Galectin-9 activates platelet ITAM receptors glycoprotein VI and C-type lectin-like receptor-2

Zhaogong Zhi, Natalie J Jooss, Yi Sun, Martina Colicchia, Alexandre Slater, Luis A Moran, Hilaire Yam Fung Cheung, Ying Di, Julie Rayes, Natalie S Poulter, Steve P Watson, Asif J Iqbal

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Abstract

BACKGROUND: Platelets are multifunctional cellular mediators in many physiological and pathophysiological processes such as thrombosis, angiogenesis, and inflammation. Several members of galectins, a family of carbohydrate-binding proteins with a broad range of immunomodulatory actions, have been reported to activate platelets.

OBJECTIVE: In this study, we investigated the role of galectin-9 (Gal-9) as a novel ligand for platelet glycoprotein VI (GPVI) and C-type lectin-like receptor 2 (CLEC-2).

METHODS: Platelet spreading, aggregation, and P-selectin expression in response to Gal-9 were measured in washed platelet suspensions via static adhesion assay, light transmission aggregometry, and flow cytometry, respectively. Solid-phase binding assay and protein phosphorylation studies were utilized to validate the interaction between Gal-9 and GPVI, and immunoprecipitation for detecting CLEC-2 phosphorylation. Wild-type (WT), GPVI-knockout (Gp6-/-), and GPVI and CLEC-2-double knockout (Gp6-/-/Gp1ba-Cre-Clec1bfl/fl) mice were used.

RESULTS: We have shown that recombinant Gal-9 stimulates aggregation in human and mouse washed platelets dose-dependently. Platelets from both species adhere and spread on immobilized Gal-9 and express P-selectin. Gal-9 competitively inhibited the binding of human recombinant D1 and D2 domains of GPVI to collagen. Gal-9 stimulated tyrosine phosphorylation of CLEC-2 and proteins known to lie downstream of GPVI and CLEC-2 including spleen tyrosine kinase and linker of activated T cells in human platelets. GPVI-deficient murine platelets exhibited significantly impaired aggregation in response to Gal-9, which was further abrogated in GPVI and CLEC-2-double-deficient platelets.

CONCLUSIONS: We have identified Gal-9 as a novel platelet agonist that induces activation through interaction with GPVI and CLEC-2.

Original languageEnglish
Number of pages15
JournalJournal of thrombosis and haemostasis : JTH
Early online date22 Dec 2021
DOIs
Publication statusE-pub ahead of print - 22 Dec 2021

Bibliographical note

© 2021 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis.

Keywords

  • C- type lectin-like receptor 2
  • galectin-9
  • glycoprotein VI
  • immunoreceptor tyrosine-based activation motif
  • platelet

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