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Keywords:

  • endocytosis;
  • factor VIII;
  • macrophages;
  • shear stress;
  • von Willebrand factor

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. Disclosure of Conflict of Interests
  9. References
  10. Supporting Information

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.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. Disclosure of Conflict of Interests
  9. References
  10. Supporting Information

Hemophilia A is an X-linked bleeding disorder that is characterized by the functional absence of coagulation factor VIII (FVIII). Treatment for this disorder involves intravenous infusion with either plasma-derived or recombinant FVIII. Multiple infusions per week are, however, required for effective treatment [1]. This is the consequence of a particularly fast clearance mechanism that eliminates FVIII from the circulation. To protect the protein from even faster clearance, FVIII is tightly associated in plasma with its carrier protein von Willebrand factor (VWF) [1–3]. Complex formation with VWF prolongs the half-life of FVIII from about 3 h to 12–14 h.

VWF circulates in plasma as a glycoprotein that consists of multiple domains, of which the N-terminal D’-D3 domains comprise a high affinity binding region for FVIII [4,5]. A unique characteristic of VWF is that it circulates as a multimeric protein, ranging in size from dimers to extremely large multimers. The molecular configuration and conformation of these VWF multimers can be modified by local shear stress conditions imposed by blood flow, or by treatment of VWF with the glycoprotein ristocetin [4,6,7]. It has been demonstrated that shear stress can change the configuration of VWF from a globular to a stretched form, which leads to the exposure of the binding region for the platelet receptor Gp1b [4,7]. This conformational change facilitates the biological role of VWF in the initial platelet plug formation at sites of vascular injury.

The low-density lipoprotein (LDL) receptor family members LDL receptor (LDLR) and LDL receptor-related protein (LRP) have been demonstrated to contribute to the clearance of FVIII from the circulation [8–12]. Evidence of a physiological role for these receptors in maintaining the FVIII plasma level has first been provided by utilizing Receptor Associated Protein (RAP) [11–13]. The infusion of this LRP-antagonist in mice prolongs the half-life of infused FVIII. A direct role of LRP and LDLR in the clearance of FVIII has been demonstrated employing conditional LRP and LRP/LDLR-deficient mice [9,10]. These mice exhibited a reduced clearance of infused human FVIII as well as increased levels of endogenous FVIII. It has recently been described that polymorphisms in LDLR modulate the activity levels of FVIII in humans [14].

The role of VWF in the LRP/LDL receptor-mediated clearance of FVIII has remained poorly understood. On the one hand, VWF effectively inhibits the direct binding of FVIII to LRP, and the LRP-dependent cellular uptake of FVIII [8]. The notion that FVIII circulates in tight complex with VWF may seem therefore incompatible with a role for LRP in the catabolism of FVIII. On the other hand, LRP and LDLR/LRP-deficient mice display elevated FVIII plasma levels and a concomitant increase in VWF [9,10]. These findings show that it is completely unclear how VWF fits into the LRP-dependent clearance pathway of FVIII. It has recently been suggested that macrophages from liver and spleen are able to clear the FVIII-VWF complex from the circulation [15]. This prompted us to reassess the uptake of FVIII and VWF by monocyte-derived human macrophages, and the role of the LRP-like proteins therein. We now show that human monocyte-derived macrophages indeed are able to internalize FVIII and VWF in an apparently LRP-dependent manner, but that this process only occurs under conditions of shear stress.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. Disclosure of Conflict of Interests
  9. References
  10. Supporting Information

