10 Aptamers for Thrombotic Diseases
283
repair, and inflammation. Most of the thrombin antagonists targeting thrombin’s
active site are currently developed as antithrombotic agents [9].
Exosites I and II are two different regions on the surface of thrombin. They
mediate specific binding of substrates, receptors, and cofactors so as to provide
specificity to its proteolytic activity. Exosite I mainly mediates binding of fibrinogen
cleavage, proteolytically activated receptors, and some coagulation factors including
FXI, cofactors V and VIII, and thrombomodulin [22]. Exosite II is the binding site
for GP Ib-α-chain of platelet GPIb/IX. The activation of FV and FVIII mediates
the binding between heparin and platelet receptor GPIb-IX thus to activate platelets
[9, 23].
Currently, UFH is the only antidote-reversible anticoagulant with a wide-scale
use in thrombosis, and heparin is the only anticoagulant of immediate anticoagulation in PCI. Heparin, a glycosaminoglycan anticoagulant, inhibits coagulation
FX, and other coagulation factors to prevent clot formation through binding to
antithrombase-III (AT-III). However, heparin-protamine antithrombotic pair has a
number of shortcomings as follows. Firstly, aside from bleeding risk, unpredictable
pharmacokinetics of heparin makes regular APTT monitoring necessary for adjusting
dosing [24]. Secondly, the antigenic heparin–Platelet Factor 4 (PF4) complex may
elicit antibody-induced life-threatening complications, known as HIT or heparininduced thrombocytopenia with thrombosis (HITT), resulting in platelet consumption in patients undergoing CPB [24, 25]. Thirdly, administration of protamine is
associated with serious cardiovascular side effects such as systemic hypotension,
pulmonary hypertension, myocardial suppression, and anaphylaxis [24].
In 1992, Toole and colleagues reported the first thrombin-specific aptamer with
inhibitory activity [26]. For two decades, anti-thrombin aptamers and their various
analogs are the most intensively studied aptamers as both diagnostic reagents and
therapeutic agents.
10.2.1 Anti-thrombin DNA Aptamers
10.2.1.1 Thrombin-Binding Aptamer (TBA)
In 1992, Bock et al. reported that they isolated ssDNA thrombin-binding aptamers
(TBA), with binding affinities and a dissociation constant (K d ) in the range of 25–
200 nM. A highly conserved 14–17-base sequence region was identified from 32
anti-thrombin aptamers selected from a large combinatorial DNA library with 60
nucleotides of random sequence by the SELEX technique. In vitro experiments
revealed that several aptamers inhibited thrombin-catalyzed fibrin clot formation
at nM concentrations using purified fibrinogen or human plasma [26].
Later a minimal 15-mer DNA consensus sequence of human protease α-thrombinbinding DNA aptamer (5
-GGTTGGTGTGGTTGG-3
) was identified. The solution
structure of TBA is a folded antiparallel chair-like G-quadruplex structure containing
two stacked G-tetrads encircled by a T
7 G
8 T
9 loop on one side and two TT (T
3 T
4 ,
283
repair, and inflammation. Most of the thrombin antagonists targeting thrombin’s
active site are currently developed as antithrombotic agents [9].
Exosites I and II are two different regions on the surface of thrombin. They
mediate specific binding of substrates, receptors, and cofactors so as to provide
specificity to its proteolytic activity. Exosite I mainly mediates binding of fibrinogen
cleavage, proteolytically activated receptors, and some coagulation factors including
FXI, cofactors V and VIII, and thrombomodulin [22]. Exosite II is the binding site
for GP Ib-α-chain of platelet GPIb/IX. The activation of FV and FVIII mediates
the binding between heparin and platelet receptor GPIb-IX thus to activate platelets
[9, 23].
Currently, UFH is the only antidote-reversible anticoagulant with a wide-scale
use in thrombosis, and heparin is the only anticoagulant of immediate anticoagulation in PCI. Heparin, a glycosaminoglycan anticoagulant, inhibits coagulation
FX, and other coagulation factors to prevent clot formation through binding to
antithrombase-III (AT-III). However, heparin-protamine antithrombotic pair has a
number of shortcomings as follows. Firstly, aside from bleeding risk, unpredictable
pharmacokinetics of heparin makes regular APTT monitoring necessary for adjusting
dosing [24]. Secondly, the antigenic heparin–Platelet Factor 4 (PF4) complex may
elicit antibody-induced life-threatening complications, known as HIT or heparininduced thrombocytopenia with thrombosis (HITT), resulting in platelet consumption in patients undergoing CPB [24, 25]. Thirdly, administration of protamine is
associated with serious cardiovascular side effects such as systemic hypotension,
pulmonary hypertension, myocardial suppression, and anaphylaxis [24].
In 1992, Toole and colleagues reported the first thrombin-specific aptamer with
inhibitory activity [26]. For two decades, anti-thrombin aptamers and their various
analogs are the most intensively studied aptamers as both diagnostic reagents and
therapeutic agents.
10.2.1 Anti-thrombin DNA Aptamers
10.2.1.1 Thrombin-Binding Aptamer (TBA)
In 1992, Bock et al. reported that they isolated ssDNA thrombin-binding aptamers
(TBA), with binding affinities and a dissociation constant (K d ) in the range of 25–
200 nM. A highly conserved 14–17-base sequence region was identified from 32
anti-thrombin aptamers selected from a large combinatorial DNA library with 60
nucleotides of random sequence by the SELEX technique. In vitro experiments
revealed that several aptamers inhibited thrombin-catalyzed fibrin clot formation
at nM concentrations using purified fibrinogen or human plasma [26].
Later a minimal 15-mer DNA consensus sequence of human protease α-thrombinbinding DNA aptamer (5
-GGTTGGTGTGGTTGG-3
) was identified. The solution
structure of TBA is a folded antiparallel chair-like G-quadruplex structure containing
two stacked G-tetrads encircled by a T
7 G
8 T
9 loop on one side and two TT (T
3 T
4 ,