Activity of ARC19499 in TFPI-dependent assays using purified proteins. (A) Effect of ARC19499 on TFPI inhibition of FXa activity. The rate of FXa (2nM) cleavage of a chromogenic substrate is shown in the absence (solid line) and presence (dashed line) of 4nM TFPI. ARC19499 (□) and the negative control oligonucleotide (○) were tested in the presence of TFPI. The rate of substrate cleavage is plotted as a function of aptamer concentration. (B) Effect of ARC19499 on TFPI inhibition of TF/FVIIa activity. The inhibitory activity of increasing concentrations of ARC19499 was tested in the presence of 1nM TFPI. Terminal rates were determined from data obtained between 2 and 5 minutes and normalized against the rate of FXa generation in the absence of TFPI or aptamer. Inset shows representative plots of FXa generation as a function of time used to generate the normalized data. The rate of FXa generation was measured in the absence of TFPI (■), in the presence of 1nM TFPI (●), and with 0.01-1000nM ARC19499 in the presence of 1nM TFPI. Results are represented as means ± SEM (error bars or shaded area); n = 4 (A) or n = 3 (B).