SPR analysis of factor IXa and factor IX binding to immobilized LRP.

(A) LRP, immobilized onto a CM5 sensorchip at a density of 9 fmol/mm² (II) and of 26 fmol/mm² (I, III), was incubated with 100-nmol/L factor IX (I) or 100-nmol/L factor IXa (II, III) in 20-mmol/L HEPES (pH 7.4), 150-mmol/L NaCl, and 0.005% (vol/vol) Tween 20 at 25°C at a flow rate of 5-μL/min for 2 minutes. To initiate dissociation, the buffer was replaced by buffer devoid of ligand. (B) Six different concentrations (10, 20, 49, 60, 98, and 142 nmol/L) of factor IXa were passed at 25 °C with a flow rate of 20 μL/min over immobilized LRP (16 fmol/mm²). The subsequent association and dissociation are represented by the 6 data curves shown. The signal is indicated in resonance units (RU) and is corrected for aspecific binding, which was less than 5% of the binding to LRP-coated channels.