300
W. Li et al.
bivalent aptamers (RNA BA 1-4) that could inhibit FX/Xa, prothrombin/thrombin, and
anticoagulate plasma the same as the combination of individual aptamers. Detailed
characterization of these bivalent aptamers indicated that each aptamer retained all
the binding and functional activity. Finally, the reversal experiments revealed that
the anticoagulant activity of the RNA BA 4 construct could be rapidly reversed with
a single oligonucleotide antidote at a 1:2 molar ratio in a dose-dependent manner.
The aptamer-antidote therapeutics pair allowed for rapid reversal of anticoagulant
activity as well as minimization bleeding risk. These studies demonstrated that bivalent anticoagulant aptamers may be a novel and potent heparin-like anticoagulation
therapeutics for active and reversible coagulation regulation with fewer side effects
[88].
Gunaratne et al. analyzed X-ray crystal complex structures of 36-mer RNA
aptamer 11F7t and FXa variant (GD-FXa
S195A ). The finding revealed that 11F7t did
not bind the catalytic site of FXa. However, 11F7t could synergize with the existing
small molecule FXa inhibitors. The result of thrombin formation demonstrated that
11F7t combined with either UFH or FXa active site inhibitor (rivaroxaban, apixaban,
or edoxaban) could enhance anticoagulation in purified reaction mixtures (containing
prothrombin, FX, FVa and FVIII supplemented with membranes) and plasma. The
thromboelastography (TEG) analysis showed that the combined inhibitory effect of
aptamer and UFH could be compared with the anticoagulant potency of UFH even
in whole blood. Aptamer-drug complex prevented clot formation as effectively as
UFH in human blood during extracorporeal circulation as CPB anticoagulant and
side effects of UFH-induced HIT or the clot formation risk of bivalirudin could be
avoided. An antidote of aptamer 11F7t, Andexanet Alfa, could promptly neutralize
the anticoagulant effects of both FXa inhibitors. The results suggest that existing
drugs and aptamers can be additive to have more specific and potent effects than
either agent alone[91] (Fig. 10.6).
10.6 Aptamers Against FXI
The plasma protease FXI is a 160 kDa serine protease homodimer containing two
identical disulphide-linked polypeptide chains. It is involved in the intrinsic coagulation pathway. In the blood coagulation cascade, an inactive precursor FXI can
be converted to enzymatically active FXIa by FXIIa or thrombin-mediated specific
proteolysis of the Arg369-Il-370 bond. Subsequently, FXIa primarily catalyzed FIX
activation, activated FX to Xa and thrombin formation through feedback mechanisms, and contributed to thrombus formation in the common coagulation pathway
[92].
Studies suggested that FXIa is crucial in thrombosis but with a relatively limited
contribution to hemostasis. In clinical practice, patients with congenital FXI deficiency rarely have spontaneous bleeding except for surgery or trauma, indicating
that FXI plays little role in hemostasis [93]. Several inhibitors of FXI/XIa proteins
W. Li et al.
bivalent aptamers (RNA BA 1-4) that could inhibit FX/Xa, prothrombin/thrombin, and
anticoagulate plasma the same as the combination of individual aptamers. Detailed
characterization of these bivalent aptamers indicated that each aptamer retained all
the binding and functional activity. Finally, the reversal experiments revealed that
the anticoagulant activity of the RNA BA 4 construct could be rapidly reversed with
a single oligonucleotide antidote at a 1:2 molar ratio in a dose-dependent manner.
The aptamer-antidote therapeutics pair allowed for rapid reversal of anticoagulant
activity as well as minimization bleeding risk. These studies demonstrated that bivalent anticoagulant aptamers may be a novel and potent heparin-like anticoagulation
therapeutics for active and reversible coagulation regulation with fewer side effects
[88].
Gunaratne et al. analyzed X-ray crystal complex structures of 36-mer RNA
aptamer 11F7t and FXa variant (GD-FXa
S195A ). The finding revealed that 11F7t did
not bind the catalytic site of FXa. However, 11F7t could synergize with the existing
small molecule FXa inhibitors. The result of thrombin formation demonstrated that
11F7t combined with either UFH or FXa active site inhibitor (rivaroxaban, apixaban,
or edoxaban) could enhance anticoagulation in purified reaction mixtures (containing
prothrombin, FX, FVa and FVIII supplemented with membranes) and plasma. The
thromboelastography (TEG) analysis showed that the combined inhibitory effect of
aptamer and UFH could be compared with the anticoagulant potency of UFH even
in whole blood. Aptamer-drug complex prevented clot formation as effectively as
UFH in human blood during extracorporeal circulation as CPB anticoagulant and
side effects of UFH-induced HIT or the clot formation risk of bivalirudin could be
avoided. An antidote of aptamer 11F7t, Andexanet Alfa, could promptly neutralize
the anticoagulant effects of both FXa inhibitors. The results suggest that existing
drugs and aptamers can be additive to have more specific and potent effects than
either agent alone[91] (Fig. 10.6).
10.6 Aptamers Against FXI
The plasma protease FXI is a 160 kDa serine protease homodimer containing two
identical disulphide-linked polypeptide chains. It is involved in the intrinsic coagulation pathway. In the blood coagulation cascade, an inactive precursor FXI can
be converted to enzymatically active FXIa by FXIIa or thrombin-mediated specific
proteolysis of the Arg369-Il-370 bond. Subsequently, FXIa primarily catalyzed FIX
activation, activated FX to Xa and thrombin formation through feedback mechanisms, and contributed to thrombus formation in the common coagulation pathway
[92].
Studies suggested that FXIa is crucial in thrombosis but with a relatively limited
contribution to hemostasis. In clinical practice, patients with congenital FXI deficiency rarely have spontaneous bleeding except for surgery or trauma, indicating
that FXI plays little role in hemostasis [93]. Several inhibitors of FXI/XIa proteins