Aptamer Mediated Inhibition of Protein S

Protein S (PS) is an anticoagulant that acts as a direct allosteric inhibitor of the activated form of factor IX (FIXa); FIXa is a coagulant, in both the presence and absence of its cofactor, factor VIIIa. Inhibition of FIXa by PS results in a retarded rate of activation of factor X; FX plays an important role as the initial protease common to the two pathways of blood coagulation. Factor IX (FIX) deficiencies present in the form of varying severities of hemophilia B; additionally, elevated levels of PS result in lengthened clotting times.We intend to increase the efficacy and functional lifetime of natural and infused FIXa in hemophilia B patients by creating a high affinity aptamer that selectively inhibits PS in its interaction with FIXa. Although inhibitory aptamers that target pro-coagulant proteins have been produced in a facile manner, the hemostasis field has yet to direct its sights towards a therapeutically viable aptamer that inhibits a naturally occurring anticoagulant.Through the use of previously selected, and experimentally successful, aptamer pools targeting either Gla-domain or plasma proteomes, we were able to use SELEX to select novel PS ligands. After two rounds of aptamer selection, using the focused pools as an initial state, we have found by nitrocellulose binding assays a strong enrichment from the focused Gla domain proteome, whereas the plasma proteome showed a weaker response.Following development of an inhibitory PS aptamer, we will perform fluorescence binding and substrate cleavage studies to obtain kinetic constants, and ex vivo studies, including aPTT and thrombin generation assays, to demonstrate aptamer efficacy in a physiological system. Ultimately, we plan to test the aptamer in vivo, to demonstrate its use as a possible adjunct therapy to current treatments of hemophilia B.

Duke Scholars

Author Bruce Alan Sullenger Surgery, Surgical Sciences