High-affinity pentasaccharide forms a complex with cleaved antithrombin. (A) Fluorescence titrations of 0.5 μM native (▴ and ▵) or cleaved (• and ○) antithrombins with natural pentasaccharide (H5) (▴ and •) or the high-affinity pentasaccharide (H5*) (▵ and ○) in the presence of 10 μM TNS monitored by TNS fluorescence changes. Fluorescence changes are expressed relative to the maximal fluorescence change at saturation obtained from nonlinear regression fits to the equilibrium binding equation (solid lines). (B) Samples of 6 μM native or cleaved antithrombin in the absence or presence of 12 μM full-length heparin (H50), natural pentasaccharide (H5), or high-affinity pentasaccharide (H5*) were subjected to 10% native PAGE and stained with Coomassie blue dye.