Phosphorothioate oligonucleotides inhibit the intrinsic tenase complex by an allosteric mechanism

Phosphorothioate oligonucleotides (PS ODNs) prolong the activated partial t hromboplastin time in human plasma by inhibition of intrinsic tenase (facto r Ma-factor Vma) activity. This inhibition was characterized using ISIS 230 2, a 20-mer antisense PS ODN. ISIS 2302 demonstrated hyperbolic, mixed-type inhibition of factor X activation by the intrinsic tenase complex. The dec rease in V-max(app) was analyzed by examining complex assembly, cofactor st ability, and protease catalysis. ISIS 2302 did not inhibit factor X activat ion by the factor IXa-phospholipid complex, or significantly affect factor VIII-phospholipid affinity. Inhibitory concentrations of ISIS 2302 modestly decreased the affinity of factor Ma-factor VIIIa binding in the presence o f phospholipid (K-D 11.5 vs 4.8 nM). This effect was insufficient to explai n the reduction in V-max(app). ISIS 2302 did not affect the in vitro half-l ife of factor VIIIa, suggesting it did not destabilize cofactor activity. I n the presence of 30% ethylene glycol, the level of factor X activation by the factor IXa-phospholipid complex increased 3-fold, and the level of chro mogenic substrate cleavage by factor Ma increased more than 50-fold ISIS 23 02 demonstrated partial inhibition of factor X activation by the factor IXa -phospholipid complex, and chromogenic substrate cleavage by factor Ma, onl y in the presence of ethylene glycol. Like the intact enzyme complex, ISIS 2302 demonstrated hyperbolic, mixed-type inhibition of chromogenic substrat e cleavage by factor Ma (K-I = 88 nM). Equilibrium binding studies with flu orescein-labeled ISIS 2302 demonstrated a similar affinity (K-D = 92 nM) fo r the PS ODN-factor IX interaction. These results suggest that PS ODNs bind to an exosite on factor IXa, modulating catalytic activity of the intrinsi c tenase complex.