INHIBITION OF COAGULATION BY A PHOSPHOROTHIOATE OLIGONUCLEOTIDE

In the development of antisense therapeutics, there have been a number of hybridization-independent effects characterized for phosphorothioa te oligodeoxynucleotides. One such effect is the transient prolongatio n of clotting times following intravenous infusion of high doses, In t his study, inhibition of clotting times was characterized by determini ng the time course of both APTT and plasma oligonucleotide following i ntravenous infusion of ISIS 2302 in cynomolgus monkeys, Prolongation o f APTT was also achieved by addition of ISIS 2302 to citrated blood fr om untreated monkeys, allowing the investigation of the mechanism of i nhibition in vitro, Results from this study clearly indicate that the intrinsic pathway (APTT) was more sensitive to inhibition than the ext rinsic pathway (PT), The prolongation of APTT was also shown to be tra nsient and closely correlated with plasma oligonucleotide concentratio ns. The extent of APTT prolongation can be controlled by minimizing pe ak plasma oligonucleotide concentrations through lowering the dose or prolonging infusion duration, Direct addition of ISIS 2302 to blood pr oduced quantitatively similar inhibition of clotting times, This effec t was similar for a number of different phosphorothioate oligodeoxynuc leotides, but oligonucleotides containing phosphodiester linkages and 2'-propoxy linkages were much less inhibitory, Additional in vitro stu dies indicated that the mechanism of inhibition was independent of tha t of heparin and possibly involved selective inhibition of the intrins ic pathway as well as the common clotting pathway, Investigation of se lective clotting factors indicated that there was no direct inhibition of the enzymatic activity of factor Xa, XIa, or thrombin using chromo genic substrates, However, ISIS 2302 did produce a concentration-depen dent increase in clotting time when fibrinogen was used as the substra te for thrombin.