Heparan sulfates are critical regulators of the inhibitory megakaryocyte-platelet receptor G6b-B

  1. Timo Vögtle
  2. Sumana Sharma
  3. Jun Mori
  4. Zoltan Nagy
  5. Daniela Semeniak
  6. Cyril Scandola
  7. Mitchell J Geer
  8. Christopher W Smith
  9. Jordan Lane
  10. Scott Pollack
  11. Riitta Lassila
  12. Annukka Jouppila
  13. Alastair J Barr
  14. Derek J Ogg
  15. Tina D Howard
  16. Helen J McMiken
  17. Juli Warwicker
  18. Catherine Geh
  19. Rachel Rowlinson
  20. W Mark Abbott
  21. Anita Eckly
  22. Harald Schulze
  23. Gavin J Wright
  24. Alexandra Mazharian
  25. Klaus Fütterer
  26. Sundaresan Rajesh
  27. Michael R Douglas
  28. Yotis A Senis  Is a corresponding author
  1. University of Birmingham, United Kingdom
  2. Wellcome Sanger Institute, United Kingdom
  3. University Hospital Würzburg, Germany
  4. Université de Strasbourg, France
  5. Sygnature Discovery Limited, United Kingdom
  6. University of Helsinki, Finland
  7. Helsinki University Hospital Research Institute, Finland
  8. University of Westminster, United Kingdom
  9. Peak Proteins Limited, United Kingdom
  10. 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

The following data sets were generated
The following previously published data sets were used

Article and author information

Author details

  1. Timo Vögtle

    Institute of Cardiovascular Sciences, University of Birmingham, Birmingham, United Kingdom
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9400-4701
  2. Sumana Sharma

    Cell Surface Signalling Laboratory, Wellcome Sanger Institute, Cambridge, United Kingdom
    Competing interests
    No competing interests declared.
  3. Jun Mori

    Institute of Cardiovascular Sciences, University of Birmingham, Birmingham, United Kingdom
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6212-1604
  4. Zoltan Nagy

    Institute of Cardiovascular Sciences, University of Birmingham, Birmingham, United Kingdom
    Competing interests
    No competing interests declared.
  5. Daniela Semeniak

    Institute of Experimental Biomedicine, University Hospital Würzburg, Würzburg, Germany
    Competing interests
    No competing interests declared.
  6. Cyril Scandola

    Institut National de la Santé et de la Recherche Médicale, Etablissement Français du Sang Grand Est, Université de Strasbourg, Strasbourg, France
    Competing interests
    No competing interests declared.
  7. Mitchell J Geer

    Institute of Cardiovascular Sciences, University of Birmingham, Birmingham, United Kingdom
    Competing interests
    No competing interests declared.
  8. Christopher W Smith

    Institute of Cardiovascular Sciences, University of Birmingham, Birmingham, United Kingdom
    Competing interests
    No competing interests declared.
  9. Jordan Lane

    Sygnature Discovery Limited, Nottingham, United Kingdom
    Competing interests
    Jordan Lane, at the time of the study, was employee at Sygnature Discovery Limited, performing surface plasmon resonance experiments as part of a paid service.
  10. Scott Pollack

    Sygnature Discovery Limited, Nottingham, United Kingdom
    Competing interests
    Scott Pollack, is employee at Sygnature Discovery Limited, performing surface plasmon resonance experiments as part of a paid service.
  11. Riitta Lassila

    Coagulation Disorders Unit, University of Helsinki, Helsinki, Finland
    Competing interests
    Riitta Lassila, is CSO and shareholder of Aplagon Oy, Helsinki, Finland.
  12. Annukka Jouppila

    Coagulation Disorders Unit, Helsinki University Hospital Research Institute, Helsinki, Finland
    Competing interests
    Annukka Jouppila, receives research funding from Aplagon Oy, Helsinki, Finland.
  13. Alastair J Barr

    Department of Biomedical Science, University of Westminster, London, United Kingdom
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7738-8419
  14. Derek J Ogg

    Peak Proteins Limited, Alderley Park, United Kingdom
    Competing interests
    Derek J Ogg, is employee at Peak proteins Limited, performing crystallography and protein expression studies as part of a paid service.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7751-5913
  15. Tina D Howard