Proteins and chemicals

Fine chemicals employed in this study were all from Merck (Darmstadt, Germany) unless otherwise stated. The antibody against early endosome antigen 1 (EEA1) was from Becton and Dickinson (Breda, the Netherlands). The rabbit polyclonal antibody against lysosome-associated-protein-1 (LAMP-1) was from Abcam (Cambridge, UK). RAP, FVIII-GFP and recombinant VWF, as well as their plasma-derived counterparts, were purified as described previously [16,17]. Cell trace carboxyfluorescein diacetate succinimidyl ester (CFSE) was from Invitrogen (Breda, the Netherlands). The antibody EL-14 was cloned from its single-chain variable fragment as described [18,19]. Monoclonal antibody CLB-RAg20 is described by Stel et al. 1984 [20]. The anti-human antibodies CD80-FITC, CD83-APC, CD86-APC, CD206-APC, CD16, CD32 and CD64 were from BD Biosciences (San Jose, CA, USA). Anti-human CD14-PE was from Sanquin Reagents (Amsterdam, the Netherlands). Anti-human CD209-APC was from AbD Serotec (Düsseldorf, Germany). Anti-human CD91-PE was from Santa Cruz Biotechnology (Heidelberg, Germany).

Culturing of monocyte-derived macrophages

Monocytes were isolated from peripheral blood by centrifugation on a Ficoll-Paque gradient (GE Healthcare Biosciences, Piscataway, NJ, USA), and sorted using CD14+ magnetic beads (MACS; Miltenyi Biotec, Auburn, CA, USA) as described by Nicholson et al. [21]. Cells were seeded on glass coverslips in six-well plates in growth medium comprising RPMI 1640 (Lonza, Breda, the Netherlands) supplemented with 10% heat-inactivated FCS, 100 U mL−1 penicillin and 100 μg mL−1 streptomycin. Cells were allowed to differentiate for 6 days into macrophages in the presence of 50 ng mL−1 M-CSF. Cells were characterized by flow cytometry [16] and were found positive for CD14, CD64, CD32, CD16, CD91 (LRP) and CD206 (macrophage mannose receptor), and negative for CD209 (DC-SIGN) and CD83.

Protein uptake by macrophages

Proteins employed in these experiments were from recombinant origin unless otherwise indicated. Glass coverslips comprising macrophages were incubated with the proteins in 10 mm HEPES (pH 7.4), 135 mm NaCl, 10 mm KCl, 5 mm CaCl2 and 2 mm MgSO4 for 30 min at 37 °C. Cells were subsequently washed with 10 mm HEPES (pH 7.4), 135 mm NaCl, 10 mm KCl, 5 mm CaCl2 and 2 mm MgSO4, followed by a wash with phosphate buffered saline (PBS) (Fresenius Kabi, Den Bosch, the Netherlands). Cells were then fixed for 30 min at 4 °C with 4% (v/v) paraformaldehyde in PBS [16,17]. For protein uptake experiments under conditions of shear stress, the glass coverslips comprising macrophages were mounted in the parallel-plate flow chamber assembly as described by the manufacture (Glyco Tech, Gaithersburg, MD, USA). Cells were subsequently perfused in the flow chamber with FVIII-GFP and/or VWF in the presence and absence of RAP in the above-described buffer at different shear rates for 30 min at 37 °C. The employed shear rate was calculated according to the instructions provided by the manufacturer. The cells were subsequently washed and fixed as described above.

Protein detection in or on macrophages with confocal microscopy

Fixed cells were washed with PBS and incubated for 1 h at room temperature with anti-EEA1 IgG1 antibody, anti-LAMP1 polyclonal antibody or anti-LRP IgG1 antibody or anti-VWF IgG2b (CLB-Rag20) antibody or combinations thereof. To this end, anti-EEA1, anti-LRP and anti-LAMP1 antibody were diluted 500-fold in PBS, 1% (v/v) HSA (Sanquin Plasma Products, Amsterdam, the Netherlands) and 0.02% (w/v) saponin, which permeabilizes the cells. Anti-VWF antibody CLB-Rag20 was diluted 100-fold in the same buffer. After removal of the unbound antibody by washing with PBS, the cells were incubated for 1 h at room temperature with the secondary antibodies Alexa Fluor 563 conjugated anti-IgG1 antibody, Alexa Flour 563 anti-IgG2b antibody, Alexa Fluor 633 conjugated anti-IgG2b antibody or Alexa fluor 488 anti-Rabbit IgG, or combinations thereof (Invitrogen). These antibodies were diluted 500-fold in PBS, 1% (v/v) HSA and 0.02% (w/v) saponin. Finally, cells were washed with PBS, mounted in Mowiol, and examined on a Zeiss LSM510 employing appropriate filter settings to detect GFP fluorescence, Alexa Fluor 563 fluorescence, Alexa Fluor 633 fluorescence and Alexa Fluor 488 fluorescence, and by using a Plan-Neofluar 40 ×/1.3 Oil immersion lens (Carl Zeiss BV, Sliedrecht, the Netherlands) as described [16,17]. To discriminate between cell-surface bound FVIII-GFP and intracellular FVIII-GFP, fixed cells were incubated with anti-FVIII Alexa Fluor 563 conjugated antibody EL14 in PBS and 1% (v/v) HSA with and without 0.02% (w/v) saponin. After washing the cells with PBS, the macrophages were mounted in Mowiol and Alexa 563 anti-FVIII EL14 and FVIII-GFP fluorescence was detected as described above.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. Disclosure of Conflict of Interests
  9. References
  10. Supporting Information

