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

Castro-Núñez, Lydia; Dienava-Verdoold, I.; Herczenik, Eszter; Mertens, Koen; Meijer, Alexander B.

doi:10.1111/j.1538-7836.2012.04860.x

Cited by 53 publications

(55 citation statements)

References 35 publications

(74 reference statements)

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“…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.…”

Section: Accelerated Clearance Of Vwf N1515q and Vwf N1574q Is Mediatsupporting

confidence: 91%

“…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).…”

Section: Resultsmentioning

confidence: 99%

“…[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.…”

Section: Introductionmentioning

confidence: 99%

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

Chion

O’Sullivan

Drakeford

et al. 2016

Blood

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Key Points• The A1 domain of VWF contains a cryptic binding site that plays a key role in regulating macrophage binding and clearance.• The N-linked glycans presented at N1515 and N1574 within the A2 domain of VWF modulate macrophage-mediated clearance.Enhanced von Willebrand factor (VWF) clearance is important in the etiology of von Willebrand disease. However, the molecular mechanisms underlying VWF clearance remain poorly understood. In this study, we investigated the role of VWF domains and specific glycan moieties in regulating in vivo clearance. Our findings demonstrate that the A1 domain of VWF contains a receptor-recognition site that plays a key role in regulating the interaction of VWF with macrophages. In A1-A2-A3 and full-length VWF, this macrophage-binding site is cryptic but becomes exposed following exposure to shear or ristocetin. Previous studies have demonstrated that the N-linked glycans within the A2 domain play an important role in modulating susceptibility to ADAMTS13 proteolysis. We further demonstrate that these glycans presented at N1515 and N1574 also play a critical role in protecting VWF against macrophage binding and clearance. Indeed, loss of the N-glycan at N1515 resulted in markedly enhanced VWF clearance that was significantly faster than that observed with any previously described VWF mutations. In addition, A1-A2-A3 fragments containing the N1515Q or N1574Q substitutions also demonstrated significantly enhanced clearance. Importantly, clodronate-induced macrophage depletion significantly attenuated the increased clearance observed with N1515Q and N1574Q in both full-length VWF and A1-A2-A3.Finally, we further demonstrate that loss of these N-linked glycans does not enhance clearance in VWF in the presence of a structurally constrained A2 domain. Collectively, these novel findings support the hypothesis that conformation of the VWF A domains plays a critical role in modulating macrophage-mediated clearance of VWF in vivo. (Blood. 2016;128(15):1959-1968

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Section: Accelerated Clearance Of Vwf N1515q and Vwf N1574q Is Mediatsupporting

confidence: 91%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

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

Chion

O’Sullivan

Drakeford

et al. 2016

Blood

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“…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.…”

Section: Discussionsupporting

confidence: 90%

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

et al. 2016

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T he development of inhibitory antibodies to therapeutic factor VIII is the major complication of replacement therapy in patients with hemophilia A. The first step in the initiation of the anti-factor VIII immune response is factor VIII interaction with receptor(s) on antigenpresenting cells, followed by endocytosis and presentation to naïve CD4 + T cells. Recent studies indicate a role for the C1 domain in factor VIII uptake. We investigated whether charged residues in the C2 domain participate in immunogenic factor VIII uptake. Co-incubation of factor VIII with BO2C11, a monoclonal C2-specific immunoglobulin G, reduced factor VIII endocytosis by dendritic cells and presentation to CD4 + T cells, and diminished factor VIII immunogenicity in factor VIII-deficient mice. The mutation of basic residues within the BO2C11 epitope of C2 replicated reduced in vitro immunogenic uptake, but failed to prevent factor VIII immunogenicity in mice. BO2C11 prevents factor VIII binding to von Willebrand factor, thus potentially biasing factor VIII immunogenicity by perturbing its half-life. Interestingly, a factor VIII Y1680C mutant, that does not bind von Willebrand factor, demonstrated unaltered endocytosis by dendritic cells as well as immunogenicity in factor VIII-deficient mice. Co-incubation of factor VIII Y1680C with BO2C11, however, resulted in decreased factor VIII immunogenicity in vivo. In addition, a previously described triple C1 mutant showed decreased uptake in vitro, and reduced immunogenicity in vivo, but only in the absence of endogenous von Willebrand factor. Taken together, the results indicate that residues in the C1 and/or C2 domains of factor VIII are implicated in immunogenic factor VIII uptake, at least in vitro. Conversely, in vivo, the binding to endogenous von Willebrand factor masks the reducing effect of mutations in the C domains on factor VIII immunogenicity.

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“…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.…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2015

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It has been proposed that von Willebrand factor might affect factor VIII immunogenicity by reducing factor VIII uptake by antigen presenting cells. Here we investigate the interaction of recombinant von Willebrand factor with immature monocyte-derived dendritic cells using flow cytometry and confocal microscopy. Surprisingly, von Willebrand factor was not internalized by immature dendritic cells, but remained bound to the cell surface. As von Willebrand factor reduces the uptake of factor VIII, we investigated the repertoire of factor VIII presented peptides when in complex with von Willebrand factor. Interestingly, factor VIII-derived peptides were still abundantly presented on major histocompatibility complex class II molecules, even though a reduction of factor VIII uptake by immature dendritic cells was observed. Inspection of peptide profiles from 5 different donors showed that different core factor VIII peptide sequences were presented upon incubation with factor VIII/von Willebrand factor complex when compared to factor VIII alone. No von Willebrand factor peptides were detected when immature dendritic cells were pulsed with different concentrations of von Willebrand factor, confirming lack of von Willebrand factor endocytosis. Several von Willebrand factor derived peptides were recovered when cells were pulsed with von Willebrand factor/factor VIII complex, suggesting that factor VIII promotes endocytosis of small amounts of von Willebrand factor by immature dendritic cells. Taken together, our results establish that von Willebrand factor is poorly internalized by immature dendritic cells. We also show that von Willebrand factor modulates the internalization and presentation of factor VIII-derived peptides on major histocompatibility complex class II.

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

Cited by 53 publications

References 35 publications

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

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

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

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

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