10 Aptamers for Thrombotic Diseases
303
inflammatory activation in a well-established flow model. They found that ssDNA
aptamers activated FXII, kallikrein, and prothrombin in a dose-dependent manner.
The study suggested that in view of aptamers’ influence on the coagulation system,
the use of any aptamer-based therapeutics should be carefully evaluated [101].
Although the oral non-vitamin antagonist anticoagulants produce less intracranial
bleeding than warfarin, serious bleeding is still unavoidable. Since thrombosis is
attenuated in mice deficient in FXI or FXII, and the patients with congenital FXI and
FXII deficiency rarely have spontaneous bleeding [90, 102, 103]. FXII and FXI are
promising drug targets with the potential to prevent thrombosis without disruption of
hemostasis, including antisense oligonucleotides, antibodies or aptamers, and small
molecule inhibitors. These strategies alleviated thrombosis in various animal models
and FXI knockdown with an antisense oligonucleotide reduced postoperative venous
thromboembolism to a greater extent than enoxaparin without increasing bleeding
in patients undergoing knee replacement surgery. Therefore, the efficacy and safety
of FXII and FXI directed anticoagulant strategies should be evaluated [104].
10.8 Aptamers Against Kallikrein
The inactive precursor of kallikrein, prekallikrein (PK), is a single-chain monomeric
glycoprotein and an important component of many biological systems, for example,
the contact activation system, kallikrein-kinin system, renin-angiotensin system
[105–107], fibrinolytic system, and the alternative complement pathway [108, 109].
Prekallikrein is usually complexed with HMWK, which is the substrate of kallikrein
for generating the pro-inflammatory peptide, namely bradykinin (BK). Plasma
kallikrein, a product of FXIIa-mediated zymogen cleavage, is a serine protease
associated with many biological processes such as coagulation, inflammation, and
fibrinolysis. In the coagulation process, kallikrein amplifies activated FXIIa generation and then results in thrombin generation and fibrin clot formation [110].
Abnormal prekallikrein/kallikrein activity is associated with thrombosis, hereditary angioedema, diabetic retinopathy, intracerebral hemorrhage, and septicemia
[111, 112].
Steen and his colleagues developed a 54-mer RNA aptamer (Kall1-T4) modified
with 2
-fluoropyrimidine for kallikrein. APTT coagulation assay showed that aptamer
Kall1-T4 bound both plasma prekallikrein and kallikrein with similar subnanomolar
binding affinities and dose-dependently slowed down fibrin clot formation. The
results suggested that anticoagulant activity of Kall1-T4 did not stem from binding
and blocking kallikrein’s active site or from disturbing active site function in an
allosteric manner but maybe through inhibition of binding between macromolecular substrates and/or cofactor, prekallikrein, and/or kallikrein. In a purified in vitro
system, Kall1-T4 inhibited the reciprocal activation between prekallikrein and FXII
by reducing FXIIa-mediated prekallikrein activation rate. In addition, Kall1-T4
significantly reduced both kallikrein-mediated HK cleavage and BK release. It was
suggested that aptamer Kall1-T4 can be used to elucidate the mechanism of kallikrein
303
inflammatory activation in a well-established flow model. They found that ssDNA
aptamers activated FXII, kallikrein, and prothrombin in a dose-dependent manner.
The study suggested that in view of aptamers’ influence on the coagulation system,
the use of any aptamer-based therapeutics should be carefully evaluated [101].
Although the oral non-vitamin antagonist anticoagulants produce less intracranial
bleeding than warfarin, serious bleeding is still unavoidable. Since thrombosis is
attenuated in mice deficient in FXI or FXII, and the patients with congenital FXI and
FXII deficiency rarely have spontaneous bleeding [90, 102, 103]. FXII and FXI are
promising drug targets with the potential to prevent thrombosis without disruption of
hemostasis, including antisense oligonucleotides, antibodies or aptamers, and small
molecule inhibitors. These strategies alleviated thrombosis in various animal models
and FXI knockdown with an antisense oligonucleotide reduced postoperative venous
thromboembolism to a greater extent than enoxaparin without increasing bleeding
in patients undergoing knee replacement surgery. Therefore, the efficacy and safety
of FXII and FXI directed anticoagulant strategies should be evaluated [104].
10.8 Aptamers Against Kallikrein
The inactive precursor of kallikrein, prekallikrein (PK), is a single-chain monomeric
glycoprotein and an important component of many biological systems, for example,
the contact activation system, kallikrein-kinin system, renin-angiotensin system
[105–107], fibrinolytic system, and the alternative complement pathway [108, 109].
Prekallikrein is usually complexed with HMWK, which is the substrate of kallikrein
for generating the pro-inflammatory peptide, namely bradykinin (BK). Plasma
kallikrein, a product of FXIIa-mediated zymogen cleavage, is a serine protease
associated with many biological processes such as coagulation, inflammation, and
fibrinolysis. In the coagulation process, kallikrein amplifies activated FXIIa generation and then results in thrombin generation and fibrin clot formation [110].
Abnormal prekallikrein/kallikrein activity is associated with thrombosis, hereditary angioedema, diabetic retinopathy, intracerebral hemorrhage, and septicemia
[111, 112].
Steen and his colleagues developed a 54-mer RNA aptamer (Kall1-T4) modified
with 2
-fluoropyrimidine for kallikrein. APTT coagulation assay showed that aptamer
Kall1-T4 bound both plasma prekallikrein and kallikrein with similar subnanomolar
binding affinities and dose-dependently slowed down fibrin clot formation. The
results suggested that anticoagulant activity of Kall1-T4 did not stem from binding
and blocking kallikrein’s active site or from disturbing active site function in an
allosteric manner but maybe through inhibition of binding between macromolecular substrates and/or cofactor, prekallikrein, and/or kallikrein. In a purified in vitro
system, Kall1-T4 inhibited the reciprocal activation between prekallikrein and FXII
by reducing FXIIa-mediated prekallikrein activation rate. In addition, Kall1-T4
significantly reduced both kallikrein-mediated HK cleavage and BK release. It was
suggested that aptamer Kall1-T4 can be used to elucidate the mechanism of kallikrein