    Peak Proteins Limited, Alderley Park, United Kingdom
    Competing interests
    Tina D Howard, is employee at Peak proteins Limited, performing crystallography and protein expression studies as part of a paid service.
  16. Helen J McMiken

    Peak Proteins Limited, Alderley Park, United Kingdom
    Competing interests
    Helen J McMiken, is employee at Peak proteins Limited, performing crystallography and protein expression studies as part of a paid service.
  17. Juli Warwicker

    Peak Proteins Limited, Alderley Park, United Kingdom
    Competing interests
    Juli Warwicker, is employee at Peak proteins Limited, performing crystallography and protein expression studies as part of a paid service.
  18. Catherine Geh

    Peak Proteins Limited, Alderley Park, United Kingdom
    Competing interests
    Catherine Geh, is employee at Peak proteins Limited, performing crystallography and protein expression studies as part of a paid service.
  19. Rachel Rowlinson

    Peak Proteins Limited, Alderley Park, United Kingdom
    Competing interests
    Rachel Rowlinson, is employee at Peak proteins Limited, performing crystallography and protein expression studies as part of a paid service.
  20. W Mark Abbott

    Peak Proteins Limited, Alderley Park, United Kingdom
    Competing interests
    W Mark Abbott, is CEO of Peak proteins Limited, performing crystallography and protein expression studies as part of a paid service.
  21. Anita Eckly

    Institut National de la Santé et de la Recherche Médicale, Etablissement Français du Sang Grand Est, Université de Strasbourg, Strasbourg, France
    Competing interests
    No competing interests declared.
  22. Harald Schulze

    Institute of Experimental Biomedicine, University Hospital Würzburg, Würzburg, Germany
    Competing interests
    No competing interests declared.
  23. Gavin J Wright

    Cell Surface Signalling Laboratory, Wellcome Trust Sanger Institute, Cambridge, United Kingdom
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0537-0863
  24. Alexandra Mazharian

    Institute of Cardiovascular Sciences, University of Birmingham, Birmingham, United Kingdom
    Competing interests
    No competing interests declared.
  25. Klaus Fütterer

    School of Biosciences, University of Birmingham, Birmingham, United Kingdom
    Competing interests
    No competing interests declared.
  26. Sundaresan Rajesh

    Institute of Cancer and Genomic Sciences, University of Birmingham, Birmingham, United Kingdom
    Competing interests
    No competing interests declared.
  27. Michael R Douglas

    Institute of Inflammation and Ageing, University of Birmingham, Birmingham, United Kingdom
    Competing interests
    No competing interests declared.
  28. Yotis A Senis

    Institute of Cardiovascular Sciences, University of Birmingham, Birmingham, United Kingdom
    For correspondence
    y.senis@bham.ac.uk
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0947-9957

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).

Reviewing Editor

  1. Pamela J Bjorkman, California Institute of Technology, United States

Publication history

  1. Received: March 14, 2019
  2. Accepted: August 21, 2019
  3. Accepted Manuscript published: August 22, 2019 (version 1)
  4. Version of Record published: September 12, 2019 (version 2)

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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  1. Timo Vögtle
  2. Sumana Sharma
  3. Jun Mori
  4. Zoltan Nagy
  5. Daniela Semeniak
  6. Cyril Scandola
  7. Mitchell J Geer
  8. Christopher W Smith
  9. Jordan Lane
  10. Scott Pollack
  11. Riitta Lassila
  12. Annukka Jouppila
  13. Alastair J Barr
  14. Derek J Ogg
  15. Tina D Howard
  16. Helen J McMiken
  17. Juli Warwicker
  18. Catherine Geh
  19. Rachel Rowlinson
  20. W Mark Abbott
  21. Anita Eckly
  22. Harald Schulze
  23. Gavin J Wright
  24. Alexandra Mazharian
  25. Klaus Fütterer
  26. Sundaresan Rajesh
  27. Michael R Douglas
  28. Yotis A Senis
(2019)
Heparan sulfates are critical regulators of the inhibitory megakaryocyte-platelet receptor G6b-B
eLife 8:e46840.
https://doi.org/10.7554/eLife.46840

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