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Uncoupling of the ITIM receptor G6b-B from the tyrosine phosphatases Shp1 and Shp2 disrupts platelet homeostasis in mice

Mitchell J. Geer, Johanna P. van Geffen, Piraveen Gopalasingam, Timo Vögtle, Christopher W. Smith, Silke Heising, Marijke J. E. Kuijpers, Bibian M. E. Tullemans, Gavin E. Jarvis, Johannes A. Eble, Mark Jeeves, Michael Overduin, Johan W. M. Heemskerk, Alexandra Mazharian and Yotis A. Senis

Key points

  • Uncoupling of G6b-B from Shp1 and Shp2 results in severe macrothrombocytopenia and aberrant platelet function.

  • G6b-B inhibits CLEC-2 signaling primarily through the protein-tyrosine phosphatase Shp2.

Abstract

The immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing receptor G6b-B has emerged as a key regulator of platelet homeostasis. However, it remains unclear how it mediates its effects. Tyrosine phosphorylation of the ITIM and immunoreceptor tyrosine-based switch motif (ITSM) within the cytoplasmic tail of G6b-B provides a docking site for SH2 domain-containing protein-tyrosine phosphatases Shp1 and Shp2, which are also critical regulators of platelet production and function. In this study, we investigate the physiological consequences of uncoupling G6b-B from Shp1 and Shp2. To address this, we generated a transgenic mouse model expressing a mutant form of G6b-B in which tyrosine (Y) residues 212 and 238 within the ITIM and ITSM were mutated to phenylalanine (F), respectively. Mice homozygous for the mutation (G6b-B diY/F) were macrothrombocytopenic, due to a reduction in platelet production, had large clusters of megakaryocytes and myelofibrosis at sites of hematopoiesis, similar to that observed in G6b knockout (G6b KO) mice. Platelets from G6b-B diY/F mice were hypo-responsive to collagen, due to a significant reduction in expression of the immunoreceptor tyrosine-based activation motif (ITAM)-containing collagen receptor complex GPVI-FcR γ-chain, and thrombin, that could be partially rescued by co-stimulating the platelets with ADP. In contrast, platelets from G6b-B diY/F, G6b KO and megakaryocyte-specific Shp2 KO mice were hyper-responsive to antibody-mediated cross-linking of the hemi-ITAM-containing podoplanin receptor CLEC-2, suggesting that G6b-B inhibits CLEC-2-mediated platelet activation through Shp2. Findings from this study demonstrate that G6b-B must engage with Shp1 and Shp2 in order to mediate its regulatory effects on platelet homeostasis.

  • Submitted October 6, 2017.
  • Accepted June 5, 2018.