Disease-causing mutations in the serpin antithrombin reveal a key domain critical for inhibiting protease activities

Abstract

Antithrombin mainly inhibits factor Xa and thrombin. The reactive center loop (RCL) is crucial for its interactions with its protease targets and is fully inserted into the A-sheet after its cleavage, causing translocation of the covalently linked protease to the opposite end of the A-sheet. Antithrombin variants with altered RCL hinge residues behave as substrates rather than inhibitors, resulting in stoichiometries of inhibition greater than one. Other antithrombin residues have been suggested to interfere with RCL insertion or the stability of the antithrombin-protease complex, but available crystal structures or mutagenesis studies have failed to identify such residues. Here, we characterized two mutations, S365L and I207T, present in individuals with type II antithrombin deficiency and identified a new antithrombin functional domain. S365L did not form stable complexes with thrombin or factor Xa, and the I207T/I207A variants inhibited both proteases with elevated stoichiometries of inhibition. Close proximity of Ile-207 and Ser-365 to the inserted RCL suggested that the preferred reaction of these mutants as protease substrates reflects an effect on the rate of the RCL insertion and protease translocation. However, both residues lie within the final docking site for the protease in the antithrombin-protease complex, supporting the idea that the enhanced substrate reactions may result from an increased dissociation of the final complexes. Our findings demonstrate that the distal end of the antithrombin A-sheet is crucial for the last steps of protease inhibition either by affecting the rate of RCL insertion or through critical interactions with proteases at the end of the A-sheet.

Keywords: antithrombin (AT); complex; factor Xa; inhibition; inhibition mechanism; kinetics; stoichiometry; thrombin.

Figure 1.

Ribbon diagram of Ile-207 and Ser-365 localization in latent antithrombin. Shown is a latent antithrombin structure (Protein Data Bank (PDB) code 2BEH) displaying the localization of Ile-207 (red spheres) and Ser-365 (green spheres) residues. The central A strands are shown in blue and the reactive center loop forming the new strand 4A in the A-sheet is displayed in yellow. All structures were rendered in PyMOL (Schrödinger, LLC). The right panel is a zoom image of the bottom of the A-sheet.

Figure 2.

Disulfide-linked dimers of S365L antithrombin variant. A, electrophoretic separation by SDS-PAGE under nonreducing conditions and Western blotting of plasma sample from a pool of healthy subjects (C) and plasma sample from the patient carrying the S365L mutation (S365L). B, electrophoretic separation by SDS-PAGE under nonreducing conditions and Western blotting of recombinant antithrombins from HEK-EBNA cells transfected with wild-type (C) and S365L mutant antithrombin plasmids (S365L). Native AT and disulfide-linked dimers are shown by arrows.

Figure 3.

Formation of covalent complexes between recombinant antithrombin and target proteases. Shown is electrophoretic separation by SDS-PAGE under nonreducing conditions and Western blotting of recombinant antithrombins produced and secreted by HEK-EBNA after 24 h of transfection. Conditioned media of cells of the wild-type antithrombin (Control) and S365L variant were incubated with proteases, thrombin (A) or factor Xa (B), in the absence and presence of its cofactor, unfractionated heparin or pentasaccharide, respectively. AT, native antithrombin: AT*, cleaved antithrombin; FIIa-AT, thrombin–antithrombin complex; FXa-AT, factor Xa–antithrombin complex. Polymers and disulfide-linked dimers are shown by arrows. The line indicates that two different parts of the same gel were groupings in the image.