that in the potassium case. This reduces the affinity and functionality of TBA. Similar situation can be found in the case of mTBA. There are no interactions between mTBA and His71, which results in the reduction of anticoagulant activity. The results of In silico calculations with molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method, suggest that the calculated binding energy (ΔG) of TBA to thrombin exosite I is slightly stronger is the presence of K+ (-66.73 kcal.mol-1) than in the case of Na+ (-60.29kcal.mol-1), however both states are likely to coexist. Therapeutic applications It has been demonstrated that TBA can inhibit the thrombin-induced platelet aggregation and clot-bound thrombin activity. The IC50 of TBA for the inhibition of platelet aggregation (0.5 U/mL thrombin) is around 70 to 80 nmol/L, which is much lower than that of hirudin (~1.7 umol/L). Also, compared with heparin, TBA is more efficient in the inhibition of clot-bound thrombin. Furthermore, TBA recognizes and inhibits prothrombin with similar affinity against alpha-thrombin. As a result, TBA prolongs the prothrombin time when interacting with prothrombin. TBA entered the phase I clinical trial for coronary artery bypass graft surgery by Archemix and Nuvelo (now ARCA Biopharma) around 2005. Although it showed a rapid onset response with desired anticoagulation activity, the activity requires significantly high dosage of TBA. Thus, the companies redesigned the sequence of TBA and developed a second-generation 26-mer DNA aptamer known as NU172, which is now under phase II clinical trial. Aptamer HD22 (the exosite II-binding aptamer) The aptamer HD22 (also known as HTDQ) is an optimized aptamer with 29 (5-AGTCCGTGGTAGGGCAGGTTGGGGTGACT-3) or 27 (lacking the first and the last nucleotides of 29-mer form) nucleotides. This aptamer recognizes the exosite II of thrombin, which is involved in the activation of factor V and factor VIII and mediates the heparin binding. Therefore, HD22 inhibits the activations of factors V/VIII rather than that of fibrinogen. Despite that this aptamer only shows moderate effect on fibrinogen regulation, the affinity of this aptamer is slightly higher than TBA (KD~0.5 nM), and nowadays this aptamer is widely used for developments of aptamer sensor. HD22 structure Unlike TBA, HD22 holds a duplex/G-quadruplex mixed structure. The X-ray crystallographic image of HD22 (27mer form) with 2.4 Å resolution was reported recently (4I7Y). The nucleotides 1-3 and 25-27 with an additional C4-G23 form a duplex motif, and the sequence ranging from G5 to G20 folds into a G-quadruplex structure with