The effect of BT200 on VWF clearance in VWF^–/– mice and ability to attenuate VWF interaction with macrophage MGL and SR-AI receptors. (A-C) In vivo clearance experiments were performed in VWF^−/− mice weighing 20 to 25 g on a C57BL/6J background. Mice were infused with 7.5 μg rVWF or rVWF-4A with or without 0.9 μM BT200 via tail-vein injection. Blood was collected via submandibular bleeding or cardiac puncture into lithium-heparin–coated microtainers. Three to 6 mice per time point were used. Residual plasma VWF:Ag levels were determined at specific time points by VWF:Ag enzyme-linked immunosorbent assay, using polyclonal rabbit anti-human VWF (Dako). (D) Binding of full-length rVWF to WT BMDMs (red) or MX1cre⁺LRP^flox/flox BMDMs (MacLRP1^–/– blue) was assessed using flow cytometry in the presence of increasing concentrations of BT200. Relative macrophage 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 error of the mean of the fluorescent intensities. P values are outcomes of extra-sum-of-squares F test. (E-F) Binding of full-length rVWF to HEK-MGL (E) and HEK-SR-AI cells (F) was assessed by flow cytometry in the presence or absence of increasing concentrations of BT100 (blue) and BT200 (red). (E-F) HEK-MGL binding (%) (E) and HEK-SR-AI binding (%) (F) on the y-axis are calculated from mean fluorescent intensities at different BT100 and 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 compare the binding capacity of BT100 and BT200 with HEK-MGL (E) or HEK-SR-AI (F).