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Blood, Vol. 92 No. 5 (September 1), 1998:
pp. 1617-1625
Phosphorothioate Oligonucleotides Inhibit the Intrinsic Tenase
Complex
John P. Sheehan and
Hao-Chang Lan
From the University of Texas Health Science Center at San Antonio,
Department of Medicine/Hematology, San Antonio, TX.
Systemic administration of ISIS 2302, a 20-mer antisense
phosphorothioate oligonucleotide targeting human intercellular adhesion molecule-1 mRNA, causes prolongation of plasma clotting times in both
monkey and human studies. The anticoagulant effects of ISIS 2302 were
investigated with both in vitro coagulation assays in human plasma and
purified enzyme systems. At high oligonucleotide plasma concentrations
(>100 µg/mL), prolongation of the prothrombin and
thrombin times was observed. In a thrombin time assay using purified
components, high concentrations of ISIS 2302 inhibited thrombin
clotting activity both by stimulating inhibition by heparin cofactor II
and directly competing with fibrinogen for binding to anion binding
exosite I. In contrast, low concentrations of ISIS 2302 (<100
µg/mL) showed a selective, linear prolongation of the activated
partial thromboplastin time (PTT). The rate limiting effect of 50 µg/mL ISIS 2302, which prolonged the PTT to 1.5 times control, was
identified by sequential modification of the clotting assay. Delaying
addition of oligonucleotide until after contact activation failed to
correct prolongation of the PTT. The calcium-dependent steps of the
intrinsic pathway were individually assessed by adding sufficient
activated coagulation factor to correct the PTT in plasma deficient in
that specific factor. Addition of factor XIa, IXa, VIIIa, or Va failed
to correct the PTT in the presence of ISIS 2302. In contrast, 0.2 nmol/L factor Xa corrected prolongation of the PTT in factor
X-deficient plasma with or without oligonucleotide present. ISIS 2302 (50 µg/mL) did not prolong a modified Russel viper venom time,
suggesting no significant inhibition of prothrombinase. Thus, 50 µg/mL ISIS 2302 prolonged the PTT by selectively inhibiting intrinsic
tenase activity. ISIS 2302 showed partial inhibition of intrinsic
tenase activity (to approximately 35% of control) at clinically
relevant oligonucleotide concentrations in a chromogenic assay. This
activity was oligonucleotide sequence-independent but required the
phosphorothioate backbone, suggesting that inhibition of intrinsic
tenase is a general property of this class of oligonucleotides. These
results are relevant to both the therapeutic use of phosphorothioate oligonucleotides and the potential design of inhibitors of the intrinsic tenase complex, a novel target for anticoagulation.
© 1998 by The American Society of Hematology.
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