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.

Antithrombin controls tumor migration, invasion and angiogenesis by inhibition of enteropeptidase

“…Antithrombin requires the following two functional domains to inhibit its target proteases in the coagulation cascade: the residue R393 or P1 at the reactive center loop 13 14 and the heparin binding domain 15 16 . To identify functional domains of antithrombin involved in enteropeptidase inhibition, two variants were tested.…”

“…Mutagenesis of plasmid containing cDNA coding for antithrombin, transfection of HEK-EBNA cells and purification of variants were carried out as previously described1315.…”

Homozygous Antithrombin Deficiency in Adolescents Presenting With Lower Extremity Thrombosis and Renal Complications

“…These polymers are retained inside the cell, and the secretion of monomers is therefore impaired, reducing antithrombotic potential. 9 There was kidney loss in the second patient. Renal involvement is not a typical presentation in inherited AT deficiency.…”

Biochemical and cellular consequences of the antithrombin p.Met1? mutation identified in a severe thrombophilic family

“…2) Mutations inducing the folding of antithrombin into hyperstable conformations (latent or polymers) might also reduce the anticoagulant capacity by impairing the function of wild-type molecules. The latent antithrombin forms dimers with native antithrombin [ 28 ] and mutations inducing the formation of antithrombin polymers incorporate wild-type monomers into the growing polymers [ 8 ]. We speculated that the cytoplasmic aberrant antithrombins generated by the p.Met1?…”

“…Alternatively, when the mutation does not significantly impair the secretion of the variant molecule but reduces or abolish its anticoagulant activity causes a qualitative deficiency that is called type II deficiency. Type II deficiencies are more frequent in the general population and usually associate with milder risk of VTE [ 7 ], although for certain mutations, the variant associates with stronger clinical severity than mutations causing type I deficiency, because the mutation may also have a dominant negative effect [ 8 ]. The characterization of families with antithrombin deficiency and very severe clinical phenotype might assist to identify new mechanisms associated with the risk of VTE.…”