Shear stress is required for the endocytic uptake of the factor VIII‐von Willebrand factor complex by macrophages

Abstract

Summary. Background: Low‐density lipoprotein (LDL) receptor family members contribute to the cellular uptake of factor VIII. How von Willebrand factor fits into this endocytic pathway has remained poorly understood. Objectives: It has been suggested that macrophages contribute to the clearance of the factor VIII (FVIII)‐von Willebrand factor (VWF) complex. We now assessed the mechanisms of uptake employing human monocyte‐derived macrophages. Methods: A confocal microscopy study was employed to study the uptake by monocyte‐derived macrophages of a functional green fluorescent FVIII‐GFP derivative in the presence and absence of VWF. Results: The results revealed that FVIII‐GFP is internalized by macrophages. We found that FVIII‐GFP co‐localizes with LDL receptor‐related protein (LRP), and that the LRP antagonist Receptor Associated Protein (RAP) blocks the uptake of FVIII‐GFP. However, FVIII‐GFP was not detected in the macrophages in the presence of VWF, suggesting that the FVIII‐VWF complex is not internalized by these cells at all. Apart from static conditions, we also investigated the effect of shear stress on the uptake of FVIII‐GFP in presence of VWF. Immunofluorescence studies demonstrated that VWF does not block endocytosis of FVIII‐GFP under flow conditions. Moreover, VWF itself was also internalized by the macrophages. Strikingly, in the presence of RAP, endocytosis of FVIII‐GFP and VWF was inhibited. Conclusion: The results show that shear stress is required for macrophages to internalize both constituents of the FVIII‐VWF complex.

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

“…In keeping with previous reports, 21,22 confocal microscopy studies performed following incubation at 4°C demonstrated that VWF-WT, VWF-N1515Q, and VWF-N1574Q all bound to differentiated THP-1 cells in vitro (supplemental Figure 2). When incubation studies were performed at 37°C, confocal studies showed colocalization of VWF-WT, VWF-N1515Q, and VWF-N1574Q with early endosomes (supplemental Figure 2), demonstrating that both VWF-N1515Q and VWF-N1574Q are taken up by macrophages.…”

“…Recent studies have shown that hepatic macrophages contribute to the clearance of full-length VWF. [21][22][23][24][25][26] To determine whether macrophages modulate A1-A2-A3 clearance in vivo, clearance studies were repeated following clodronate-induced macrophage depletion. In keeping with previous studies, clearance of full-length rVWF was significantly reduced following macrophage depletion ( Figure 1B).…”

“…[15][16][17][18][19][20] Recent evidence further suggests that hepatic and splenic macrophages may play key roles in mediating VWF clearance. [21][22][23][24][25][26] For example, differentiated primary human macrophages can bind and endocytose purified VWF in vitro. 21 Furthermore, macrophage depletion significantly prolonged the in vivo survival of VWF infused into VWF 2/2 mice.…”

The C1 and C2 domains of blood coagulation factor VIII mediate its endocytosis by dendritic cells

“…42 In addition, under shear stress, VWF fails to block FVIII internalization by macrophages, and both VWF and FVIII co-localize within the same cells. 43 This is in agreement with the observation that exogenous FVIII and exogenous VWF may be co-detected in splenic macrophages after injection into double FVIII/VWF-deficient mice. 44 The internalization of FVIII and VWF was, however, inhibited by the LRP antagonist receptor-associated protein (RAP) in the case of macrophages under shear stress, but not in the case of MoDCs in static conditions, 8 suggesting that this process is more relevant to FVIII catabolism than immunogenicity.…”

von Willebrand factor binds to the surface of dendritic cells and modulates peptide presentation of factor VIII

“…Human serum albumin was supplied by Sanquin Blood Supply, Amsterdam, the Netherlands. B-domain-deleted FVIII 14 and recombinant VWF 15 were cultured in 6320 cm 2 factories (Nunc, Roskilde, Denmark) in DMEM/F12 (Lonza, Walkerville, MD, USA) supplemented with 10% heat inactivated fetal calf serum (Bodinco, Alkmaar, the Netherlands), which is stepwise reduced to 8% FCS. Medium was concentrated using the hemoflow F5 HPS system (Fresenius Medical Care, Bad Homburg vor der Höhe, Germany) before protein purification.…”