Binding of VWF to rPSGL–immunoglobulin fusion protein. Biotinylated F(ab′)2 goat anti–human Fcγ antibodies were immobilized (2.5 kRU) onto 2 adjacent channels of a streptavidin-coated sensor chip. Purified recombinant rPSGL–immunoglobulin (0.3 mg/mL) was then applied to the second of these channels to reach a density of 0.5 kRU, whereas channel 1 was used as a reference. (A, inset) The various sensorgrams obtained with these channels are shown. Dotted lines indicate perfusion of recombinant wt-VWF (300 nM) or botrocetin (600 nM) alone. Solid lines represent sensorgrams of various concentrations of VWF/botrocetin complexes (25-300 nM). Complexes were obtained by incubating VWF with a 2-fold molar excess of botrocetin for 15 minutes before SPR analysis. (A) The response of the VWF/botrocetin complex at equilibrium was determined and plotted against the concentration applied. Using the resultant isotherm, the apparent affinity constant was calculated to be 60 ± 10 nM. (B) Various sensorgrams obtained through the perfusion of VWF deletion mutants (100 nM) complexed with botrocetin (200 nM). Line I, VWF/ΔA1. Line II, VWF/ΔA3. Line III, VWF/ΔA2. (C) Various concentrations of recombinant VWF/A1-A2-A3 fragment (0-4.2 μM; solid lines) or recombinant VWF/D′-D3 fragment (4.5 μM; dotted line) were passed over rPSGL–immunoglobulin, and resultant sensorgrams are depicted. Perfusions were performed in 100 mM NaCl, 0.005% Tween-20, 2.5 mM CaCl2, and 25 mM HEPES (pH 7.4) at a flow rate of 5 μL/min at 25°C. Association was allowed for 2 minutes, after which ligand solution was replaced by buffer. Dissociation was then followed for a 2-minute period. rPSGL–immunoglobulin dissociation during the association period was less than 5%. Before each ligand injection, channel 2 was reloaded with rPSGL–immunoglobulin to reach a density of 0.5 kRU.
