decreased affinity for heparin. At pH 6.0, however, the abnormal molecule bound more avidly to heparin-Sepharose and was eluted like normal AT III at pH 7.4. Similarly, the heparin enhancement of intrinsic fluorescence of the variant AT III, markedly reduced at pH 7.4, was normalized at pH 6.0. The abnormal AT III showed a normal antiprotease activity, a normal molecular weight by SDS-PAGE, but displayed only a partial immunological identity with the normal protein. Analysis of amplified genomic DNA from this patient by dot-blot demonstrates a heterozygous substitution of arginine by histidine at position 47.

The Role of Arg46 and Arg47 of Antithrombin in Heparin Binding

“…c nd, not determined. heparin affinity reinforced this conclusion (14,15,17). The recent X-ray structure of a complex of antithrombin with a heparin pentasaccharide further supported the involvement of Arg47 in heparin binding and also implicated the neighboring Arg46 residue in this binding (22).…”

“…The N135A and R46A/N135A variants were purified by heparin affinity chromatography at pH 7.4 on 5-mL Econo-Pac Heparin (Bio-Rad, Hercules, CA) or HiTrap Heparin (Amersham Pharmacia Biotech, Uppsala, Sweden) columns, as described previously ( , ). An initial purification of the R47H/N135A variant was done by the same procedure, but at pH 6 to increase heparin affinity ( , ). Nevertheless, this variant required further purification on a 1-mL HiTrap Heparin column, eluted with a 35-mL gradient from 0.02 to 3 M NaCl in 20 mM phosphate, pH 7.4, 0.1% (w/v) poly(ethylene glycol) 8000.…”

“…Although the heparin-binding site of antithrombin thus appears reasonably well defined, the importance of individual residues of this site for the affinity of the interaction with heparin and for the heparin-induced conformational change remains essentially unknown. Congenital antithrombin variants with mutations of Arg47 to Cys, Ser, or His are known to have an impaired heparin binding that is associated with thrombosis in the homozygous state (13)(14)(15)17), but the binding defect has not been quantified. Moreover, the Arg46 residue of antithrombin has been implicated in heparin binding only by the crystallographic studies.…”

“…The site on antithrombin to which heparin binds has been the focus of considerable research in recent years. Basic residues, in particular Arg47, Arg129, Lys125, and Lys114, have been implicated in the binding by studies of natural and recombinant variants of the inhibitor ( − ). The recent crystal structure of antithrombin in complex with a heparin pentasaccharide () confirmed these conclusions and defined the heparin-binding site in greater detail.…”

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

“…Further characterization showed reduced specific heparin-cofactor activity (1.5 U/mL instead of 5 to 10 U/mL for normal AT) and a normal ability to form complexes with tnrombin (not shown), as already described for this mutation. 31 -34 ' 38 The other variant, which did not bind to heparin sepharose, migrated as a high-molecular-mass species in the absence of reducing agent but had the normal molecular mass of AT after reduction. As in the other two cases described here, this suggests that the mutated AT has a free Cys available for disulfide bonding with other molecules.…”