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Growth factor receptor-bound protein 2 contributes to (hem)immunoreceptor tyrosine-based activation motif-mediated
Sebastian Dütting1, Timo Vögtle1, Martina Morowski1
1Department of Experimental Biomedicine, University Hospital Würzburg (S.D., T.V., M.M., S.S., B.N.) and Rudolf Virchow Center for Experimental Biomedicine (S.D., T.V., C.M.S., H.M.H., B.N.), University of Würzburg, Würzburg, Germany; Centre for Cardiovascular Sciences, Institute for Biomedical Research, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom (S.K.W., C.E.H., S.P.W.); and Department of Biology, Division of Genetics, University of Erlangen, Erlangen, Germany (J.A.A., D.R., L.N.).
Rationale:
Platelets are anuclear cell fragments derived from bone marrow megakaryocytes (MKs) that safeguard vascular integrity but may also cause pathological vessel occlusion. One major pathway of platelet activation is triggered by 2 receptors that signal through an (hem)immunoreceptor tyrosine-based activation motif (ITAM), the activating collagen receptor glycoprotein (GP) VI and the C-type lectin-like receptor 2 (CLEC-2). Growth factor receptor-bound protein 2 (Grb2) is a ubiquitously expressed adapter molecule involved in signaling processes of numerous receptors in different cell types, but its function in platelets and MKs is unknown.
Objective:
We tested the hypothesis that Grb2 is a crucial adapter protein in (hem)immunoreceptor tyrosine-based activation motif signaling in platelets.
Methods And Results:
Here, we show that genetic ablation of Grb2 in MKs and platelets did not interfere with MK differentiation or platelet production. However, Grb2-deficiency severely impaired glycoprotein VI-mediated platelet activation because of defective stabilization of the linker of activated T-cell (LAT) signalosome and activation of downstream signaling proteins that resulted in reduced adhesion, aggregation, and coagulant activity on collagen in vitro. Similarly, CLEC-2-mediated signaling was impaired in Grb2-deficient platelets, whereas the cells responded normally to stimulation of G protein-coupled receptors. In vivo, this selective (hem)immunoreceptor tyrosine-based activation motif signaling defect resulted in prolonged bleeding times but affected arterial thrombus formation only after concomitant treatment with acetylsalicylic acid, indicating that defective glycoprotein VI signaling in the absence of Grb2 can be compensated through thromboxane A2-induced G protein-coupled receptor signaling pathways.
Conclusions:
These results reveal an important contribution of Grb2 in (hem)immunoreceptor tyrosine-based activation motif signaling in platelets in hemostasis and thrombosis by stabilizing the LAT signalosome.
Insights
Growth factor receptor-bound protein 2 (Grb2) is essential for platelet activation via glycoprotein VI and CLEC-2 signaling, impacting hemostasis and thrombosis. Its absence impairs collagen-mediated responses but can be compensated by other pathways.
Area of Science:
- Hematology
- Molecular Biology
- Cell Signaling
Background:
- Platelets are critical for vascular integrity but can cause pathological occlusion.
- Platelet activation involves receptors like glycoprotein VI (GPVI) and CLEC-2, signaling via (hem)immunoreceptor tyrosine-based activation motifs (ITAMs).
- The role of the adapter protein Growth factor receptor-bound protein 2 (Grb2) in platelets and megakaryocytes (MKs) was previously unknown.
Purpose of the Study:
- To investigate the function of Grb2 in platelet and MK signaling.
- To test the hypothesis that Grb2 is crucial for ITAM signaling in platelets.
Main Methods:
- Genetic ablation of Grb2 in MKs and platelets.
- Assessment of MK differentiation and platelet production.
- Analysis of GPVI- and CLEC-2-mediated platelet activation.
- Evaluation of platelet adhesion, aggregation, and coagulant activity.
- In vivo studies of bleeding times and arterial thrombus formation.
Main Results:
- Grb2 deficiency did not affect MK differentiation or platelet production.
- Grb2-deficient platelets showed impaired GPVI- and CLEC-2-mediated activation due to defective LAT signalosome stabilization.
- Reduced platelet adhesion, aggregation, and coagulant activity on collagen were observed.
- In vivo, Grb2 deficiency led to prolonged bleeding times.
- Arterial thrombus formation was affected only after aspirin treatment, suggesting compensatory pathways.
Conclusions:
- Grb2 plays a significant role in ITAM signaling in platelets.
- Grb2 stabilizes the LAT signalosome, which is crucial for GPVI and CLEC-2 signaling.
- These findings highlight Grb2's importance in hemostasis and thrombosis.
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