Studies on the mechanism of action of the aptamer BAX499, an inhibitor of tissue factor pathway inhibitor.

INTRODUCTION: Promoting thrombin generation by inhibiting tissue factor pathway inhibitor (TFPI) is a potentially viable therapeutic approach to the prevention and/or treatment of bleeding in hemophilia. In this report, we studied the interaction between an aptamer (BAX499; formerly ARC19499) and TFPI that resulted in inhibition of TFPI-mediated regulation of the tissue factor pathway.
MATERIALS AND METHODS: Enzyme kinetic analyses were performed to study the interaction between BAX499 and recombinant TFPI against factor Xa, the extrinsic Xase and prothrombinase activities. Diluted prothrombin time assay was used to investigate the effects of BAX499 on factor VIII-deficient plasma collected from hemophilia patients.
RESULTS: Our results indicate that after binding of BAX499 to TFPI, the TFPI/ BAX499 complex retains factor Xa inhibitory activity, albeit with reduced affinity. When tested in an extrinsic Xase activity assay, BAX499 delayed TFPI-mediated inhibition of extrinsic Xase activity. In addition, BAX499 reversed TFPI inhibition of the prothrombinase complex. BAX499 shortened the dilute prothrombin time in factor VIII-deficient plasma, and when added to freshly drawn hemophilia A blood either with or without a factor VIII inhibitor, the whole blood clotting time was also shortened. These results suggest that BAX499 may be a useful addition to the armamentarium of bypassing agents to control bleeding in hemophilic patients with inhibitors.