Effect of glycoPEGylation on the binding of N9 to endothelial cells. (A) Competitive displacement of 1nM ¹²⁵I-rFIX with increasing concentrations of unlabeled rFIX, N9, or N9-GP (0.6-600nM). Monolayers of HUVEC cells were incubated with compound for 3 hours at 4°C before counting of bound radioactivity. Binding of rFIX was analyzed according to a homologous 1-site binding model and the estimated apparent K_d of 1.6nM used to derive K_i values for N9 (2.4nM) and N9-GP (48nM). (B) HUVECs were incubated with 0.5nM ¹²⁵I-rFIX with or without molar excess of anti-FIX Gla domain Ab (Gla-Ab; 33nM), FX (400nM), or RAP (500nM) for 3 hours at 4°C. All data are shown as mean ± SD (n = 4).