VWF blocks the endocytic uptake of FVIII by human macrophages

To study the uptake of FVIII by human macrophages, we employed monocyte-derived macrophages and a functional fluorescent FVIII-GFP derivative [16,17]. The macrophages were incubated with 20 nm FVIII-GFP for 30 min at 37 °C. Confocal microscopy analysis revealed that the majority of FVIII-GFP was targeted to vesicles that are positive for the early endosomal marker EEA1 (Fig. 1A). As LRP mediates FVIII endocytosis by cells [8,11,16], we also stained the cells for LRP. These data showed that macrophages express LRP, and that this receptor co-localizes with FVIII (Fig. 1B). We subsequently incubated FVIII-GFP with the macrophages in the presence of RAP, which effectively antagonizes LRP and its related receptors [8,11,13,16]. Confocal microscopy studies now showed an almost complete absence of FVIII-GFP fluorescence (Fig. 1C). This observation suggests that LRP may mediate the uptake of FVIII by macrophages. Because previous studies suggested that macrophages may also internalize the FVIII-VWF complex [15], we assessed uptake of FVIII-GFP also in the presence of VWF. After incubation of the complex with the macrophages, however, we did not detect any FVIII-GFP fluorescence (Fig. 1D,E). Replacing the B-domain-deleted FVIII-GFP with that of plasma-derived FVIII also did not result in detection of FVIII (via immunofluorescence staining) in the macrophages in the presence of plasma-derived VWF (Fig. S1). These findings are in full agreement with our initial report that VWF effectively blocks the LRP-dependent FVIII uptake by other cells [8]. It suggests that FVIII is internalized by macrophages via an LRP-dependent pathway that is effectively blocked by VWF.

image

Figure 1.  Monocyte-derived macrophages internalize FVIII in a RAP-sensitive manner. Macrophages were incubated for 30 min at 37 °C with 20 nm FVIII-GFP in the (A,B) absence or (C) presence of 1 μm RAP or (D,E) 240 nm VWF. FVIII-GFP fluorescence is shown in green in all panels. LRP is immunostained in red in panel B. The early endosomes in the panels A, C, D and E are displayed in red employing immunofluorescence staining of EEA1. Sites of co-localization appear yellow. The scale bars represent 10 μm in panels A–D and 20 μm in panel E. A split view of a zoomed-in area of the macrophages is shown in panels A–D.

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VWF interacts with macrophages under flow conditions

We assessed whether or not VWF itself interacts with macrophages. To this end, these cells were incubated with 75 nm VWF for 30 min at 37 °C. However, VWF-positive cells could not be identified by confocal microscopy (Fig. 2A). This shows that under the employed conditions neither the FVIII-VWF complex nor VWF alone is internalized by macrophages. As the conformation of VWF is susceptible to flow conditions [4,6,7], we subsequently evaluated the effect of shear stress on the interaction between macrophages and VWF. For this purpose, 75 nm VWF was perfused over the macrophages for 30 min at 37 °C under increasing conditions of shear stress (Fig. 2B–F). Interestingly, VWF was now identified by confocal microscopy in or on the macrophages, especially at shear stress levels up to and above 4 dyn cm−2. This finding shows that VWF interacts with macrophages under conditions of shear stress, but not under static conditions.

image

Figure 2.  VWF interacts with primary human macrophages under conditions of shear stress. (A–F) 75 nm VWF was perfused over macrophages at the indicated level of shear stress. Immunofluorescence staining of VWF is shown in green. Cells were stained in red employing celltrace CFSE. The white scale bars represent 10 μm.

