Glycocalicin interacts with rFVIIa. (A) rFVIIa was immobilized on a microtiter plate at concentrations indicated. Subsequently, wells were incubated with GC (5 μg/mL) or vehicle, and bound GC was detected by using a polyclonal antibody against GPIb (2 μg/mL). (B) rFVIIa (3400 RU) was immobilized on a CM5 sensor chip, and binding of GC was investigated by SPR. After adjusting for binding to a blank channel, the response of GC at equilibrium was determined and plotted against the concentration applied. Inset shows representative SPR traces of indicated concentrations of GC. (C) GC (1100 RU) was immobilized on a CM5 sensor chip, and binding of rFVIIa was investigated by SPR. (D) Representative traces of GC (67 nM) binding to a rFVIIa-coated channel, a channel coated with factor X (FX, 5400 RU) or an uncoated channel (neg). (E) Binding of GC (67 nM) to rFVIIa was investigated in the absence or presence of 2 μM rFVIIa. Representatives of at least 3 experiments are shown.