Recently we designed the first small organic ligands, sulfated flavanoids and flavonoids, that act as activators of antithrombin for accelerated inhibition of factor Xa, a key proteinase of the coagulation cascade [Gunnarsson and Desai, Bioorg. Med. Chem. Lett. (2003) 13:579]. To better understand the binding properties of these activators at a molecular level, we have utilized computerized hydropathic interaction (HINT) analyses of the sulfated molecules interacting in two plausible electropositive regions, the pentasaccharide- and extended heparin-binding sites, of antithrombin in its native and activated forms. HINT analyses indicate favorable multi-point interactions of the activators in both binding sites of the two forms of antithrombin. Yet, HINT predicts better interaction of most activators, except for (-)-catechin sulfate, with the activated form of antithrombin than with the native form supporting the observation in solution that these molecules function as activators of the inhibitor. Further, whereas (+)-catechin sulfate recognized the activated form of antithrombin better in both the pentasaccharide- and extended heparin- binding sites, the native form was better recognized by (-)-catechin sulfate, thus explaining its weaker binding and activation potential in solution. A reasonable linear correlation between the overall HINT score and the solution free energy of binding of the sulfated activators was evident. This investigation indicates that HINT is a useful tool in understanding interactions of antithrombin with small sulfated organic ligands at a molecular level, has some good predictive properties, and is likely to be useful for rational design purposes.