title: The infective polymerization of conformationally unstable antithrombin mutants may play a role in the clinical severity of antithrombin deficiency.
abstract: Mutations affecting mobile domains of antithrombin induce conformational instability resulting in protein polymerization that associates with a severe clinical phenotype, probably by an unknown gain of function. By homology with other conformational diseases, we speculated that these variants might infect wild-type (WT) monomers reducing the anticoagulant capacity. Infective polymerization of WT polymers and different P1 mutants (p.R425del, p.R425C and p.R425H) were evaluated by using native gels and radiolabeled WT monomers and functional assays. Human embryonic kidney cells expressing the Epstein-Barr nuclear antigen 1 (HEK-EBNA) cells expressing inducible (p.R425del) or two novel constitutive (p.F271S and p.M370T) conformational variants were used to evaluate intracellular and secreted antithrombin under mild stress (pH 6.5 and 39°C for 5 h). We demonstrated the conformational sensitivity of antithrombin London (p.R425del) to form polymers under mild heating. Under these conditions purified antithrombin London recruited WT monomers into growing polymers, reducing the anticoagulant activity. This process was also observed in the plasma of patients with p.R425del, p.R425C and p.R425H mutations. Under moderate stress, coexpression of WT and conformational variants in HEK-EBNA cells increased the intracellular retention of antithrombin and the formation of disulfide-linked polymers, which correlated with impaired secretion and reduction of anticoagulant activity in the medium. Therefore, mutations inducing conformational instability in antithrombin allow its polymerization with the subsequent loss of function, which under stress could sequestrate WT monomers, resulting in a new prothrombotic gain of function, particularly relevant for intracellular antithrombin. The in vitro results suggest a temporal and severe plasma antithrombin deficiency that may contribute to the development of the thrombotic event and to the clinical severity of these mutations.
authors.affiliation: Department of Internal Medicine, Centro Regional de Hemodonación, University of Murcia, Murcia, Spain.
authors.forename: Irene
authors.initials: I
authors.lastname: Martínez-Martínez
authors.forename: José
authors.initials: J
authors.lastname: Navarro-Fernández
authors.forename: Sonia
authors.initials: S
authors.lastname: Aguila
authors.forename: Antonia
authors.initials: A
authors.lastname: Miñano
authors.forename: Nataliya
authors.initials: N
authors.lastname: Bohdan
authors.forename: María Eugenia
authors.initials: ME
authors.lastname: De La Morena-Barrio
authors.forename: Adriana
authors.initials: A
authors.lastname: Ordóñez
authors.forename: Constantino
authors.initials: C
authors.lastname: Martínez
authors.forename: Vicente
authors.initials: V
authors.lastname: Vicente
authors.forename: Javier
authors.initials: J
authors.lastname: Corral
mesh_terms: D020152:Antithrombin III Deficiency / Q000378:metabolism*; D058833:Antithrombin Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D006801:Humans; D050505:Mutant Proteins / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D055503:Protein Multimerization; D055550:Protein Stability; D013312:Stress, Physiological
chemical_list: D058833:Antithrombin Proteins; D050505:Mutant Proteins; C474022:antithrombin London