BACKGROUND: Tissue factor pathway inhibitor (TFPI) is a multi-domain protein that negatively regulates the coagulation cascade. TFPI inhibits the tissue factor (TF)/factor VIIa /factor Xa complex during TF-mediated coagulation initiation. The aptamer BAX 499 binds specifically to TFPI and inhibits its function, mediating a procoagulant effect in both in vitro and in vivo models of hemophilia.
OBJECTIVES: This study sought to identify the regions of TFPI that are critical for BAX 499 binding and to understand how binding mediates aptamer inhibition of TFPI.
METHODS AND RESULTS: In vitro biochemical methods were used to evaluate the BAX 499 interaction with and inhibition of TFPI. Binding experiments indica... More
BACKGROUND: Tissue factor pathway inhibitor (TFPI) is a multi-domain protein that negatively regulates the coagulation cascade. TFPI inhibits the tissue factor (TF)/factor VIIa /factor Xa complex during TF-mediated coagulation initiation. The aptamer BAX 499 binds specifically to TFPI and inhibits its function, mediating a procoagulant effect in both in vitro and in vivo models of hemophilia.
OBJECTIVES: This study sought to identify the regions of TFPI that are critical for BAX 499 binding and to understand how binding mediates aptamer inhibition of TFPI.
METHODS AND RESULTS: In vitro biochemical methods were used to evaluate the BAX 499 interaction with and inhibition of TFPI. Binding experiments indicate that the full-length TFPI protein is required for tight aptamer binding. Binding-competition experiments implicate the Kunitz 1, Kunitz 3, and C-terminal domains of TFPI in aptamer binding, a finding that is supported by hydrogen-deuterium exchange experiments, and indicate that aptamer and factor Xa can bind simultaneously to TFPI. In enzymatic assays, BAX 499 inhibits TFPI in a manner that is distinct from domain-specific antibodies, and aptamer inhibitory activity is reduced in the presence of TFPI cofactor protein S.
CONCLUSIONS: These studies demonstrate that BAX 499 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 factor Xa. With this unique inhibitory mechanism, BAX 499 provides a useful tool to study TFPI biology in health and disease. ©2013 International Society on Thrombosis and Haemostasis.