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VWF is internalized by macrophages under conditions of shear stress

The possibility exists that shear stress alters macrophages, leading to the expression of a cellular mechanism for interaction with VWF. However, subjecting the cells to a shear stress of 9 dyn cm−2 for 30 min at 37 °C prior to immediate incubation with VWF under static conditions did not result in any detection of VWF on these cells (not shown). This suggests that there is either a particularly short-lived transient expression of this shear-dependent mechanism on macrophages, or it is not present at all. Incubation of plasma-derived or recombinant VWF with ristocetin, which induces a shear-like dependent conformational change in VWF [6], did result in effective staining of VWF on the macrophage (Fig. 3). Co-localization studies employing a marker of the early endosomes revealed that part of VWF was inside the cells. To establish whether, apart from ristocetin, shear stress mediates the cellular uptake of VWF as well, we perfused VWF over the macrophages at 9 dyn cm−2 for 30 min at 37 °C. The results revealed again that VWF co-localizes with the early endosomes (Fig. 4A,B). This suggests that VWF is indeed taken up by the macrophages. Figure 4(A,B) further shows that VWF staining is partially outside the early endosomes. Co-localization studies employing lysosome-associated protein-1, which is a marker for both late endosomes and lysosomes, revealed that the majority of VWF was outside these vesicles (Fig. S2). Apparently, after 30 min of shear stress, VWF is still located at the cell surface, or in the early endosomes. Intriguingly, there was no co-localization between VWF and the early endosomes in the presence of RAP (Fig. 4C). This finding suggests that LRP or related receptors contribute to the transfer of VWF into the early endocytic compartment of the cell. These findings together imply that shear stress exposes a binding site within VWF that mediates internalization of VWF by macrophages.

image

Figure 3.  VWF is internalized by macrophages in the presence of ristocetin. (A) 75 nm plasma-derived VWF or (B) 75 nm recombinant VWF were incubated with macrophages for 30 min at 37 °C in the presence of 1 mg mL−1 ristocetin. Immunofluorescence staining of VWF is shown in green, and the early endosomes are immunostained via EEA1 in red. Sites of co-localization appear yellow. The white scale bars represent 10 μm.

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image

Figure 4.  RAP-sensitive receptors contribute to VWF internalization under shear stress. 50 nm VWF was perfused over macrophages at 9 dyn cm−2 for 30 min at 37 °C in the (A,B) absence or (C) presence of 1 μm RAP. Immunofluorescence staining of VWF is shown in green, and the early endosomes are immunostained via EEA1 in red. Sites of co-localization appear yellow. The white scale bars represent 10 μm. Panel A shows a side-view of the displayed macrophage. This view was obtained employing z-stack analysis of the macrophage. Panels B and C display a split view of a zoomed-in area of the displayed macrophage.

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Shear stress is required to mediate the uptake of FVIII by macrophages in the presence of VWF

