284
W. Li et al.
T
12 T
l3 ) loops on the opposite side as determined by nuclear magnetic resonance
(NMR) and X-ray crystallography analysis. Padmanabhang et al. further revealed that
the ssDNA aptamers are sandwiched in the aptamer-thrombin complex between two
different positively charged regions, with exosites I and II of two symmetry-related
thrombin molecules in the crystal structure, which provides ionic and hydrophobic
interactions [27, 28].
In the preclinical studies, a short-acting TBA was administrated in a canine CPB
model to determine its anticoagulant efficacy and the potential to be an alternative
therapeutic of heparin in CPB and other clinical settings was evaluated [29]. The
results showed that the aptamer exhibited a rapid anti-coagulation effect with safe
and predictable pharmacokinetics, indicating its potential as an alternative to heparin
[30].
Li et al. applied 15-mer DNA TBA to block clot-bound thrombin activity and
platelet thrombus formation ex vivo in a whole artery angioplasty model. The aptamer
was found to inhibit thrombin-induced platelets aggregation in a dose-dependently
manner in human platelet-rich plasma, with an IC 50 of about 70–80 nmol/L. In
vitro clot-bound thrombin assay showed that the thrombin aptamer inhibited clotbound thrombin at a clinically relevant anticoagulant concentration (0.2 U/mL
and 0.4 U/mL) as compared with heparin. The anti-thrombin aptamer significantly
suppressed fibrinopeptide A (FPA) generation in the whole artery angioplasty model.
In contrast, heparin at a concentration of 2 U/ml was ineffective.In addition, the
aptamer reduced platelet deposition by 34.5% ± 5% at low shear rates and 61.3% ±
11% at high shear rates compared with a scrambled ssDNA control [31].
Based on the C15-mer thrombin aptamer, Mendelboum et al. synthesized three
different modified thrombin aptamers with the replacement of 4-thio-deoxyuridylates
at positions 3, 7, 9, and 13 of thymidylate residues. Then they examined the
effects of these thrombin aptamer analogs on the hemostatic/thrombotic activities of thrombin. As compared with the original C15-mer, the analog with
the sequence GG(s4dU)TGG(s4dU)G(s4dU)GGT(s4dU)GG(UC15-mer) showed a
twofold greater inhibition on thrombin-catalyzed fibrin clot formation, FPA release,
platelet aggregation, and secretion in human plasma and thrombus formation on
thrombin-treated fibrinogen surfaces under flow conditions. UC15-mer aptamer was
threefold and twelve-fold more effective than C15-mer on thrombin-induced fibrin
formation from purified fibrinogen and activation of washed platelets, respectively.
Except for more stability in a biological environment, the result showed that replacement of the thymidylate residues of C15-mer aptamer increased anticoagulant and
antithrombotic activity, indicating the potential application of this new anti-thrombin
aptamer as an antithrombotic agent [32].
Pica et al. conducted a crystallographic characterization of the complexes between
thrombin and two TBA mutants, TBAT3, and TBAT12, which lack a thymine
nucleobase and the human enzyme to evaluate the thrombin-TBA interaction. Structural analysis showed that exosite I of the two complexes is actually splitted into two
regions, which contribute differently to TBA recognition. This study provides the
W. Li et al.
T
12 T
l3 ) loops on the opposite side as determined by nuclear magnetic resonance
(NMR) and X-ray crystallography analysis. Padmanabhang et al. further revealed that
the ssDNA aptamers are sandwiched in the aptamer-thrombin complex between two
different positively charged regions, with exosites I and II of two symmetry-related
thrombin molecules in the crystal structure, which provides ionic and hydrophobic
interactions [27, 28].
In the preclinical studies, a short-acting TBA was administrated in a canine CPB
model to determine its anticoagulant efficacy and the potential to be an alternative
therapeutic of heparin in CPB and other clinical settings was evaluated [29]. The
results showed that the aptamer exhibited a rapid anti-coagulation effect with safe
and predictable pharmacokinetics, indicating its potential as an alternative to heparin
[30].
Li et al. applied 15-mer DNA TBA to block clot-bound thrombin activity and
platelet thrombus formation ex vivo in a whole artery angioplasty model. The aptamer
was found to inhibit thrombin-induced platelets aggregation in a dose-dependently
manner in human platelet-rich plasma, with an IC 50 of about 70–80 nmol/L. In
vitro clot-bound thrombin assay showed that the thrombin aptamer inhibited clotbound thrombin at a clinically relevant anticoagulant concentration (0.2 U/mL
and 0.4 U/mL) as compared with heparin. The anti-thrombin aptamer significantly
suppressed fibrinopeptide A (FPA) generation in the whole artery angioplasty model.
In contrast, heparin at a concentration of 2 U/ml was ineffective.In addition, the
aptamer reduced platelet deposition by 34.5% ± 5% at low shear rates and 61.3% ±
11% at high shear rates compared with a scrambled ssDNA control [31].
Based on the C15-mer thrombin aptamer, Mendelboum et al. synthesized three
different modified thrombin aptamers with the replacement of 4-thio-deoxyuridylates
at positions 3, 7, 9, and 13 of thymidylate residues. Then they examined the
effects of these thrombin aptamer analogs on the hemostatic/thrombotic activities of thrombin. As compared with the original C15-mer, the analog with
the sequence GG(s4dU)TGG(s4dU)G(s4dU)GGT(s4dU)GG(UC15-mer) showed a
twofold greater inhibition on thrombin-catalyzed fibrin clot formation, FPA release,
platelet aggregation, and secretion in human plasma and thrombus formation on
thrombin-treated fibrinogen surfaces under flow conditions. UC15-mer aptamer was
threefold and twelve-fold more effective than C15-mer on thrombin-induced fibrin
formation from purified fibrinogen and activation of washed platelets, respectively.
Except for more stability in a biological environment, the result showed that replacement of the thymidylate residues of C15-mer aptamer increased anticoagulant and
antithrombotic activity, indicating the potential application of this new anti-thrombin
aptamer as an antithrombotic agent [32].
Pica et al. conducted a crystallographic characterization of the complexes between
thrombin and two TBA mutants, TBAT3, and TBAT12, which lack a thymine
nucleobase and the human enzyme to evaluate the thrombin-TBA interaction. Structural analysis showed that exosite I of the two complexes is actually splitted into two
regions, which contribute differently to TBA recognition. This study provides the