Page 192 - Haematologica-April 2018

N. Wohmer et al.
VWF to LRP1 (data not shown), indicating that binding sites in these domains do not overlap. Second, introduction of the VWD-type 2B mutation leaves binding of VWF to SR- AI unaffected, as does the addition of ristocetin. Hence, VWF does not need to be in its active conformation to interact with SR-AI, whereas it does need to be for bind- ing to LRP1. As for the role of glycans present on the VWF molecule in the interaction with SR-AI, this could be the subject of further studies. However, neither the A1 domain nor the D4 domain contains glycan structures, suggesting that the interaction with SR-AI is mainly gly- can-independent. This does not exclude the possibility that glycans elsewhere in the protein could modulate this interaction, akin to what has previously been reported for the binding of the A1 domain to LRP1.11
Apart from binding to purified recombinant soluble SR- AI, we also observed a specific binding of VWF to cellular SR-AI (Figure 3). First, we used SR-AI-transfected HEK293 cells as a model system, and both classical immunofluo- rescent staining and the Duolink-PLA revealed selective binding of VWF to SR-AI. Second, the association of VWF with THP1-derived macrophages (as depicted in Figure 1) is at least in part mediated by SR-AI, as illustrated by the Duolink-PLA approach (Figure 3). Moreover, immuno- staining for VWF on THP1-derived macrophages was strongly reduced in the presence of antibodies Mab723 and Mab540, which interfere with SR-AI binding (Figure 4). Finally, when binding of VWF to primary bone mar- row-derived murine macrophages was tested, binding was reduced to near background levels for SR-AI-deficient macrophages compared to wt-macrophages (Figure 4). Being able to interact with SR-AI expressed on the cell sur- face supports a role of SR-AI as a clearance receptor for VWF. We analyzed this possibility by measuring VWFpp/VWF:Ag ratios of human VWF expressed in wt- and SR-AI-deficient mice. There were several reasons for choosing this approach over measuring classical VWF sur- vival. First, in a recent study we compared the clearance of two mutants in parallel via protein survival and via meas- uring VWFpp/VWF:Ag ratios.9 This analysis revealed that the VWFpp/VWF:Ag approach was clearly more sensitive than the classic protein survival experiments in detecting differences in VWF clearance, due to a markedly smaller error margin between mice. Second, this smaller error margin also favors the use of fewer mice in this type of experiments. For a classical clearance experiment, approx- imately 15 mice are included per molecule to be tested, whereas with the VWFpp/VWF:Ag approach fewer than ten mice per molecule are needed. Thus, from an animal ethical perspective this latter approach is to be preferred. Finally, expression in hepatocytes allows more homoge- nous post-translational processing compared to the pro- duction of proteins in distinct stable cells lines. Indeed, the lectin binding profile of hepatic VWF is similar to that of endothelial VWF.30 One might fear interference of clear- ance of hepatic VWF by endogenous endothelial-derived VWF. However, VWF clearance is similar in wt- and VWF- deficient mice,3 and even at VWF levels of 1500%, clear- ance remains unsaturated.
Compared to VWFpp/VWF:Ag ratios obtained for wt- mice (ratio=1.3) and LRP1-deficient mice (ratio=1.1), these ratios were strongly reduced in SR-AI-deficient mice (ratio=0.6). This not only points to SR-AI being a clearance
receptor for VWF, but also to SR-AI being more dominant in VWF clearance than LRP1. We anticipated that endoge- nous VWF levels would be increased in SR-AI-deficient mice compared to those in wt-mice. However, analysis of VWF levels did not reveal a statistically significant differ- ence between SR-AI-deficient and wt-mice. We believe that the lack of difference is due to the fact that the SR-AI- deficient and wt-mice were not true littermates, which complicates a direct comparison. Indeed, even among mice with a similar genetic background, the variation in VWF levels is substantial (e.g. 0.3-1.9 U/mL),12,31 which may explain the lack of difference between SR-AI-defi- cient and wt-mice. Of note, we observed that murine VWF efficiently interacts with murine SR-AI, indicating that the lack of difference is not because murine VWF is unable to interact with this receptor.
An intriguing aspect of VWF receptor interactions is how these are modulated by mutations in VWF, in partic- ular those mutations that are associated with increased clearance. We previously showed that VWD-type 2B mutations promote spontaneous binding to LRP1, explain- ing the increased clearance of these mutants. Here we examined two clearance mutants: VWF/p.R1205H and VWF/p.S2179F.1,3,5,8 Both mutants are known to be associ- ated with increased VWFpp/VWF:Ag ratios; in humans for VWF/p.S2179F and in human and mice for VWF/p.R1205H.5,7,8 Here we show that both mutants dis- play increased binding to SR-AI, both to purified SR-AI and SR-AI expressed on THP1-cells (Figure 6). Increased binding of the VWF/p.R1205H is in agreement with data reported by O’Donnell et al., who also observed increased binding of this mutant to macrophages. Increased binding to SR-AI may suggest that SR-AI contributes to the accel- erated removal of these mutants from the circulation. Indeed, VWFpp/VWF:Ag ratios for VWF/p.R1205H and VWF/p.S2179F were significantly reduced in SR-AI-defi- cient mice compared to wt-mice (Figure 7). However, even in the SR-AI-deficient mice, these ratios were substantially higher compared to wt-VWF, indicating that SR-AI is not the only receptor that mediates increased clearance of these mutants. We considered the option that LRP1 could play a role in the enhanced clearance of these mutants, and preliminary experiments revealed that both mutants did indeed display enhanced binding to LRP1 (data not shown). Apparently, enhanced receptor binding due to such clearance mutations is not always restricted to a sin- gle receptor, but may involve several receptors simultane- ously, thereby multiplying the clearance rate of the mutant proteins.
In summary, we identify SR-AI as a macrophage-specif- ic receptor for VWF, and this receptor may contribute to the increased clearance of certain VWF clearance mutants.
Acknowledgments
This study was supported by grants from the Agence Nationale de la Recherche (ANR-13-BSV1-0014; PJL), and the Fondation pour la Recherche Médicale (FRM-SPF20130526717; NW & PJL). We would like to thank Pascal Roux & Dr. Audrey Salles (Pasteur Institute, Paris, France) for their help in accessing the con- focal microscope facility and interpretation of optical microscopy data and Emilie Bouvier & Alexandre Diet (Center for Breeding & Distribution of Transgenic Animals, Orléans, France) for their tech- nical assistance.
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