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.
Highlights
The broad ranges of procoagulant serine proteases that are inhibited by antithrombin, together with its strong and efficient mechanism of inhibition, explain the embryonic lethality observed in knock-out mice and the high risk of thrombosis associated with the antithrombin heterozygous deficiency [1,2]
We studied six different antithrombin mutants detected in unrelated patients with venous or arterial thrombosis associated with antithrombin deficiency (Supplementary Table S1) from a selected investigation of self-referred families with thrombophilia
Purification of antithrombin London from plasma of carriers of ∆393 mutation by heparin affinity chromatography was facilitated by the increased heparin affinity associated with the deletion of P1 [22]
Summary
The broad ranges of procoagulant serine proteases that are inhibited by antithrombin, together with its strong and efficient mechanism of inhibition, explain the embryonic lethality observed in knock-out mice and the high risk of thrombosis associated with the antithrombin heterozygous deficiency [1,2]. As a consequence of the high energy required for keeping the native metastable conformation of serpins, which is essential for the inhibitory activity of these molecules [7], these mutations cause aberrant conformational transitions, mainly resulting in a unique hyperstable ordered protein–protein linkage (polymerization) [8,9]. These ordered polymers that are retained within the cell and result in protein overload, plasma deficiency and cell death are associated with a RESEARCH ARTICLE variety of diseases, called serpinopathies [10]
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