Isoform composition of antithrombin in a covalent antithrombin–heparin complex

Abstract

Antithrombin (AT) circulates in two isoforms, α- (90–95%) and β-AT (5–10%). AT inhibits clotting factors such as thrombin and factor Xa, a reaction catalyzed by heparin. Heparin has been used in many clinical situations but suffers from limitations such as a short intravenous half-life, bleeding risk, and the inability to inhibit thrombin bound to fibrin clots. In order to overcome some of heparin’s limitations, we prepared a covalent AT–heparin complex (ATH) that has increased intravenous half-life, reduced bleeding risk, and can directly inhibit clot-bound thrombin. However, structural analysis is required to further develop this promising antithrombotic agent. It was found that the proportion of isoforms in ATH (55% α-AT, and 45% β-AT) was significantly different than that in the commercial AT starting material (80% α-AT and 20% β-AT). Further analysis of the rate of heparin-catalyzed inhibition of thrombin by AT isoforms prepared from ATH revealed that the β-variant reacted ∼2-fold faster.