We evaluated the role of flow on the uptake of FVIII in the presence and absence of VWF. Macrophages were perfused with FVIII-GFP or the (FVIII-GFP)-VWF complex at 9 dyn cm−2 for 30 min at 37 °C. Confocal microscopy revealed a fluorescent FVIII-GFP punctate pattern in the absence of VWF (Fig. 5A,B) and in the presence of VWF (Fig. 5C). This observation shows that FVIII-GFP interacts with macrophages regardless of the presence of VWF. To assess whether FVIII-GFP is inside the cells, we made optimal use of the fact that the fluorescent anti-FVIII antibody EL14 can only associate with intracellular FVIII-GFP when saponin is included in the staining procedure. This compound is required to permeabilize the cells, which only then allows the transfer of EL14 to the inside of the cell for optimal association to FVIII-GFP. The green intrinsic fluorescence of FVIII-GFP will always be visible irrespective of whether the cells are permeabilized by saponin or not. In Fig. 5(A), the cells were permeabilized with saponin, resulting in co-localization of the fluorescence of EL14 and FVIII-GFP. In Fig. 5(B), we excluded saponin in the staining procedure. EL14 can now only bind FVIII-GFP that is located at the cell surface. Figure 5(B) shows that most of the green fluorescence of FVIII-GFP does not co-localize with the red fluorescence of EL14. This observation demonstrates that FVIII-GFP is inside the cell in the absence of VWF. In Fig. 5(C), we incubated the (FVIII-GFP)-VWF complex with the macrophages under conditions of shear stress, and we stained FVIII-GFP with the red fluorescent EL14 in the absence of saponin. The result showed again that most of the fluorescence derived from FVIII-GFP does not co-localize with that of the fluorescence of EL14. This finding suggests that FVIII-GFP is also inside the cells in the presence of VWF, as FVIII-GFP is unreachable for EL14 for effective association.

image

Figure 5.  FVIII is internalized by macrophages in the presence of VWF and shear stress. 20 nm FVIII-GFP was perfused over macrophages for 30 min at 9 dyn cm−2 in the (A,B) absence or (C) presence of 240 nm VWF or (D,E) 240 nm VWF and 1 μm RAP. FVIII-GFP fluorescence is shown in green in the panels A–E. In panel A, the cells were permeabilized with saponin to allow for intracellular staining of FVIII-GFP with the red fluorescent monoclonal anti-FVIII antibody EL14. In panels B–D only extracellular FVIII can be stained with the fluorescent EL14 antibody as no saponin was employed to permeabilize the cells in the staining procedure. Panel E shows immunofluorescence staining of VWF in red employing saponin in the staining procedure. The white scale bars in panels A and D represent 5 μm, and in panels B, C and E 10 μm.

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To address the role of LRP or related receptors in this process, we subsequently perfused the (FVIII-GFP)-VWF complex over the cells in the presence of RAP. The results revealed that FVIII-GFP is still detectable in or on the macrophages under these conditions. However, permeabilization of the cells by saponin was now not required for effective association of the fluorescent EL14 to FVIII-GFP (Fig. 5D). Apparently, FVIII-GFP is located at the cell surface in the presence of VWF and RAP. Visualizing VWF and FVIII-GFP after perfusing the (FVIII-GFP)-VWF complex over the macrophages in the presence of RAP revealed an almost complete co-localization of both proteins (Fig. 5E). The results shown in Fig. 5(D,E) imply that VWF is together with FVIII at the cell surface when LRP and related receptors are blocked for interaction with their ligands. This fits with the finding that VWF is not in the early endosomes of the cells in the presence of RAP (Fig. 4C). These data suggest that the constituents of the FVIII-VWF complex are internalized by the macrophages in a RAP-sensitive manner under conditions of shear stress.

VWF and FVIII are both inside the macrophages under conditions of shear stress

To obtain additional evidence that both VWF and FVIII are taken up by macrophages under conditions of shear stress, we performed co-localization studies with early endosomes, FVIII, and VWF. Figure 6 displays macrophages over which the (FVIII-GFP)-VWF complex was perfused for 30 min at 9 dyn cm−2. The result shows a considerable overlap between the fluorescent staining for VWF, the early endosome marker EEA1 and the GFP fluorescence of FVIII-GFP. This finding implies that both proteins are indeed internalized by macrophages. We also observed a partial co-localization of plasma-derived FVIII with the early endosomes in the presence of plasma-derived VWF or recombinant VWF under these conditions (Fig. S1), which implies that the recombinant proteins behave the same as their plasma-derived counterparts. The results of this study suggest that under shear stress conditions, macrophages can internalize FVIII, VWF and the complex thereof in a RAP-sensitive manner, suggesting the involvement of LRP or related receptors.

