Lack of VWF binding to LRP1during SPR analysis and under static adhesion. (A) LRP1 immobilized at a CM5-sensorchip (12 fmol/mm²) was incubated with FVIII (40nM) in the presence of various concentrations of VWF (0-430nM) in 0.15M NaCl, 2mM CaCl₂, 0.005% Tween 20, and 20mM HEPES (pH 7.4) at a flow of 20 μL/min at 25°C. Residual FVIII binding at equilibrium (femtomoles per square millimeter) versus VWF concentration (nanomolar) is plotted. Inset, LRP1 (0.5 μg/well) was immobilized in microtiter wells and incubated with recombinant FVIII (2nM) in the absence or presence of recombinant murine VWF (0-360nM). Residual FVIII bound was detected using monoclonal anti-FVIII antibody D4H1. Percentage of residual FVIII bound compared with binding in the absence of VWF is plotted. (B) LRP1 and anti-VWF antibody 539 (both 0.5 μg/well) were immobilized in microtiter wells and subsequently incubated with VWF- or BSA-coated microspheres (Fluoresbrite-YG, 2 μm) for 1 hour at room temperature. Wells were washed, and adhered microspheres were quantified via light microscopy. Data represent the number of microspheres per field ± SD. Twelve fields (125 × 95 μm) were examined, with a maximum of 4 fields/well. Images display a representative field of VWF-coated beads incubated with LRP1 (inset I) or anti-VWF antibody 539 (inset II).