Thrombin is a major component of blood clotting and involved in the formation of a fibrin clot. One of the precursors during thrombin maturation is prethrombin-2, with the presence of Arg363-Ile364 bond between the light and heavy chain of protein, the only distinction from thrombin. Prethrombin-2 is able to interact with less efficiency with a 15-mer thrombin-binding aptamer (TBA). We investigate the interaction of both known conformers of TBA with thrombin and prethrombin-2 by simulation of molecular dynamics. It was shown that TBA could interact with thrombin in both conformations with similar efficiency, although a stable complex of prethrombin-2 with TBA was found only in conformation identical with the aptamer structure, pdb 1HAO. Analysis of molecular dynamics of complexes offered an assumption that the motion of the exosite-1 forming loop Lys428-Ile438 determined the difference in affinity of the complexes of TBA with thrombin and prethrombin-2.