10 Aptamers for Thrombotic Diseases
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of thrombin were associated with platelet activation. The authors suggested that
aptamers targeting exosite I and exosite II may be a particularly effective approach
to prevent thrombosis in a variety of clinical settings due to the additive inhibitory
effects [59].
In short, the conversion of prothrombin to thrombin in the only activation step that
is not duplicated toward thrombin formation, suggesting it is an important drug target.
In total, several DNA and RNA aptamers targeting exosite I (HD1, ARC183, RE31)
and exosite II (HD22, NU172) or both exosites (HD1-22) of thrombin have been
screened for therapeutics. These aptamers are usually used as thrombin inhibitors,
which can compete for thrombin exosite binding substrates such as fibrinogen and
platelet receptors. Among them, HD1 and NU172 have finished phase 1 and phase
2 clinical trials, respectively, for CABG surgery. Additionally, thrombin is associated with the formation of atherosclerotic plaque, injury repair, and inflammation,
therefore these thrombin aptamers are promising agents for these diseases [16].
10.3 Aptamers Against FVII
TF is on the outside of blood vessels. After vessel injury, it is exposed to the bloodstream and circulating FVII. FVII is activated to form FVIIa by different proteases
after binding to TF and in conjunction with TF to form TF/FVIIa complex, which
initiates the process of the coagulation cascade. Then FVIIa-TF complexes convert
the blood coagulation FIX and X into FIXa and FXa, respectively [60]. So FVII
activation and FVIIa-TF complexes formation are the initiating steps in thrombin
generation.
Coagulation FVII is a plasma serine protease with a significant role in natural
human hemostasis, and recombinant FVII such as AryoSeven™ has been applied
in the clinical treatment of bleeding disorders. The possible therapeutic mechanism
of rFVIIa is the production of a “thrombin burst” via activation of FIX, X, and II
on the surface of activated platelets to accelerate clot formation. When congenital
factor deficiency is absent, rFVIIa may produce excess thrombin resulting in fatal
thromboembolic complications in more than 27% of patients [61, 62].
Layzer et al. identified an aptamer to FVII with inhibitory activity in vitro at
a relatively low concentration [63]. To generate new inhibitors of TF/factor VIIa
activity, a combinatorial RNA library and in vitro selection was used to generate high
affinity, nuclease-resistant RNA aptamers targeting coagulation FVII/VIIa. After 16
rounds of selection, RNA aptamer to FVIIa was obtained and two similar motifs,
AGARrGA (R: purine, r: an additional purine) and AUCCCUNG (N: any base substitution) were identified. The highest K d of RNA 16.3 aptamer was 11.3 nM. The
kinetic analyses experiments revealed that RNA aptamers inhibited FVIIa activity
mainly by preventing a functional TF/FVII complex formation, and thus inhibited
TF-dependent activation of FX. RNA aptamer reduced FX activation by approximately 95% at 1 μM. The PT assay showed that 10 μM RNA aptamer prolonged the
clotting time of human plasma by 175% [64]. Furthermore, the results showed that
293
of thrombin were associated with platelet activation. The authors suggested that
aptamers targeting exosite I and exosite II may be a particularly effective approach
to prevent thrombosis in a variety of clinical settings due to the additive inhibitory
effects [59].
In short, the conversion of prothrombin to thrombin in the only activation step that
is not duplicated toward thrombin formation, suggesting it is an important drug target.
In total, several DNA and RNA aptamers targeting exosite I (HD1, ARC183, RE31)
and exosite II (HD22, NU172) or both exosites (HD1-22) of thrombin have been
screened for therapeutics. These aptamers are usually used as thrombin inhibitors,
which can compete for thrombin exosite binding substrates such as fibrinogen and
platelet receptors. Among them, HD1 and NU172 have finished phase 1 and phase
2 clinical trials, respectively, for CABG surgery. Additionally, thrombin is associated with the formation of atherosclerotic plaque, injury repair, and inflammation,
therefore these thrombin aptamers are promising agents for these diseases [16].
10.3 Aptamers Against FVII
TF is on the outside of blood vessels. After vessel injury, it is exposed to the bloodstream and circulating FVII. FVII is activated to form FVIIa by different proteases
after binding to TF and in conjunction with TF to form TF/FVIIa complex, which
initiates the process of the coagulation cascade. Then FVIIa-TF complexes convert
the blood coagulation FIX and X into FIXa and FXa, respectively [60]. So FVII
activation and FVIIa-TF complexes formation are the initiating steps in thrombin
generation.
Coagulation FVII is a plasma serine protease with a significant role in natural
human hemostasis, and recombinant FVII such as AryoSeven™ has been applied
in the clinical treatment of bleeding disorders. The possible therapeutic mechanism
of rFVIIa is the production of a “thrombin burst” via activation of FIX, X, and II
on the surface of activated platelets to accelerate clot formation. When congenital
factor deficiency is absent, rFVIIa may produce excess thrombin resulting in fatal
thromboembolic complications in more than 27% of patients [61, 62].
Layzer et al. identified an aptamer to FVII with inhibitory activity in vitro at
a relatively low concentration [63]. To generate new inhibitors of TF/factor VIIa
activity, a combinatorial RNA library and in vitro selection was used to generate high
affinity, nuclease-resistant RNA aptamers targeting coagulation FVII/VIIa. After 16
rounds of selection, RNA aptamer to FVIIa was obtained and two similar motifs,
AGARrGA (R: purine, r: an additional purine) and AUCCCUNG (N: any base substitution) were identified. The highest K d of RNA 16.3 aptamer was 11.3 nM. The
kinetic analyses experiments revealed that RNA aptamers inhibited FVIIa activity
mainly by preventing a functional TF/FVII complex formation, and thus inhibited
TF-dependent activation of FX. RNA aptamer reduced FX activation by approximately 95% at 1 μM. The PT assay showed that 10 μM RNA aptamer prolonged the
clotting time of human plasma by 175% [64]. Furthermore, the results showed that