Figure 1
Figure 1. The A domains of VWF modulate macrophage-mediated clearance. (A) The in vivo clearance of a monomeric A1-A2-A3 VWF fragment in VWF−/− mice was compared with that of full-length rVWF. At each time point, the residual circulating VWF concentration was determined by VWF:antigen ELISA. All results are plotted as percentage residual VWF levels relative to the amount injected. Data are presented as mean ± SEM. In some cases, the SEM cannot be seen due to its small size. Mean residence times for full-length and A1-A2-A3 were 11.3 ± 0.6 and 10.0 ± 0.61 minutes, respectively. (B) To study the role of macrophages in modulating clearance of A1-A2-A3 and full-length rVWF, in vivo clearance studies were repeated in VWF−/− mice 24 hours following clodronate-induced macrophage depletion. Blood was collected at 3- and 10-minute time points, and residual VWF quantified by ELISA. (C) The in vitro binding of A1-A2-A3 to macrophages was assessed using THP-1 macrophage cells as detailed in “Materials and methods.” (D) Individual A-domain proteins A1, A2, and A3 were examined for binding to THP-1 macrophages. Significant binding was observed for the A1 domain compared with the A2 and A3 domains (*P < .05, **P < .01, and ***P < .001, respectively; negative control is no VWF). (E) To investigate the role of VWF carbohydrate determinants in modulating VWF clearance, rVWF was treated with PNGase F (PNG-rVWF). N-linked glycan removal was confirmed using a specific lectin ELISA. In vivo survival was then measured in VWF−/− mice as before. Results are plotted as percentage residual VWF:antigen levels relative to the amount injected. Data are represented as mean ± SEM. (F) To assess a potential role for VWF N-linked glycans in the A domains in regulating macrophage binding, A1-A2-A3 was treated with PNGase to remove the N-linked glycans in the A2 domain at N1515 and N1574 (PNG-A1A2A3). The ability of PNG-A1A2A3 to bind to THP-1 macrophages in the presence of ristocetin was then compared with WT A1-A2-A3 using HiContent image analysis as before. Data are graphed as percentage binding relative to maximal (mean ± SEM) (****P < .001).

The A domains of VWF modulate macrophage-mediated clearance. (A) The in vivo clearance of a monomeric A1-A2-A3 VWF fragment in VWF−/− mice was compared with that of full-length rVWF. At each time point, the residual circulating VWF concentration was determined by VWF:antigen ELISA. All results are plotted as percentage residual VWF levels relative to the amount injected. Data are presented as mean ± SEM. In some cases, the SEM cannot be seen due to its small size. Mean residence times for full-length and A1-A2-A3 were 11.3 ± 0.6 and 10.0 ± 0.61 minutes, respectively. (B) To study the role of macrophages in modulating clearance of A1-A2-A3 and full-length rVWF, in vivo clearance studies were repeated in VWF−/− mice 24 hours following clodronate-induced macrophage depletion. Blood was collected at 3- and 10-minute time points, and residual VWF quantified by ELISA. (C) The in vitro binding of A1-A2-A3 to macrophages was assessed using THP-1 macrophage cells as detailed in “Materials and methods.” (D) Individual A-domain proteins A1, A2, and A3 were examined for binding to THP-1 macrophages. Significant binding was observed for the A1 domain compared with the A2 and A3 domains (*P < .05, **P < .01, and ***P < .001, respectively; negative control is no VWF). (E) To investigate the role of VWF carbohydrate determinants in modulating VWF clearance, rVWF was treated with PNGase F (PNG-rVWF). N-linked glycan removal was confirmed using a specific lectin ELISA. In vivo survival was then measured in VWF−/− mice as before. Results are plotted as percentage residual VWF:antigen levels relative to the amount injected. Data are represented as mean ± SEM. (F) To assess a potential role for VWF N-linked glycans in the A domains in regulating macrophage binding, A1-A2-A3 was treated with PNGase to remove the N-linked glycans in the A2 domain at N1515 and N1574 (PNG-A1A2A3). The ability of PNG-A1A2A3 to bind to THP-1 macrophages in the presence of ristocetin was then compared with WT A1-A2-A3 using HiContent image analysis as before. Data are graphed as percentage binding relative to maximal (mean ± SEM) (****P < .001).

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