Phosphorothioate Oligonucleotides Inhibit the Intrinsic Tenase Complex

Abstract

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.

Tumor regression by combination antisense therapy against Plk1 and Bcl-2

“…However, we cannot exclude that downmodulation of PLK1 expression affects expression of BCL-2 through nonantisense mechanism. Known side effects of phosphorothioate ODN administration are hypotension, thrombocytopenia and increased clotting times (Henry et al, 1997;Sheehan and Lan, 1998). None of these effects have yet been problematic in animal trials and ODN treatment also caused no apparent side effects in the present study.…”

Identification of a boron nitride nanosphere-binding peptide for the intracellular delivery of CpG oligodeoxynucleotides

“…However, there is concern over undesirable side effects associated with the use of CpG ODNs with a phosphorothioate backbone. [15][16][17] Since CpG ODNs are negatively charged, it is difficult for them to bind to the negatively charged cell surface. This electrostatic repulsion is believed to limit the efficiency of CpG ODNs uptake.…”

Making Sense of Antisense

“…In primates, toxicity has been primarily limited to transient prolongation of the intrinsic pathway of coagulation, as assessed by the activated partial thromboplastin time (APTT), and alternative pathway complement activation with dose regimens that produce peak plasma drug levels of greater than 50 µg/mL. Nonspecific protein binding appears to be responsible for these effects; inhibition of APTT appears to be mediated through the inhibition of intrinsic tenase (factors IXa, VIIIa, phospholipid and calcium) activity (13) and complement activation through interaction, either directly or indirectly, with factor H, an inhibitory protein of the alternative pathway (14).…”