Antithrombin-III-Stockholm: a codon 392 (Gly----Asp) mutation with normal heparin binding and impaired serine protease reactivity

Abstract

Antithrombin-III-Stockholm is a new structural variant of antithrombin- III (AT-III) with normal heparin affinity but defective serine protease inhibitory activity. The proposita, a white female born in 1966, was diagnosed to have developed a pulmonary embolus while on oral contraceptives at age 19. The proposita, as well as her father, were diagnosed to have a type 2 AT-III deficiency as they had normal levels of immunoreactive AT-III associated with decreased (approximately 60%) functional AT-III when measured with either alpha-thrombin or factor Xa as the substrate, either in the presence or absence of heparin. There was no evidence of abnormal electrophoretic mobility of AT-III from the proposita either in the presence or absence of heparin. Genomic DNA was prepared and all seven AT-III exons were polymerase chain reaction (PCR)-amplified and sequenced in both directions using nested primers. Only exon 7 provided evidence for the presence of a mutation, with the second base of codon 392 having a G----A substitution. Such a mutation would cause the substitution of aspartic acid at the site of the normally appearing glycine in the translated product. Furthermore, this mutation caused the destruction of an Hae III restriction site at this point in the AT-III gene. The absence of this Hae III site was confirmed using restriction fragment length polymorphism analysis of PCR-amplified material from the proposita. Experiments with AT-III from the proposita together with experiments with cell-free translated AT- III-Stockholm provided evidence that the mutant AT-III protein does not efficiently form a stable covalent inhibitory complex with alpha- thrombin, although it exhibits normal heparin affinity. The minimal thrombin-complexing ability of the mutant AT-III protein that was observed was accelerated by heparin, but to subnormal levels.

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 .…”

Regulation of Thrombin Activity by Antithrombin and Heparin

“…4) was replaced with Asp was found to have a reduced reactivity with throm bin. 104 A number of recombinant antithrombins with other substitutions at the P2 position similarly showed impaired binding of both thrombin and Factor Xa, amino acids with hydrophobic or negatively charged side chains having the most pronounced effects. 105 In contrast, re placement of Gly 392 with Pro insignificantly affected the reaction with either proteinase, suggestive of a bend in the polypeptide chain in this position being of importance for proper interaction with the target proteinase.…”

Antithrombin: The Principal Inhibitor of Thrombin

“…It should be appreciated, though, that the proposed model is based on reactive site cleaved α 1 -antitrypsin and nei ther reactive site cleaved antithrombin nor α 1 -antitrypsin actually binds heparin with high affinity. Nevertheless, the crystal structures of both cleaved and intact an tithrombin provided support for a surface-orientated pri mary contact site comprised of elements of helices D, A, and perhaps C. From a consideration of the cleaved bo vine antithrombin structure, it has been proposed that Arg 13 , Lys 39 , Arg 46 , Arg 47 , Arg 129 , and Arg 132 comprise the primary pentasaccharide contact surface. 17 Although specific antithrombin residues are impor tant in the heparin-binding site providing the points of contact with the pentasaccharide, it is apparent that the overall conformation of the heparin-binding domain must also be considered along with any structural changes that may occur in the heparin-induced conformational change.…”

“…A similar phenotype has been observed with a substitution at the P2 position, Gly 392 to Asp, in antithrombin Stockholm. 129 The Arg 393 to Cys substitution is particularly interesting, as Cys 393 is able to form a disulfide bond with albumin 122 circulating as a disulfide-linked heterodimer between albumin and antithrombin. Substitution at the P1' position, Ser 394 to Leu *This information has been taken from the revised antithrombin mutation database.…”

“…A similar mechanism could explain the reduction in heparin affinity that occurs with the recently reported substitution of Ser 116 to Pro. 141 An interesting type II HBS variant (antithrombin Geneva Arg 129 Glu) has been reported at position 129, the second heparin-binding region of antithrombin. Gandrille et al 142 and Church et al 143 propose that Lys 125 , Arg 129 , Arg 132 , Lys 133 , Lys 136 , and Lys 139 are all on the same side of helix D and constitute an essential part of the primary heparin-binding domain, a proposal well supported by the chemical modification studies and by the characterization of antithrombin Geneva.…”