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N-linked glycans within the A2 domain of von Willebrand factor modulate macrophage-mediated clearance

Alain Chion, Jamie M. O’Sullivan, Clive Drakeford, Gudmundur Bergsson, Niall Dalton, Sonia Aguila, Soracha Ward, Padraic G. Fallon, Teresa M. Brophy, Roger J. S. Preston, Lauren Brady, Orla Sheils, Michael Laffan, Thomas A. J. McKinnon and James S. O’Donnell

Data supplements

Article Figures & Data

Figures

  • 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).

  • Figure 2

    N-linked glycans at N1515 and N1574 are critical determinants of VWF clearance in vivo. (A) A model of the VWF A2 domain was prepared as previously described.66 Mass spectrometry analysis of human pd-VWF has provided extensive information regarding the N-glycome of VWF. Utilizing this information, a model of the VWF A2 domain with its associated glycans was constructed using Glycam Glycoprotein Builder software. N1515 and N1574 glycans structures were mapped onto the A2 domain crystal structure using this glycan modeling. This in silico analysis revealed that the complex glycans at N1515 and N1574 were both of significant size, spanning ∼33 Å and ∼36 Å in length, respectively. (B) To investigate a potential role for specific glycan sites in influencing VWF clearance, N1515 and N1574 in the A2 domain were targeted for removal by site-directed mutagenesis (VWF-N1515Q and VWF-N1574Q, respectively). In vivo clearance studies of these VWF glycan variants were performed as before and compared with WT rVWF. (C) Given that the glycans N1515 and N1574 reside within the A2 domain of VWF, we further sought to examine if these glycans could also influence the in vivo survival of an A1A2A3 VWF truncated fragment. To this end, site-directed mutagenesis was performed to eliminate the glycan at N1515 (A1A2A3-N1515Q) and N1574 (A1A2A3-N1574Q). Clearance examined in VWF−/− mice as before. All results are plotted as percentage residual VWF:antigen levels relative to the amount injected. Data are presented as mean ± SEM.

  • Figure 3

    N-linked glycans within the VWF A2 domain regulate enhanced clearance. Our findings suggest that the N-linked glycan within A2 may have a specific role in modulating VWF clearance. To examine if glycans outside the A2 domain may also influence VWF survival, a fragment of VWF with the A2 domain deleted was constructed. (A) Consequently, this VWF variant (ΔA2-VWF) fails to express the N-linked glycans N1515 and N1574. (B) This VWF variant was subjected to PNGase F treatment (PNG ΔA2-VWF) to remove all remaining N-linked glycans. (C) Clearance was assessed in VWF−/− mice as before. All results are plotted as percentage residual VWF:antigen levels relative to the amount injected. Data are presented as mean ± SEM.

  • Figure 4

    Accelerated clearance of VWF N1515Q and VWF N1574Q is mediated by macrophages. (A) In order to assess the potential contribution of macrophages in modulating the enhanced clearance of VWF glycan variants, clearance of VWF N1515Q and VWF N1574Q was repeated in VWF−/− mice 24 hours after clodronate-induced macrophage depletion. (B) To determine whether macrophages play a role in regulating the reduced survival of A1A2A3-N1515Q and A1A2A3-N1574Q, in vivo clearance studies were also re-assessed in VWF−/− mice following clodronate treatment. Data are graphed as percentage residual VWF relative to the amount injection (*P < .05, **P < .01, and ***P < .001).

  • Figure 5

    N-linked glycans N1515 and N1574 modulate in vitro binding of VWF to macrophages. To examine the biological mechanisms mediating the enhanced clearance of A1A2A3-N1515Q/N1574Q, we assessed binding to THP-1 macrophages in vitro. The binding of A1A2A3 VWF and the glycan variants A1A2A3-N1515Q and A1A2A3-N1574Q to THP-1 macrophages was examined in the presence or absence of 1mg/ml ristocetin. Additionally, all the A1A2A3 variants were subjected to PNGase treatment to remove both N-linked glycans (black columns) and THP-1 macrophage binding was measured. Data are graphed as percentage binding relative to maximal binding (mean ± SEM).

  • Figure 6

    Glycan structures at N1515 and N1574 in the A2 domain influence LRP1-mediated clearance. Recent studies have shown that macrophage LRP1 plays an important role in regulating in vivo clearance of VWF. Moreover, RAP prolongs VWF survival in vivo predominantly by inhibiting this macrophage LRP1 mediated clearance. To investigate whether the effect of VWF glycans on macrophage-mediated clearance were modulated via LRP1, clearance studies for wild type rVWF and glycan variants N1515Q and N1574Q were repeated in VWF−/− mice in the presence or absence of the LRP1 antagonist RAP. Blood was collected at 3 and 10 minutes after injection, and data are graphed as percentage residual VWF relative to the amount injected (*P < .05, **P < .01, and ***P < .001; ns, not significant).

  • Figure 7

    Removal of the N-linked glycans at N1515 does not enhance clearance in VWF with a structurally constrained A2 domain. (A) To examine a potential role for A2 domain conformation in modulating clearance VWF, a previously described cysteine-clamp mutation (N1493C/C1670S) was inserted into full-length rVWF (rVWF-CC) and VWF-N1515Q (VWF-N1515Q-CC). This mutation creates a structurally constrained A2 due to the presence of a long-range disulfide bridge, homologous to those present in the A1 and A3 domains. (B) Clearance was assessed in VWF−/− mice. All results are plotted as percentage residual VWF:antigen levels relative to the amount injected. Data are presented as mean ± SEM.