An acyclic pyrimidine analogue, containing a five-member cycle fused on the pyrimidine ring, was synthesized and introduced at position 7 or 12 of the 15-mer oligodeoxynucleotide GGTTGGTGTGGTTGG, known as thrombin-binding aptamer (TBA). Characterization by 1H NMR and CD spectroscopies of the resulting aptamers, TBA-T7b and TBA-T12b, showed their ability to fold into the typical antiparallel chairlike G-quadruplex structure formed by TBA. The apparent CD melting temperatures indicated that the introduction of the acyclic residue, mainly at position 7, improves the thermal stability of resulting G-quadruplexes with respect to TBA. The anticoagulant activity of the new molecules was then valued in PT assay, and it resulted that TBA-T7b is more potent than TBA in prolonging clotting time. On the other hand, in purified fibrinogen assay the thrombin inhibitory activity of both modified sequences was lower than that of TBA using human enzyme, whereas the potency trend was again reversed using bovine enzyme. Obtained structure-activity relationships were investigated by structural and computational studies. Taken together, these results reveal the active role of TBA residues T7 and T12 and the relevance of some amino acids located in the anion binding exosite I of the protein in aptamer-thrombin interaction.