Multiple covalent forms of Antithrombin: implications in thrombosis

Abstract

Antithrombin is a key anticoagulant protein that regulates the proteolytic activity of several procoagulant proteases, and its deficiency is associated with pathological thrombosis. Our study of coagulation proteins has revealed that they are often constitutively produced as multiple partially disulphide-bonded states that can be important for protein function. Antithrombin contains three disulphide bonds and their state and function in the mature protein was investigated. Disulphide-bonded states of plasma antithrombin from healthy human donors were quantified using differential cysteine alkylation and mass spectrometry. The C21-C95, C247-C430, and C8-C128 disulphide bonds are unformed in ~1 in 3, ~1 in 5, and ~1 in 10 antithrombin molecules, respectively. Heparin selectively binds a subset of antithrombin molecules with significantly more unformed disulphides and greater conformational flexibility measured by intrinsic protein fluorescence. Force spectroscopy studies revealed enhanced heparin-antithrombin bond lifetimes for the heparin-selected subset. The functions of the most prevalent antithrombin states, containing an unpaired C21-C95 or C247-C430 disulphide bond, were investigated by replacing both cysteines of the disulphide with alanine or valine. Ablation of the bonds affected uncatalysed and heparin-catalysed inhibition of factor Xa and thrombin, rate of conversion to the latent antithrombin state, structural flexibility, hydrodynamic radius and molecular dynamics. These findings indicate that antithrombin circulates in different disulphide-bonded states and a subset of molecules with more unformed disulphides, greater conformational flexibility, and subtly different functional properties selectively binds to heparin.