(A) (I) Native-PAGE of aliquots of fractions 1, 2, and 3 (Fig 2B) from 0- and 72-hour incubations of plasma at 37°C. Bands with the characteristic fast mobility of both the latent and cleaved forms of antithrombin are seen faintly in the 0-hour sample and strongly in the 72-hour sample. (II) Western blot of SDS-PAGE of the eluted band 1 and an intact normal control () shows the predominant presence of intact antithrombin in the 72-hour sample with the presence of some cleaved (Cld) antithrombin at 0 and 72 hours. The combined results (I) and (II), together with the absence of inhibitory activity, confirm the identity of the latent component. (B) Western immunostaining of a native PAGE of plasma shows that normal plasma (P) has a major band that aligns with -antithrombin standards (). The addition of latent antithrombin (L) in near equimolar proportions to the control plasma (P + L) fails to show the presence of the free latent band, but results in a quantitative shift of the major band from the  position to the characteristic slower mobility of the /latent heterodimer. (C) Commercial antithrombin concentrate. (I) SDS-PAGE confirms that the concentrate (conc) described in Chang and Harper12 has a single major component with the molecular weight of intact antithrombin. (II) Native PAGE illustrates how the electrophoretic mobility of the concentrate could be readily mistaken for that of active -antithrombin; alignment with an  + L standard (with excess ) confirms that the concentrate is predominantly /latent dimer. (D) Native PAGE showing addition of increasing amounts of β-antithrombin (to 4.3 μg) to preformed -dimer (3 μg latent:3 μg , with incubation for 5 minutes) to give complete displacement of the -antithrombin with formation of the β-dimer (βd). The position of the -dimer (d) is indicated by the dotted arrow.