Critical redundant functions of the adapters Grb2 and Gads in platelet (hem)ITAM signaling in mice

Abstract

Platelets are essential for normal hemostasis; however, pathological conditions can also trigger unwanted platelet activation precipitating thrombosis and ischemic damage of vital organs such as the heart or brain. Glycoprotein (GP)VI- and C-type lectin-like receptor 2 (CLEC-2)-mediated (hem)immunoreceptor tyrosine-based activation motif (ITAM) signaling represents a major pathway for platelet activation. The two members of the Growth-factor receptor-bound protein 2 (Grb2) family of adapter proteins expressed in platelets – Grb2 and Grb2-related adapter protein downstream of Shc (Gads) – are part of the hem(ITAM) signaling cascade by forming an adapter protein complex with linker for activation of T cells (LAT). To date, a possible functional redundancy between these two adapters in platelet activation has not been investigated. We here generated megakaryocyte- and platelet-specific Grb2/Gads double knockout (DKO) mice and analyzed their platelet function in vitro and in vivo. The DKO platelets exhibited virtually abolished (hem)ITAM signaling whereas only partial defects were seen in Grb2 or Gads single-deficient platelets. This was based on impaired phosphorylation of key molecules in the (hem)ITAM signaling cascade and translated into impaired hemostasis and partially defective arterial thrombosis, thereby exceeding the defects in either Grb2 KO or Gads KO mice. Despite this severe (hem)ITAM signaling defect, CLEC-2 dependent regulation of blood-lymphatic vessel separation was not affected in the DKO animals. These results provide direct evidence for critically redundant roles of Grb2 and Gads for platelet function in hemostasis and thrombosis, but not development.

Keywords:

• Adapter proteins
• hemostasis
• ITAM
• mice
• signaling

Key Points

• The adapter proteins Grb2 and Gads have additive roles in platelet (hem)ITAM signaling, activation, and hemostasis in mice.

• Grb2 and Gads have a minor role in thrombosis and are dispensable for CLEC-2-dependent regulation of blood-lymphatic vessel separation.

Acknowledgements

This study was supported by the Deutsche Forschungsgmeinschaft (SFB/TR 240, project number 374031971). We thank Sylvia Hengst and Birgit Midloch for excellent technical assistance, Dr. Craig Hughes for advice and shipping Gads-deficient mice and Dr. Katharina Remer for support with documentation and management of animal experimentation and welfare. A.A.B. and J.V. were supported by a grant of the German Excellence Initiative to the Graduate School of Life Sciences, University of Würzburg. S.P.W is a British Heart Foundation Chair (CH003/03).

Authorship contributions

T.V. and A.A.B. performed experiments, analyzed data and wrote the manuscript. J.V., T.B.D., and S.D. performed experiments and analyzed data. I.P. analyzed data and critiqued the manuscript, L.N. provided key resources, S.P.W. provided key resources, analyzed data and critiqued the manuscript. B.N. conceived the study, designed research, analyzed data and wrote the manuscript.

Declaration of interest statement

The authors declare no competing financial interests.

Supplementary material

Supplemental data for this article can be accessed here.

Additional information

Funding

This work was supported by the Deutsche Forschungsgemeinschaft [SFB/TR 240, 374031971]; British Heart Foundation [CH003/03]; German Excellence Initiative and Graduate School of Life Sciences