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W. Li et al.
10.2.1.3 HD1, HD22, and HD1-22
HD1 and HD22 are two distinct aptamers targeting exosites I and II of the human
thrombin, respectively, with anticoagulant potential.
HD1 is a 15-mer minimal motif of antithrombin aptamer. It is one of the most
intensively studied aptamers, initially reported in 1992 [26]. Apart from clinical trials
as an anticoagulant, HD1 has been intensively used for structural and diagnostic
investigations due to its unique G-quadruplex structure. NMR, circular dichroism
(CD) spectroscopy, native polyacrylamide gel electrophoresis, molecular dynamic
simulation, and crystallography have been employed to illustrate the G-quadruplex
conformation and the structural basis for HD1-thrombin interaction. The results
showed that HD1 folds into a quadruplex with an antiparallel orientation of the
strands in a chair-like conformation. HD1 interacted with exosite I of thrombin
and inhibited conversion of fibrinogen to fibrin and platelet activation [38]. As an
anticoagulant agent, a phase 1 clinical trial of HD1 (Archemix and Nuvelo) during
bypass surgery was conducted but failed to show its therapeutic efficacy [39]. In
addition, HD1 was found to recognize the precursor of thrombin, prothrombin, with
approximate binding affinity and inhibitory activation [40].
HD22 is another 29-mer thrombin-binding DNA aptamer containing a Gquadruplex structure with a substitution of three bases outside of the conserved
two G-quartets targeting exosite II of thrombin [23].
HD1-22 is a bivalent 61-mer anti-thrombin aptamer containing two G-quadruplex
structures made by connecting HD1 and HD22 through a poly-nucleotidic linker.
Müller and his group [41] revealed that HD1-22 bound thrombin with high affinity
(K d = 0.65 nM) and took up both exosite I and exosite II without blocking the enzyme
active centre. Surface plasmon resonance (SPR) showed that the affinity of bivalent
aptamer HD1-22 to human α-thrombin was significantly improved compared to the
monovalent aptamers HD1 and HD22 (Fig. 10.2).
A series of clotting experiments demonstrated that the anticoagulant activity
of HD1-22 was more effective than argatroban and as effective as bivalirudin.
Meanwhile, HD1-22 was obviously more effective than argatroban and also had
a slightly stronger inhibition on thrombin-induced platelet aggregation compared
with bivalirudin. Thus, the authors suggested that HD1-22 may be an ideal candidate anticoagulant agent when both effective anticoagulation and rapid reversal of
anticoagulant effect is clinically required [41, 42].
In another study, an inverted deoxythymidine nucleotide at the 3
-end position was introduced into the two aptamers to increase nuclease resistance and
improve the pharmacokinetic properties. Then bivalent anti-thrombin aptamers were
constructed by connecting the HD1 and HD22 either through a triethylene glycol
(TEG) linkage chemistry or four consecutive deoxythymidines together with an
inverted deoxythymidine nucleotide at the 3
-end position to increase nuclease
resistance. Thrombin clotting time (TCT) assay, nuclease degradation assay, and
human serum degradation assay showed that bivalent chimeric aptamer RNV220
had significantly greater anticoagulant activity in blood plasma and greater nuclease
stability compared to the original aptamers HD1 and HD22. Furthermore, a bivalent
W. Li et al.
10.2.1.3 HD1, HD22, and HD1-22
HD1 and HD22 are two distinct aptamers targeting exosites I and II of the human
thrombin, respectively, with anticoagulant potential.
HD1 is a 15-mer minimal motif of antithrombin aptamer. It is one of the most
intensively studied aptamers, initially reported in 1992 [26]. Apart from clinical trials
as an anticoagulant, HD1 has been intensively used for structural and diagnostic
investigations due to its unique G-quadruplex structure. NMR, circular dichroism
(CD) spectroscopy, native polyacrylamide gel electrophoresis, molecular dynamic
simulation, and crystallography have been employed to illustrate the G-quadruplex
conformation and the structural basis for HD1-thrombin interaction. The results
showed that HD1 folds into a quadruplex with an antiparallel orientation of the
strands in a chair-like conformation. HD1 interacted with exosite I of thrombin
and inhibited conversion of fibrinogen to fibrin and platelet activation [38]. As an
anticoagulant agent, a phase 1 clinical trial of HD1 (Archemix and Nuvelo) during
bypass surgery was conducted but failed to show its therapeutic efficacy [39]. In
addition, HD1 was found to recognize the precursor of thrombin, prothrombin, with
approximate binding affinity and inhibitory activation [40].
HD22 is another 29-mer thrombin-binding DNA aptamer containing a Gquadruplex structure with a substitution of three bases outside of the conserved
two G-quartets targeting exosite II of thrombin [23].
HD1-22 is a bivalent 61-mer anti-thrombin aptamer containing two G-quadruplex
structures made by connecting HD1 and HD22 through a poly-nucleotidic linker.
Müller and his group [41] revealed that HD1-22 bound thrombin with high affinity
(K d = 0.65 nM) and took up both exosite I and exosite II without blocking the enzyme
active centre. Surface plasmon resonance (SPR) showed that the affinity of bivalent
aptamer HD1-22 to human α-thrombin was significantly improved compared to the
monovalent aptamers HD1 and HD22 (Fig. 10.2).
A series of clotting experiments demonstrated that the anticoagulant activity
of HD1-22 was more effective than argatroban and as effective as bivalirudin.
Meanwhile, HD1-22 was obviously more effective than argatroban and also had
a slightly stronger inhibition on thrombin-induced platelet aggregation compared
with bivalirudin. Thus, the authors suggested that HD1-22 may be an ideal candidate anticoagulant agent when both effective anticoagulation and rapid reversal of
anticoagulant effect is clinically required [41, 42].
In another study, an inverted deoxythymidine nucleotide at the 3
-end position was introduced into the two aptamers to increase nuclease resistance and
improve the pharmacokinetic properties. Then bivalent anti-thrombin aptamers were
constructed by connecting the HD1 and HD22 either through a triethylene glycol
(TEG) linkage chemistry or four consecutive deoxythymidines together with an
inverted deoxythymidine nucleotide at the 3
-end position to increase nuclease
resistance. Thrombin clotting time (TCT) assay, nuclease degradation assay, and
human serum degradation assay showed that bivalent chimeric aptamer RNV220
had significantly greater anticoagulant activity in blood plasma and greater nuclease
stability compared to the original aptamers HD1 and HD22. Furthermore, a bivalent