Abstract Number: PB0214
Meeting: ISTH 2020 Congress
Background: The C-terminal end (C-term) of antithrombin (p.Val432-Lys464) has no evident functional role, but it has been suggested to be crucial for the folding and secretion of this hemostatic serpin.
Aims: To characterize C-term natural variants of antithrombin.
Methods: Selection of C-term antithrombin variants from 340 unrelated cases (collected from 1998 to 2020). Plasma antithrombin was characterized by functional and antigenic methods. A recombinant model (HEK-EBNA) was used for expressing natural and artificial variants. Intracellular trafficking of antithrombin was analyzed by electron and confocal microscopy.
Results: Nine C-term variants where identified in 14 unrelated cases: 5 missense, 4 small deletions; 6 caused severe type-I deficiency, whilst 3, all missense, caused type-II pleiotropic deficiency (increase of latent antithrombin).
The recurrent p.Arg445Serfs*17 (N=5) caused an aberrant full-length C-term antithrombin. This variant had a new dominant-negative effect through endoplasmic reticulum aggregation and retention (Figure 1), which explained the severe type-I deficiency and its clinical severity.
Seven additional frameshift variants, with different extension of the aberrant C-term, were analysed, identifying a pleomorphic behaviour: variants affecting Val432-Phe440 were efficiently secreted in an inactive form, whilst frameshift mutants with shorter aberrant C-term had a severely impaired secretion, like p.Arg445Serfs*17. A common aberrant proline shared by the variants efficiently secreted (Pro441) was introduced in two non-secreted variants, resulting in secretory rescue (Figure 2).
Conclusions: The consequences of natural C-term antithrombin variants are highly heterogeneous, strongly related with the secondary structures affected, thus supporting the key structural role of the C-term of this plasma inhibitor. A completely aberrant C-term caused by frameshift may be secreted in an inactive form, but the resulting protein is folded into polymers if the frameshift takes place after Phe440. The generation of a Pro441 in mutants located between Val432-Phe440 seems to improve their secretion, and may promote novel targets for serpinopathies.
To cite this abstract in AMA style:Bravo-Perez C, Toderici M, de la Morena-Barrio B, Padilla J, Miñano A, Palomo A, Perez-Sanchez H, Martinez-Menarguez J, Chambers J, Marciniak S, Vicente V, Corral J, de la Morena-Barrio ME. Bisecting the Role of the C-terminal End of Antithrombin: Natural Variants due to a Frameshift at Different Secondary Structures May Exacerbate or Ameliorate a New Dominant-negative Effect [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/bisecting-the-role-of-the-c-terminal-end-of-antithrombin-natural-variants-due-to-a-frameshift-at-different-secondary-structures-may-exacerbate-or-ameliorate-a-new-dominant-negative-effect/. Accessed January 26, 2022.
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