Figure 2.
Figure 2. MGL facilitates VWF clearance in vivo. To investigate other receptors and/or cell types that modulate the enhanced clearance of hyposialylated VWF, α2-3 Neu-VWF (A) and α2-3,6,8,9 Neu-VWF (B) clearance studies in VWF−/−Asgr1−/− mice were repeated in the presence of ASOR, or following clodronate-induced macrophage depletion. The enhanced clearance of both α2-3 Neu-VWF and α2-3,6,8,9 Neu-VWF was still inhibited by ASOR (blue lines) even in the absence of the AMR (α2-3 Neu-VWF t1/2 = 8.2 ± 1.4 minutes vs 16.8 ± 1.6 minutes, P < .005; and α2-3,6,8,9 Neu-VWF t1/2 = 3.7 ± 0.7 minutes vs 7.7 ± 1.3 minutes, P < .05, respectively). In addition, clodronate-induced macrophage depletion (green lines) also significantly attenuated the enhanced clearance of hyposialylated VWF (α2-3 Neu-VWF t1/2 = 8.2 ± 1.4 minutes vs 24.0 ± 1.1 minutes, P < .05; and α2-3,6,8,9 Neu-VWF t1/2 = 3.7 ± 0.7 minutes vs 9.6 ± 4.1 minutes, P < .05, respectively). Three to 5 mice were used per time point, and data are represented as mean ± SEM. (C) SPR was used to evaluate the binding of immobilized purified pd-VWF to recombinant human MGL. Dose-dependent binding were observed, with Kd (app) of 18.4 ± 3 µg/mL. (D) In mice, there are 2 homologs of human MGL, mMGL1 and mMGL2. Murine MGL1 shares significant sequence homology with human MGL and binds oligosaccharides with multiple terminal Gal residues including the T antigen. Interestingly, the markedly enhanced clearance of both α2-3 Neu-VWF and α2-3,6,8,9 Neu-VWF in VWF−/−Asgr1−/− mice was significantly attenuated in the presence of an mMGL blocking antibody vs isotype immunoglobulin G (IgG) control antibody, respectively (α2-3 Neu-VWF t1/2 = 21.9 ± 11.8 minutes vs 9.1 ± 1.5 minutes, P < .05; and α2-3,6,8,9 Neu-VWF t1/2 = 24.4 ± 8.1 minutes vs 5.7 ± 2.1 minutes, P < .05, respectively). (E) THP1 macrophages incubated with VWF demonstrated VWF-MGL colocalization detected by Duolink-PLA, visualized as red spots via immunofluorescence microscopy. No signal was observed from cells incubated with phosphate-buffered saline (PBS) alone. (F) Plasma VWF:Ag levels were significantly elevated in MGL1−/− mice compared with wild-type (WT) littermate controls (P < .05). (G) The clearance of endogenous murine VWF in MGL1−/− mice was significantly attenuated compared with wild-type controls at all time points measured (P < .05). (H) In vivo clearance of wild-type pd-VWF in VWF−/−Asgr1−/− mice was significantly attenuated in the presence of an mMGL blocking antibody compared with isotype control IgG (t1/2 = 64.6 ± 18.4 minutes vs 42.8 ± 10.7 minutes; P < .005). A minimum of 3 mice were used per time point; data are plotted as mean ± SEM.

MGL facilitates VWF clearance in vivo. To investigate other receptors and/or cell types that modulate the enhanced clearance of hyposialylated VWF, α2-3 Neu-VWF (A) and α2-3,6,8,9 Neu-VWF (B) clearance studies in VWF−/−Asgr1−/− mice were repeated in the presence of ASOR, or following clodronate-induced macrophage depletion. The enhanced clearance of both α2-3 Neu-VWF and α2-3,6,8,9 Neu-VWF was still inhibited by ASOR (blue lines) even in the absence of the AMR (α2-3 Neu-VWF t1/2 = 8.2 ± 1.4 minutes vs 16.8 ± 1.6 minutes, P < .005; and α2-3,6,8,9 Neu-VWF t1/2 = 3.7 ± 0.7 minutes vs 7.7 ± 1.3 minutes, P < .05, respectively). In addition, clodronate-induced macrophage depletion (green lines) also significantly attenuated the enhanced clearance of hyposialylated VWF (α2-3 Neu-VWF t1/2 = 8.2 ± 1.4 minutes vs 24.0 ± 1.1 minutes, P < .05; and α2-3,6,8,9 Neu-VWF t1/2 = 3.7 ± 0.7 minutes vs 9.6 ± 4.1 minutes, P < .05, respectively). Three to 5 mice were used per time point, and data are represented as mean ± SEM. (C) SPR was used to evaluate the binding of immobilized purified pd-VWF to recombinant human MGL. Dose-dependent binding were observed, with Kd (app) of 18.4 ± 3 µg/mL. (D) In mice, there are 2 homologs of human MGL, mMGL1 and mMGL2. Murine MGL1 shares significant sequence homology with human MGL and binds oligosaccharides with multiple terminal Gal residues including the T antigen. Interestingly, the markedly enhanced clearance of both α2-3 Neu-VWF and α2-3,6,8,9 Neu-VWF in VWF−/−Asgr1−/− mice was significantly attenuated in the presence of an mMGL blocking antibody vs isotype immunoglobulin G (IgG) control antibody, respectively (α2-3 Neu-VWF t1/2 = 21.9 ± 11.8 minutes vs 9.1 ± 1.5 minutes, P < .05; and α2-3,6,8,9 Neu-VWF t1/2 = 24.4 ± 8.1 minutes vs 5.7 ± 2.1 minutes, P < .05, respectively). (E) THP1 macrophages incubated with VWF demonstrated VWF-MGL colocalization detected by Duolink-PLA, visualized as red spots via immunofluorescence microscopy. No signal was observed from cells incubated with phosphate-buffered saline (PBS) alone. (F) Plasma VWF:Ag levels were significantly elevated in MGL1−/− mice compared with wild-type (WT) littermate controls (P < .05). (G) The clearance of endogenous murine VWF in MGL1−/− mice was significantly attenuated compared with wild-type controls at all time points measured (P < .05). (H) In vivo clearance of wild-type pd-VWF in VWF−/−Asgr1−/− mice was significantly attenuated in the presence of an mMGL blocking antibody compared with isotype control IgG (t1/2 = 64.6 ± 18.4 minutes vs 42.8 ± 10.7 minutes; P < .005). A minimum of 3 mice were used per time point; data are plotted as mean ± SEM.

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