Shorter forms of VWF are adhesive to erythrocytes and cleared in a desialylation-dependent manner. (A) Representative confocal microscopic images comparing VWF clearance (left) and vaso-occlusion (middle and right) in the liver of ADAMTS13-treated S/S mice with fetuin or asialofetuin coinjection. White arrow indicates shorter forms of VWF. The sections were stained with primary antibodies to erythrocytes (Ter119), macrophages (F4/80), and VWF. Data represent at least 4 independent experiments. DAPI, cell nuclear staining. (B) Quantification of VWF clearance and VWF-positive vaso-occlusion in panel A, showing that asialofetuin injection reduced colocalization of VWF with macrophages and abolished the ADAMTS13-mediated reduction of VWF-rich vaso-occlusions. ∗∗P < .01; ∗∗∗P < .001, 2-tailed, unpaired Student t test. (C) Multimer analysis of purified mouse VWF with or without ADAMTS13 cleavage. Lanes 1 and 2 represent reaction mix without ADAMTS13 before and after overnight incubation at 37 °C, respectively. Lanes 3 and 4 represent reaction mix with ADAMTS13 before and after overnight incubation at 37°C, respectively. (D) Comparison of erythrocyte adhesion to flow chambers coated with ADAMTS13 (ATS)-treated or untreated VWF. Some sickle erythrocytes were preincubated with integrin blockers (20 μg/mL bocking antibody to mouse β2 [clone GAME-46], 20 μg/mL arginylglycylaspartic acid (RGD) peptide, or 20 μg/mL bocking antibody to mouse β1 [clone HM β1-1]). Data represent 3 independent experiments. (E) Quantification of adherent sickle erythrocytes per field of view (FOV). ∗∗∗∗P < .0001; 1-way ANOVA. mAb, monoclonal antibody; ns, nonsignificant.