Three novel missense mutations in the antithrombin III (AT3) gene causing recurrent venous thrombosis

Millar, David; Wacey, A. I.; Ribando, Jennifer; Melissari, E.; Laursen, Birgitte Schantz; Woods, Paul; Kakkar, Vijay V.; Cooper, David Neil

doi:10.1007/bf00211016

Cited by 21 publications

(14 citation statements)

References 17 publications

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“…However, substitution of either isoleucine or leucine for Phe372, and either serine or threonine for Pro36 not only fail to disrupt PAI-I stability, but actually markedly enhance it (Table TI). In contrast, a proline to threonine substitution in ATIII (P80T), the position homologous to Pro36 in PAI-1, is associated with a type I deficiency state manifesting reduced synthesis of ATIII (Millar et al, 1994). This conserved proline is thought to initiate formation of a-helix B.…”

Section: Discussionmentioning

confidence: 99%

Molecular evolution of plasminogen activator inhibitor-1 functional stability.

1995

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Plasminogen activator inhibitor‐1 (PAI‐1) is a member of the serine protease inhibitor (serpin) supergene family and a central regulatory protein in the blood coagulation system. PAI‐1 is unique among serpins in exhibiting distinct active and inactive (latent) conformations in vivo. Though the structure of latent PAI‐1 was recently solved, the structure of the short‐lived, active form of PAI‐1 is not known. In order to probe the structural basis for this unique conformational change, a randomly mutated recombinant PAI‐1 expression library was constructed in bacteriophage and screened for increased functional stability. Fourteen unique clones were selected, and shown to exhibit functional half‐lives (T1/2S) exceeding that of wild‐type PAI‐1 by up to 72‐fold. The most stable variant (T1/2 = 145 h) contained four mutations. Detailed analysis of these four mutations, individually and in combination, demonstrated that the markedly enhanced functional stability of the parent compound mutant required contributions from all four substitutions, with no individual T1/2 exceeding 6.6 h. The functional stability of at least eight of the remaining 13 compound mutants also required interactions between two or more amino acid substitutions, with no single variant increasing the T1/2 by > 10‐fold. The nature of the identified mutations implies that the unique instability of the PAI‐1 active conformation evolved through global changes in protein packing and suggest a selective advantage for transient inhibitor function.

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Section: Discussionmentioning

confidence: 99%

Molecular evolution of plasminogen activator inhibitor-1 functional stability.

1995

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“…We selected pleiotropic mutations identified in patients with antithrombin deficiency from available databases (www.hgmd.org) [3,4,[10][11][12][13][14]: K241E, M251I, M315K, F402L, and P429L. Each one of these mutations was located at one of the strands of the C sheet (s1C-s4C), except for the P429L mutation, which was located at the C-terminal extreme of antithrombin.…”

Section: Pleiotropic Mutations Selectedmentioning

confidence: 99%

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

Águila

Navarro-Fernández

Bohdan

et al. 2014

Journal of Thrombosis and Haemostasis

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To cite this article:Aguila S, Navarro-Fern andez J, Bohdan N, Guti errez-Gallego R, de la Morena-Barrio ME, Vicente V, Corral J, Mart ınez- Summary. Background: The characterization of natural mutants identified in patients with antithrombin deficiency has helped to identify functional domains or regions of this key anticoagulant and the mechanisms involved in the deficiency, as well as to define the clinical prognosis. Recently, we described an abnormal glycosylation in a pleiotropic mutant (K241E) that explained the impaired heparin affinity and the mild risk of thrombosis in carriers. Objectives: To evaluate the effects of different natural pleiotropic mutations on the glycosylation of antithrombin and their functional effects. Methods: Five pleiotropic mutations identified in patients with antithrombin deficiency and located at each one of the strands of the C-sheet were selected (K241E, M251I, M315K, F402L, and P429L). Recombinant mutants were generated and purified. Glycoform heterogeneity and conformational sensitivity were studied with electrophoresis, proteomic analysis, and glycomic analysis. Heparin affinity was evaluated from intrinsic fluorescence. Reactivity assays with factor Xa, thrombin and neutrophil elastase in the presence or absence of heparin were also performed. Results and Conclusions: Pleiotropic mutants, except for that with the M315K mutation, which affects a non-exposed residue, showed two glycoforms. Variant 1, with abnormal glycosylation, had reduced heparin affinity and severely affected reactivity with the target proteases. In contrast, variant 2, with similar electrophoretic mobility and heparin affinity to wild-type antithrombin, had impaired inhibitory activity that was partially compensated for by activation with heparin. Our results suggest the C-sheet of antithrombin as a new region that is relevant for proper maturation of the N-glycans. Therefore, pleiotropic mutations lead to glycosylation defects that are responsible for the reduced heparin affinity.

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“…It would be predicted to result in a rapid conversion to latent. In the literature the reported association with antithrombin deficiency was said to be controversial for p.Tyr190Cys mutation as it did not cosegregate with the phenotype in the pedigrees from a previous study (Millar et al , ). However, our study showed a complete segregation and strong association with a type II.…”

Section: Discussionmentioning

confidence: 96%

SERPINC1 gene mutations in antithrombin deficiency

Mulder

Huntington

et al. 2017

Br J Haematol

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Existing evidence suggests that in most cases antithrombin deficiency can be explained by mutations in its gene, SERPINC1. We investigated the molecular background of antithrombin deficiency in a single centre family cohort study. We included a total of 21 families comprising 15 original probands and sixty-six relatives, 6 of who were surrogate probands for the genetic analysis. Antithrombin activity and antigen levels were measured. The heparin-antithrombin binding ratio assay was used to distinguish between the different subtypes of type II antithrombin deficiency. SERPINC1 mutations were detected by direct sequencing of all 7 exons and regulatory regions, and multiplex ligation-dependent probe amplification. Eighty-six per cent of the families had a detrimental SERPINC1 gene mutation that segregated in the family. We detected 13 different SERPINC1 gene mutations of which 5 were novel. Among all these mutations, 44% was associated with type I deficiency, whereas the remainder was associated with type II heparin binding site (11%), type II pleiotropic effect (33%), type II reactive site (6%) or had the antithrombin Cambridge II mutation (6%). The current study reports several novel SERPINC1 mutations, thereby adding to our knowledge of the molecular background of antithrombin deficiency. Finally, our results point out the importance of future research outside the conventional SERPINC1 gene approach.

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Three novel missense mutations in the antithrombin III (AT3) gene causing recurrent venous thrombosis

Cited by 21 publications

References 17 publications

Molecular evolution of plasminogen activator inhibitor-1 functional stability.

Molecular evolution of plasminogen activator inhibitor-1 functional stability.

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

SERPINC1 gene mutations in antithrombin deficiency

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