Isolation and characterization of an antithrombin III variant with reduced carbohydrate content and enhanced heparin binding
Abstract
Two distinct forms of antithrombin II were isolated by chromatography of normal human plasma on heparin-Sepharose. The predominant antithrombin species present (AT-IIIα), which eluted from the affinity column in 1 M NaCl, was identified as the antithrombin III form which has been previously characterized. Ionic strength of the buffer was increased to elute a variant form of antithrombin III, designated as AT-IIIβ. The molecular weight of AT-IIIβ is less than that of AT-IIIα, but physicochemical studies do not indicate measurable differences in the polypeptide portion of the proteins. Carbohydrate determination revealed the sole detectable structural difference in the two anti-thrombins: levels of hexosamine, neutral sugars, and sialic acid in AT-IIIβ were all 25-30% less than in AT-IIIα. Kinetic studies of thrombin inactivation by both antithrombins, in the presence of nonsaturating amounts of heparin, indicated that AT-IIIβ inhibited thrombin more rapidly. AT-IIIβ is also distinguishable from AT-IIIα on the basis of heparin-binding affinity estimated from titration of protein fluorescence with heparin. Thus, antithrombin III exists as two molecular entities in human plasma which differ both structurally, in carbohydrate content, and functionally, in their heparin-binding behavior.