An immunochemical method for quantitative determination of latent antithrombin, the reactive center loop-inserted uncleaved form of antithrombin

Abstract

Antithrombin (AT) is a serine protease inhibitor that has thrombin, factors IXa and Xa as target proteases. In addition to active native AT, two other forms have been identified in plasma: the reactive center loop inserted cleaved and latent, uncleaved forms. Both have been shown to be present in normal human blood. Latent AT forms a dimer with native AT in vitro, thus inactivating the native form. Here we describe a mouse monoclonal antibody, 8C8, that is specific for latent AT. The affinity of 8C8 was found to be 500-fold higher for latent than for native AT and 5000-fold higher for latent than for cleaved AT. A sandwich assay was developed to measure the concentration of latent AT in plasma, which was found to be ∼4.8 mg L-1 in healthy individuals. The KD of the interaction between native and latent AT was found to be 51 μm, i.e. far above the plasma concentration of both native and latent AT, indicating a negligible complex formation in blood. © 2007 International Society on Thrombosis and Haemostasis.