Aptamer BAX 499 mediates inhibition of tissue factor pathway inhibitor via interaction with multiple domains of the protein

Abstract

Tissue factor pathway inhibitor (TFPI) is a multidomain protein that negatively regulates the coagulation cascade. TFPI inhibits the tissue factor (TF)-activated factorVII-activated FX (FXa) complex during TF-mediated coagulation initiation. The aptamer BAX499 binds specifically to TFPI and inhibits its function, mediating a procoagulant effect in both invitro and invivo models of hemophilia. This study sought to identify the regions of TFPI that are critical for BAX499 binding, and to determine how binding mediates aptamer inhibition of TFPI. Invitro biochemical methods were used to evaluate the BAX499 interaction with and inhibition of TFPI. Binding experiments indicated that the full-length TFPI protein is required for tight aptamer binding. Binding-competition experiments implicated the Kunitz1, Kunitz3 and C-terminal domains of TFPI in aptamer binding, a finding that is supported by hydrogen-deuterium exchange experiments, and indicated that aptamer and FXa can bind simultaneously to TFPI. In enzymatic assays, BAX499 inhibited TFPI in a manner that is distinct from domain-specific antibodies, and aptamer inhibitory activity is reduced in the presence of the TFPI cofactor proteinS. These studies demonstrate that BAX499 binds to TFPI via multiple domains of the protein in a manner that is distinct from other TFPI inhibitors, mediating a mechanism of inhibition that does not involve direct competition with FXa. With this unique inhibitory mechanism, BAX499 provides a useful tool for studying TFPI biology in health and disease.