image

Figure 6.  FVIII-GFP and VWF are internalized under flow conditions. (A,B) 20 nm FVIII-GFP and 240 nm VWF were perfused over macrophages at 9 dyn cm−2 for 30 min at 37 °C. Immunostaining of VWF is shown in white, FVIII-GFP fluorescence is shown in green, and the early endosomes are immunostained via EEA1 in red. Sites of co-localization appear yellow. Panel B shows an overlay of the fluorescence of FVIII-GFP (green), VWF (white) and EEA1 (red) from two macrophages. Two side views from the area of the cell, indicated by the white frame, are depicted at the right side of the panel. The side views were obtained employing z-stack analysis of the cells. The white scale bars represent 10 μm.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. Disclosure of Conflict of Interests
  9. References
  10. Supporting Information

Our previous studies have shown that the level of VWF is, like FVIII, elevated in mice that are deficient in LRP and/or LDLR [9,10]. This would be compatible with a role of these receptors not only in the catabolism of FVIII, but also of VWF. In the same time, however, this would seem incompatible with the observation that purified VWF is not a ligand of LRP, and with the present finding that VWF effectively blocks the uptake of FVIII by macrophages (Fig. 1D,E) and other LRP-expressing cells [8]. In this regard it is intriguing that VWF does not protect FVIII from LRP-dependent endocytosis under conditions of shear stress (Figs 5 and 6). Activation of VWF by shear stress may therefore represent an essential step in the molecular mechanism behind the LRP-dependent clearance of FVIII. Strikingly, as RAP blocks the trafficking of VWF to the early endosomes, VWF itself seems to be a ligand for LRP on macrophages under these conditions (Figs 4 and 5). This finding is independently confirmed by a very recent study that was published during the review period of the present study [22]. Rastegarlari et al. showed that selective knockdown of LRP from macrophages leads to a reduced clearance of VWF from the circulation. Moreover, VWF-coated beads adhered to LRP but only when exposed to shear forces exceeding 2.5 dyn cm−2 [22]. These and our observations show that LRP, LDL receptor and related receptors may be constituents of a previously unrecognized pathway that contributes to the clearance of not only FVIII but of VWF as well. This explains why VWF levels are elevated in LRP/LDLR-deficient mice [9,10].

Under conditions of high shear stress (> 30 dyn cm−2), the multimeric VWF undergoes a structural rearrangement of its monomers as well as in its internal domains [4,6,7]. Our results now show that VWF exposes functional regions involved in the cellular uptake of VWF at a markedly lower level of shear stress (Figs 2–6). The exposure of this region in VWF also seems to promote the interaction of FVIII with the macrophages, as VWF blocks the interaction between the cells and FVIII in the absence of sheer stress. It even seems possible that VWF mediates an indirect assembly of FVIII with LRP as VWF is known to shield the LRP binding sites on FVIII [8]. This would explain the finding that both VWF and FVIII-GFP are found in the same early endosomes (Fig. 6). It cannot be excluded, however, that part of the FVIII-VWF complex dissociates at the cell surface, and that FVIII-GFP is internalized by macrophages independently of VWF.

The results show that FVIII and VWF remain at the cell surface in the presence of RAP (Fig. 5D,E). This finding suggests that there is a two-step mechanism that facilitates the uptake of the FVIII-VWF complex. One step involves the binding of sheared-VWF to the cell surface, and the second step involves the internalization of the constituents of the complex via LRP and related receptors. The notion that FVIII is detected at the cell surface in the presence of RAP but not in its absence suggests that cell surface binding of VWF is the rate limiting step (Fig. 5C,D). Blocking LRP by RAP allows for concentration of the complex at the cell surface, which facilitates detection of FVIII-GFP thereon (Fig. 5D). Such a two-step endocytic uptake mechanism is not uncommon for ligands of the LDL receptor family. These ligands usually comprise an overlapping binding region for the receptor and for cell surface structural elements, like sulphated glycans [8,16,23–25]. Intriguingly, VWF has been suggested to also comprise distinct binding regions for glycosulphatides and heparin within the A1 domain, which becomes exposed under conditions of shear stress [26,27]. It is tempting to speculate that this cryptic region contributes to the binding of sheared VWF to the cell surface prior to the transfer of VWF and FVIII to LRP. Alternatively, shear stress may mediate the binding of VWF to activated integrins on the cell surface [22,28]. Under such conditions, VWF may also bind to activated integrins on the macrophage surface prior to VWF internalization via LRP.

