Research Article

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

Aug 22, 2019

Open access
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Abstract
Data availability
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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

1. Vögtle et al
(2019) G6b-B in complex with dp12
Protein Data Bank, 6R0X.

http://www.rcsb.org/structure/6R0X

The following previously published data sets were used

1. Cai Z
2. Yarovoi S V
3. Zhu Z
4. Rauova L
5. Hayes V
6. Lebedeva T
7. Liu Q
8. Poncz M
9. Arepally G
10. Cines D B
11. Greene M I
(2015) Crystal structure of platelet factor 4 complexed with fondaparinux
Protein Data Bank, 4R9W.

https://www.rcsb.org/structure/4R9W
1. Dahms S O
2. Mayer M C
3. Roeser D
4. Multhaup G
5. Than M E
(2015) X-RAY STRUCTURE OF THE AMYLOID PRECURSOR PROTEIN-LIKE PROTEIN 1 (APLP1) E2 DOMAIN IN COMPLEX WITH A HEPARIN DODECASACCHARIDE
Protein Data Bank, 4RDA.

https://www.rcsb.org/structure/4RDA
(2008) Drosophila Robo IG1-2 (monoclinic form)
Protein Data Bank, 2VRA.

https://www.rcsb.org/structure/2VRA
(2000) CRYSTAL STRUCTURE OF A TERNARY FGF1-FGFR2-HEPARIN COMPLEX
Protein Data Bank, 1E0O.

https://www.rcsb.org/structure/1E0O
(2000) CRYSTAL STRUCTURE OF A TERNARY FGF2-FGFR1-HEPARIN COMPLEX
Protein Data Bank, 1FQ9.

https://www.rcsb.org/structure/1FQ9

Article and author information

Author details

Timo Vögtle

Institute of Cardiovascular Sciences, University of Birmingham, Birmingham, United Kingdom

Competing interests
No competing interests declared.

"This ORCID iD identifies the author of this article:" 0000-0002-9400-4701
Sumana Sharma

Cell Surface Signalling Laboratory, Wellcome Sanger Institute, Cambridge, United Kingdom

Competing interests
No competing interests declared.
Jun Mori

Institute of Cardiovascular Sciences, University of Birmingham, Birmingham, United Kingdom

Competing interests
No competing interests declared.

"This ORCID iD identifies the author of this article:" 0000-0002-6212-1604
Zoltan Nagy

Institute of Cardiovascular Sciences, University of Birmingham, Birmingham, United Kingdom

Competing interests
No competing interests declared.
Daniela Semeniak

Institute of Experimental Biomedicine, University Hospital Würzburg, Würzburg, Germany

Competing interests
No competing interests declared.
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.
Mitchell J Geer

Institute of Cardiovascular Sciences, University of Birmingham, Birmingham, United Kingdom

Competing interests
No competing interests declared.
Christopher W Smith

Institute of Cardiovascular Sciences, University of Birmingham, Birmingham, United Kingdom

Competing interests
No competing interests declared.
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.
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.
Riitta Lassila

Coagulation Disorders Unit, University of Helsinki, Helsinki, Finland

Competing interests
Riitta Lassila, is CSO and shareholder of Aplagon Oy, Helsinki, Finland.
Annukka Jouppila

Coagulation Disorders Unit, Helsinki University Hospital Research Institute, Helsinki, Finland

Competing interests
Annukka Jouppila, receives research funding from Aplagon Oy, Helsinki, Finland.
Alastair J Barr

Department of Biomedical Science, University of Westminster, London, United Kingdom

Competing interests
No competing interests declared.

"This ORCID iD identifies the author of this article:" 0000-0001-7738-8419
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.

"This ORCID iD identifies the author of this article:" 0000-0002-7751-5913
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.
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.
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.
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.
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.
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.
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.
Harald Schulze

Institute of Experimental Biomedicine, University Hospital Würzburg, Würzburg, Germany

Competing interests
No competing interests declared.
Gavin J Wright

Cell Surface Signalling Laboratory, Wellcome Trust Sanger Institute, Cambridge, United Kingdom

Competing interests
No competing interests declared.

"This ORCID iD identifies the author of this article:" 0000-0003-0537-0863
Alexandra Mazharian

Institute of Cardiovascular Sciences, University of Birmingham, Birmingham, United Kingdom

Competing interests
No competing interests declared.
Klaus Fütterer

School of Biosciences, University of Birmingham, Birmingham, United Kingdom

Competing interests
No competing interests declared.
Sundaresan Rajesh

Institute of Cancer and Genomic Sciences, University of Birmingham, Birmingham, United Kingdom

Competing interests
No competing interests declared.
Michael R Douglas

Institute of Inflammation and Ageing, University of Birmingham, Birmingham, United Kingdom

Competing interests
No competing interests declared.
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.

"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).

Copyright

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.