Heparan sulfates are critical regulators of the inhibitory megakaryocyte-platelet receptor G6b-B
- University of Birmingham, United Kingdom
- Wellcome Sanger Institute, United Kingdom
- University Hospital Würzburg, Germany
- Université de Strasbourg, France
- Sygnature Discovery Limited, United Kingdom
- University of Helsinki, Finland
- Helsinki University Hospital Research Institute, Finland
- University of Westminster, United Kingdom
- Peak Proteins Limited, United Kingdom
- Wellcome Trust Sanger Institute, United Kingdom
Abstract
The immunoreceptor tyrosine-based inhibition motif (ITIM)-containing receptor G6b-B is critical for platelet production and activation. Loss of G6b-B results in severe macrothrombocytopenia, myelofibrosis and aberrant platelet function in mice and humans. Using a combination of immunohistochemistry, affinity chromatography and proteomics, we identified the extracellular matrix heparan sulfate (HS) proteoglycan perlecan as a G6b-B binding partner. Subsequent in vitro biochemical studies and a cell-based genetic screen demonstrated that the interaction is specifically mediated by the HS chains of perlecan. Biophysical analysis revealed that heparin forms a high-affinity complex with G6b-B and mediates dimerization. Using platelets from humans and genetically-modified mice, we demonstrate that binding of G6b-B to HS and multivalent heparin inhibits platelet and megakaryocyte function by inducing downstream signaling via the tyrosine phosphatases Shp1 and Shp2. Our findings provide novel insights into how G6b-B is regulated and contribute to our understanding of the interaction of megakaryocytes and platelets with glycans.
Data availability
Diffraction data have been deposited in PDB under the accession code 6R0X
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Crystal structure of platelet factor 4 complexed with fondaparinuxProtein Data Bank, 4R9W.
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Drosophila Robo IG1-2 (monoclinic form)Protein Data Bank, 2VRA.
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CRYSTAL STRUCTURE OF A TERNARY FGF1-FGFR2-HEPARIN COMPLEXProtein Data Bank, 1E0O.
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CRYSTAL STRUCTURE OF A TERNARY FGF2-FGFR1-HEPARIN COMPLEXProtein Data Bank, 1FQ9.
Article and author information
Author details
Funding
British Heart Foundation (RG/15/13/31673)
- Jun Mori
- Alexandra Mazharian
- Yotis A Senis
British Heart Foundation (FS/13/1/29894)
- Yotis A Senis
Deutsche Forschungsgemeinschaft (VO 2134-1/1)
- Timo Vögtle
Medical Research Council (Confidence in Concept 2018)
- Timo Vögtle
- Yotis A Senis
Wellcome (206194)
- Gavin J Wright
British Heart Foundation (FS/15/58/31784)
- Alexandra Mazharian
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: All animal procedures were undertaken with the U.K. Home Office approval (project license No P46252127) in accordance with the Animals (Scientific Procedures) Act of 1986.
Human subjects: Blood was collected from healthy drug-free volunteers. Donors gave full informed consent according to the Helsinki declaration. Ethical approval for collecting blood was granted by Birmingham University Internal Ethical Review (ERN_11-0175 and ERN_15-0973).
Copyright
© 2019, Vögtle et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Heparan sulfates are critical regulators of the inhibitory megakaryocyte-platelet receptor G6b-B
eLife 8:e46840.
https://doi.org/10.7554/eLife.46840
Further reading
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Teichoic acids (TA) are linear phospho-saccharidic polymers and important constituents of the cell envelope of Gram-positive bacteria, either bound to the peptidoglycan as wall teichoic acids (WTA) or to the membrane as lipoteichoic acids (LTA). The composition of TA varies greatly but the presence of both WTA and LTA is highly conserved, hinting at an underlying fundamental function that is distinct from their specific roles in diverse organisms. We report the observation of a periplasmic space in Streptococcus pneumoniae by cryo-electron microscopy of vitreous sections. The thickness and appearance of this region change upon deletion of genes involved in the attachment of TA, supporting their role in the maintenance of a periplasmic space in Gram-positive bacteria as a possible universal function. Consequences of these mutations were further examined by super-resolved microscopy, following metabolic labeling and fluorophore coupling by click chemistry. This novel labeling method also enabled in-gel analysis of cell fractions. With this approach, we were able to titrate the actual amount of TA per cell and to determine the ratio of WTA to LTA. In addition, we followed the change of TA length during growth phases, and discovered that a mutant devoid of LTA accumulates the membrane-bound polymerized TA precursor.
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