Previously, van Schooten et al. [15] showed that human monocyte-derived macrophages are capable of binding VWF at 4 °C. The results from our study do not exclude this possibility. Our findings show, however, that these macrophages do not internalize VWF at 37 °C (Fig. 2A). The cellular uptake of VWF by macrophages was investigated by van Schooten et al. in more detail utilizing THP1-cell-derived macrophages as a substitute for primary macrophages. These cells internalized VWF under static conditions via a rather atypical endocytic mechanism because VWF homogeneously diffused into the cells instead of appearing in typical endocytic vesicles. Employing iodine-labeled VWF, data were obtained suggesting that THP1-cell-derived macrophages internalize and degrade VWF [15]. Using unmodified VWF, however, we were unable to detect any interaction of VWF with THP1-cell-derived macrophages under static conditions (data not shown). The reason for this discrepancy remains unclear. We believe that our current data, together with the work of Rastegarlari et al. [22], provide firm evidence that LRP-dependent uptake of VWF by human monocyte-derived macrophages requires VWF activation by shear stress (Figs 2–6).

The physiological relevance of shear stress for uptake of FVIII-VWF complex remains to be established. In vivo studies have shown that macrophages from liver and spleen are involved in the uptake of FVIII-VWF complex [15,22]. Our current study implies that shear stress could be an important determinant for endocytic uptake to occur in macrophages. At first glance, it may seem unlikely that high shear rates can be reached in the liver or in the spleen. Yet, we observed interaction between VWF and the macrophages at a shear stress level of 4 dyn cm−2, which is markedly lower than the shear force that exists in the arterial circulation. Interestingly, the sinusoidal endothelial cells, which constitute the vascular lining of the capillaries of the liver and spleen, comprise narrow fenestrae that allow the passage of only small particles to the underlying tissue [29]. Scanning electron microscopy has revealed that Kupffer cells exhibit long filopodia that penetrate these fenestrae [30]. Local shear conditions in these narrow fenestrae may be sufficiently high to trigger the endocytosis of the FVIII-VWF complex by Kupffer cells. Earlier studies have further suggested that local shear stress in the spleen can induce a deformation or even rupture of erythrocytes at the fenestrae of the sinusoids in the spleen [31]. High-shear conditions in these tissues may therefore be likely as well. Taken together, we propose that shear stress drives the uptake of the constituents of FVIII-VWF complex by macrophages.

Acknowledgement

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. Disclosure of Conflict of Interests
  9. References
  10. Supporting Information

This study was supported by research funding from Landsteiner Stichting voor Bloedtransfusie Research (grant no. LSBR-0730) to ABM.

Disclosure of Conflict of Interests

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. Disclosure of Conflict of Interests
  9. References
  10. Supporting Information

The authors state that they have no conflict of interest.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. Disclosure of Conflict of Interests
  9. References
  10. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. Disclosure of Conflict of Interests
  9. References
  10. Supporting Information

Figure S1. (A) 20 nM plasma derived FVIII was incubated with macrophages for 30 min at 37 °C under static conditions. (B,C) 20 nM plasma derived FVIII was perfused over macrophages for 30 min at 9 dyn cm−2 in the presence of (B) 240 nM plasma derived VWF or (C) 240 nM recombinant VWF. FVIII was visualized via immunofluorescence staining employing the fluorescent anti-FVIII antibody EL14. FVIII is shown in red, and the early endosomes are immunostained via EEA1 in green. Sites of co-localization appear yellow. The white scale bars represent 10 μm.

Figure S2. 75 M VWF was perfused over macrophages at 9 dyn cm−2 for 30 min at 37 °C. Immunofluorescence staining of VWF is shown in red and lysosome-associated-protein-1 (LAMP1) is immunostained in green. Sites of co-localization appear yellow. The white scale bar represents 10 μm. A side view of the macrophage is displayed below the image. This was obtained via Z-stack analysis of the displayed macrophage.

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