Impact of glycoPEGylation on the activation and functional properties of N9. The kinetics of N9 or N9-GP activation was determined in the presence of (A) 10pM FXIa or (B) 100pM lipidated TF saturated with 1nM FVIIa. After incubation for 30 minutes at pH 7.4, 5mM Ca2+, and 25°C, further activation was quenched by addition of excess EDTA and rates of FIXa generation determined from the amidolytic activity. Data (mean ± SD, n = 3) were fitted to the Michaelis-Menten equation. (C) Binding of FVIIIa to activated N9 and N9-GP was analyzed from titrations of 0.1nM FIXa with 0-3nM FVIIIa in the presence of 25μM 25:75 phosphatidylserine: phosphatidylcholine (PS:PC) vesicles and 100nM FX at pH 7.4, 5mM Ca2+, and 37°C. After 30 seconds of incubation, reactions were quenched with EDTA and initial rates of FXa generation determined from the amidolytic activity. Fit of data (mean ± SD, n = 3) to a 1:1 binding isotherm yielded apparent dissociation constants (K½,FVIIIa) of 1.14 ± 0.03 and 1.21 ± 0.06nM for activated N9 and N9-GP, respectively. (D) The kinetics of FX (0-100nM) activation by activated N9 or N9-GP (20pM) were determined in the presence of saturating FVIIIa (2.7nM) and 25μM 25:75 PS:PC vesicles. Initial rates of FX activation (mean ± SD, n = 3) were fitted to the Michaelis-Menten equation. Kinetic parameters are listed in Table 1.
