Detection of conformational transformation of antithrombin in blood with crossed immunoelectrophoresis: new application for a classical method

Abstract

The structural flexibility of antithrombin is essential for its molecular trapping mechanism but also makes it vulnerable to even minor changes affecting its conformational stability, which influences hemostasis significantly. The conformational transformation of this serpin has been poorly investigated in biologic samples because available immunologic methods hardly differentiate between different conformations of this protein. Crossed immunoelectrophoresis (CIE) in presence of heparin has been classically used to identify mutant antithrombins with low heparin affinity. We demonstrate that this method also separates native and relaxed antithrombin, permitting the analysis of conformational variations of this potent anticoagulant with just a few microliters of plasma. However, CIE does not distinguish between antithrombin conformations with reduced heparin affinity: latent, cleaved, thrombin-antithrombin complexes, or heparin-binding mutants. Therefore, clinical interpretation of CIE results should be examined with caution. Using this and other methods, and evaluating the functional activity of antithrombin, we analyzed the conformational transformation of antithrombin in biologic samples. We confirmed its transformation to the latent configuration by incubating it at 50°C. This conformational change also occurs at 37°C, supporting the idea that this process is involved in the senescence of antithrombin. However, fresh plasma contains only traces of latent antithrombin, suggesting that this conformation is rapidly cleared in vivo. Finally, small increases in temperature (to 40°C) resulted in a faster conformational transformation of antithrombin. Fever has been suggested to have key structural, functional, and clinical consequences in patients with conformational mutations in antithrombin. Our results support a role for small changes in temperature in nonmutated antithrombin, suggesting that fever is a general risk factor for thrombosis.