PHOSPHOROTHIOATE OLIGONUCLEOTIDES INHIBIT THE INTRINSIC TENASE COMPLEX

Systemic administration of ISIS 2302, a 20-mer antisense phosphorothio ate 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 investigate d with both in vitro coagulation assays in human plasma and purified e nzyme systems. At high oligonucleotide plasma concentrations (> 100 mu g/mL), prolongation of the prothrombin and thrombin times was observe d. In a thrombin time assay using purified components, high concentrat ions of ISIS 2302 inhibited thrombin clotting activity both by stimula ting inhibition by heparin cofactor II and directly competing with fib rinogen for binding to anion binding exosite I. In contrast, low conce ntrations of ISIS 2302 (<100 mu g/mL) showed a selective, linear prolo ngation of the activated partial thromboplastin time (PIT). The rate l imiting effect of 50 mu g/mL ISIS 2302, which prolonged the PTT to 1.5 times control, was identified by sequential modification of the clott ing assay. Delaying addition of oligonucleotide until after contact ac tivation failed to correct prolongation of the PTT. The calcium-depend ent steps of the intrinsic pathway were individually assessed by addin g 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 con trast, 0.2 nmol/L factor Xa corrected prolongation of the PTT in facto r X-deficient plasma with or without oligonucleotide present. ISIS 230 2 (50 mu g/mL) did not prolong a modified Russel viper venom time, sug gesting no significant inhibition of prothrombinase. Thus, 50 mu g/mL ISIS 2302 prolonged the PTT by selectively inhibiting intrinsic tenase activity. ISIS 2302 showed partial inhibition of intrinsic tenase act ivity (to approximately 35% of control) at clinically relevant oligonu cleotide concentrations in a chromogenic assay. This activity was olig onucleotide sequence-independent but required the phosphorothioate bac kbone, suggesting that inhibition of intrinsic tenase is a general pro perty 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 nov el target for anticoagulation. (C) 1998 by The American Society of Hem atology.