Page 185 - Haematologica-April 2018

SR-AI contributes to VWF clearance
ed uptake by macrophages.10,11 This is in contrast to non- modified VWF, which only binds to macrophage- expressed LRP1 when exposed to increased shear stress.12 Apart from LRP1, other receptors have also been described to contribute to VWF clearance, including asialoglycopro- tein receptor, CLEC4M and Siglec-5.13-16
Despite the various receptors having been identified, their functional absence is usually associated with a mild- to-modest effect on VWF clearance. We previously observed that the majority of VWF is targeted to macrophages, and that chemical depletion of macrophages results in a 2- to 3-fold increase in VWF lev- els.17,18 We therefore explored the hypothesis that macrophages express one or more additional receptors that contribute to VWF clearance. Here, we present data that are compatible with the macrophage-specific receptor Scavenger receptor class A member I (SR-AI) being a clear- ance-receptor for VWF. Moreover, we show that two clearance mutants (VWF/p.R1205H and VWF/p.S2179F) display enhanced binding to SR-AI, providing a potential explanation for their reduced half-life in the circulation.
Methods
Ethics statement
Animal housing and experiments were done as recommended by French regulations and the experimental guidelines of the European Community. This project was approved by the local ethical committee CEEA 26 (# 2012-036).
Proteins
A detailed description of the proteins used in this study is pro- vided in the Online Supplementary Material. The main proteins included plasma-derived (pd)-VWF purified from VWF concen- trates (Wilfactin) and recombinant full-length VWF [wild-type (wt)-VWF, VWF/p.R1205H, VWF/p.V1316M, and VWF/p.S2179F] produced in stably transfected BHK-furin cells using serum-free medium. All variants displayed a similar distribution of multimers (data not shown). Non-purified cell culture supernatants were used for protein- and cellular binding experiments. Other proteins are detailed in the Online Supplementary Material.
Cell culture
Detailed information on cell cultures is provided in the Online Supplementary Material. Briefly, human macrophages were differ- entiated from the THP1 acute monocytic leukemia cell line using phorbol 12-myristate 13-acetate, human macrophage colony- stimulating factor and human granulocyte-macrophage colony- stimulating factor as described elsewhere.18,19 Non-transfected and stable HEK293 cell lines expressing human SR-AI were cultured as described presviously.19 Murine macrophages were obtained from CD115+ cells as described by Breslin et al.20
Cellular binding experiments
A detailed description of cellular binding experiments is provid- ed in the Online Supplementary Material. Briefly, cells seeded on glass coverslips were incubated with purified pd-VWF (10 mg/mL) for 1 h at 37°C. Where indicated, culture medium containing recombinant wt-VWF, VWF/p.R1205H, VWF/p.V1316M or VWF/p.S2179F was used. In other experiments, pd-VWF was pre- incubated with monoclonal antibodies to VWF (MAb723 and MAb540, 167 mg/mL) for 30 min at room temperature. Following incubation, cells were gently washed twice and then fixed for 15 min with 4% paraformaldehyde at 37°C.
Microscopy analyses and immunofluorescence-based quantification
A detailed description of the microscopic analysis is provided in the Online Supplementary Material. Briefly, after saturation of non- specific binding sites, cells were exposed to primary antibodies for 2 h at room temperature, followed by incubation for 1 h with sec- ondary antibodies. Nuclei were counterstained with 4’,6’-diamidi- no-2-phenylindole. Alexa-Fluor647 or Alexa-Fluor488-labeled phalloïdin was used to determine cell boundaries.
For the Duolink-Proximity Ligation Assay (Duolink-PLA) to detect close proximity between different proteins, double immunostaining was performed as described above with the sec- ond antibodies replaced by PLA probes (Sigma-Aldrich). The remainder of the protocol was conducted according to the manu- facturer’s recommendations and the 550 nm wavelength detection kit was used. Hybridization between the two PLA probes leading to the fluorescent signal only occurs when the distance between the two detected antigens is less than 40 nm.
Images were analyzed using ImageJ software for quantification of fluorescence by measuring the total pixel intensity per cell. Duolink-PLA experiments were analyzed using BlobFinder soft- ware (Uppsala University, Sweden) to quantify the number of flu- orescent spots per cell. All images were assembled using ImageJ software.
Immunosorbent binding assay
Binding experiments are described in detail in the Online Supplementary Material.
Mice
Wild-type C57Bl/6 mice were purchased from Janvier Labs (Le Genest-Saint-Isle, France) and SR-AI-deficient C57Bl/6 mice (B6.Cg-Msr1tm1Csk/J) were from The Jackson Laboratory (Bar Harbor, ME, USA). Wild-type mice and SR-AI-deficient mice were not true littermates. MacLRP1-positive and macLRP1-deficient mice were described previously.12 MacLRP1 mice have a C57Bl/6 back- ground, with >12 backcross steps.
von Willebrand factor propeptide to antigen ratios
cDNA encoding human wild-type VWF, mutant VWF/p.R1205H or mutant VWF/p.S2179F was cloned into the pLIVE-plasmid (Mirus Bio LLC, Madison, WI, USA) and expressed in wild-type, macLRP1 and/or SR-AI-deficient mice following hydrodynamic gene transfer, essentially as described elsewhere.9, 21-23 Four days after injection, blood samples were taken via retro- orbital puncture and plasma was prepared for the analysis of VWF propeptide (VWFpp; cat# MW1939; Sanquin Blood Supply, Amsterdam, the Netherlands) and VWF:Ag (in-house enzyme- linked immunosorbent assay using a pool of monoclonal murine anti-VWF antibodies). Both assays are specific for human VWFpp and VWF:Ag, and do not detect murine proteins. VWF:Ag levels varied between 300% and 800% of normal for all constructs. VWF clearance remains unsaturated by VWF levels up to 1500%. The presence of endogenous VWF in the mouse strains used in this study does, therefore, leave clearance of hepatocyte-expressed human VWF unaffected.
Results
von Willebrand factor binds to macrophages in an LRP1-independent manner
Previously, we demonstrated that VWF binds to LRP1 in a shear-stress-dependent manner or when it contains type 2B gain-of-function mutations.9,12 This specific binding was
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