Clearance of hyposialylated VWF proceeds independently of AMR. (A) To study the effects of N- and O-linked sialylation on VWF clearance, purified human pd-VWF was treated with either α2-3,6,8,9 or α2-3 neuraminidase. In vivo clearance for each glycoform was then assessed in VWF^−/− mice and compared with that of wild-type pd-VWF. At each time point, residual circulating VWF concentration was determined by VWF:Ag enzyme-linked immunosorbent assay. All results are plotted as percentage residual VWF:Ag levels relative to the amount injected. Three to 5 mice were used per time point. Data are represented as mean ± SEM. In some cases, the SEM cannot be seen because of its small size. (B) In the presence of ASOR, the enhanced in vivo clearance of both α2-3 Neu-VWF and α2-3,6,8,9 Neu-VWF was significantly attenuated (α2-3 Neu-VWF t_1/2 = 8.2 ± 1.4 minutes vs 12.4 ± 2.4 minutes, P < .05; and α2-3,6,8,9 Neu-VWF t_1/2 = 3.7 ± 0.7 minutes vs 14.4 ± 2.7 minutes, P < .005, respectively). (C) To determine whether AMR-independent pathways contribute to the enhance clearance of hyposialylated VWF, in vivo clearance studies were repeated in VWF^−/−/Asgr1^−/− mice. Importantly, the markedly enhanced clearance of both α2-3 Neu-VWF and α2-3,6,8,9 Neu-VWF was still evident in the absence of the AMR (t_1/2 = 8.2 ± 0.6 and 3.2 ± 0.4 compared with 50.6 ± 2 minutes for pd-VWF; P < .05). Furthermore, the reduced half-life observed for α2-3 Neu-VWF (D) and α2-3,6,8,9 Neu-VWF (E) were not significantly different in the presence or absence of the AMR (α2-3 Neu-VWF t_1/2 = 8.2 ± 1.4 minutes vs 8.2 ± 0.6 minutes, P = .96; and α2-3,6,8,9 Neu-VWF t_1/2 = 3.7 ± 0.7 minutes vs 3.2 ± 0.4 minutes, P = .42, respectively).