title: Phosphorothioate oligonucleotides inhibit the intrinsic tenase complex by an allosteric mechanism.
abstract: Phosphorothioate oligonucleotides (PS ODNs) prolong the activated partial thromboplastin time in human plasma by inhibition of intrinsic tenase (factor IXa-factor VIIIa) activity. This inhibition was characterized using ISIS 2302, a 20-mer antisense PS ODN. ISIS 2302 demonstrated hyperbolic, mixed-type inhibition of factor X activation by the intrinsic tenase complex. The decrease in V(max(app)) was analyzed by examining complex assembly, cofactor stability, and protease catalysis. ISIS 2302 did not inhibit factor X activation by the factor IXa-phospholipid complex, or significantly affect factor VIII-phospholipid affinity. Inhibitory concentrations of ISIS 2302 modestly decreased the affinity of factor IXa-factor VIIIa binding in the presence of phospholipid (K(D) = 11.5 vs 4.8 nM). This effect was insufficient to explain the reduction in V(max(app)). ISIS 2302 did not affect the in vitro half-life of factor VIIIa, suggesting it did not destabilize cofactor activity. In 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 chromogenic substrate cleavage by factor IXa increased more than 50-fold. ISIS 2302 demonstrated partial inhibition of factor X activation by the factor IXa-phospholipid complex, and chromogenic substrate cleavage by factor IXa, only in the presence of ethylene glycol. Like the intact enzyme complex, ISIS 2302 demonstrated hyperbolic, mixed-type inhibition of chromogenic substrate cleavage by factor IXa (K(I) = 88 nM). Equilibrium binding studies with fluorescein-labeled ISIS 2302 demonstrated a similar affinity (K(D) = 92 nM) for the PS ODN-factor IX interaction. These results suggest that PS ODNs bind to an exosite on factor IXa, modulating catalytic activity of the intrinsic tenase complex.
authors: Department of Medicine/Hematology, South Texas Veteran's Health Care System (Audie Murphy Division), University of Texas Health Science Center at San Antonio, 78284, USA. sheehan@uthscsa.edu
authors: J P
authors: JP
authors: Sheehan
authors: T M
authors: TM
authors: Phan
mesh_terms: D000494:Allosteric Regulation; D001665:Binding Sites / Q000187:drug effects; D001667:Binding, Competitive; D002384:Catalysis; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D019855:Ethylene Glycol / Q000494:pharmacology; D015949:Factor IXa / Q000037:antagonists & inhibitors* / Q000378:metabolism; D015944:Factor VIIIa / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006207:Half-Life; D006801:Humans; D046911:Macromolecular Substances; D009363:Neoplasm Proteins; D020319:Oligodeoxyribonucleotides, Antisense / Q000378:metabolism / Q000494:pharmacology; D010743:Phospholipids / Q000037:antagonists & inhibitors / Q000378:metabolism; D054735:Phosphorothioate Oligonucleotides; D015842:Serine Proteinase Inhibitors / Q000145:classification / Q000494:pharmacology*; D013873:Thionucleotides / Q000378:metabolism / Q000494:pharmacology*
chemical_list: D046911:Macromolecular Substances; D009363:Neoplasm Proteins; D020319:Oligodeoxyribonucleotides, Antisense; D010743:Phospholipids; D054735:Phosphorothioate Oligonucleotides; D015842:Serine Proteinase Inhibitors; D013873:Thionucleotides; D015944:Factor VIIIa; D015949:Factor IXa; D003546:Cysteine Endopeptidases; C030910:cancer procoagulant; D019855:Ethylene Glycol; C089753:alicaforsen