BT200 inhibits VWF interaction with HEK293 cells expressing LRP1. (A) Binding of full-length recombinant human VWF to HEK293 cells stably transfected with LRP1 (HEK-LRP1) was assessed by flow cytometry in the presence (blue) or absence (red) of ristocetin (1.5 mg/mL). The y-axis represents the binding capacity normalized to the number of cells. FITC-A on the x-axis represents the fluorescence intensity absorbance at the wavelength of 490 nm, in which higher values indicate more VWF binding. (B) Binding of rVWF to HEK-LRP1 in the presence of ristocetin was assessed by flow cytometry in the presence of increasing concentrations of BT200. Blue is VWF binding in the absence of BT200; gray is background in the absence of VWF; and green to red signals for increasing BT200 concentrations. The y-axis represents the binding capacity normalized to the number of cells. BL1-A::BL1-A on the x-axis represents the fluorescence intensity absorbance at the wavelength of 488 nm, in which higher values indicate more VWF binding. (C) rVWF binding (%) to HEK-LRP1 cells by flow cytometry is concentration-dependently inhibited by BT200. HEK-LRP1 binding (%) on the y-axis is calculated from mean fluorescent intensities at different BT200 concentrations presented on the x-axis. Error bars depict the standard deviations of the fluorescent intensities. P values are outcomes of extra-sum-of-squares F test and represent the significance of the binding of rVWF to HEK-LRP1. (D-E) To investigate whether BT200 inhibits VWF interaction with LRP1 through competitive, allosteric, or mixed mechanisms, K_d values for human rVWF binding to LRP1 cluster IV were determined over a range of different BT200 concentrations. (E) A linear relationship was observed, suggesting a competitive rather than allosteric mechanism for BT200-mediated inhibition of VWF-LRP1 interaction.