Thromb Haemost 2020; 120(02): 262-276
DOI: 10.1055/s-0039-3400295
Cellular Haemostasis and Platelets
Georg Thieme Verlag KG Stuttgart · New York

A Comprehensive Tyrosine Phosphoproteomic Analysis Reveals Novel Components of the Platelet CLEC-2 Signaling Cascade

Irene Izquierdo
1  Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), Universidad de Santiago de Compostela, and Instituto de Investigación Sanitaria (IDIS), Santiago de Compostela, Spain
,
María N. Barrachina
1  Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), Universidad de Santiago de Compostela, and Instituto de Investigación Sanitaria (IDIS), Santiago de Compostela, Spain
,
Lidia Hermida-Nogueira
1  Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), Universidad de Santiago de Compostela, and Instituto de Investigación Sanitaria (IDIS), Santiago de Compostela, Spain
,
Vanessa Casas
2  CSIC/UAB Proteomics Laboratory, Institute of Biomedical Research of Barcelona, Spanish National Research Council (IIBB-CSIC/IDIBAPS), Barcelona, Spain
,
Luis A. Morán
1  Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), Universidad de Santiago de Compostela, and Instituto de Investigación Sanitaria (IDIS), Santiago de Compostela, Spain
,
Serena Lacerenza
3  Department of Pharmacy, University of Pisa, Pisa, Italy
,
Roberto Pinto-Llorente
2  CSIC/UAB Proteomics Laboratory, Institute of Biomedical Research of Barcelona, Spanish National Research Council (IIBB-CSIC/IDIBAPS), Barcelona, Spain
,
Johannes A. Eble
4  Institute of Physiological Chemistry and Pathobiochemistry, University of Münster, Münster, Germany
,
Vivian de los Ríos
5  Proteomics Facility, Centro de Investigaciones Biológicas (CIB-CSIC), Madrid, Spain
,
Eduardo Domínguez
1  Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), Universidad de Santiago de Compostela, and Instituto de Investigación Sanitaria (IDIS), Santiago de Compostela, Spain
,
María I. Loza
1  Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), Universidad de Santiago de Compostela, and Instituto de Investigación Sanitaria (IDIS), Santiago de Compostela, Spain
,
José Ignacio Casal
5  Proteomics Facility, Centro de Investigaciones Biológicas (CIB-CSIC), Madrid, Spain
,
Montserrat Carrascal
2  CSIC/UAB Proteomics Laboratory, Institute of Biomedical Research of Barcelona, Spanish National Research Council (IIBB-CSIC/IDIBAPS), Barcelona, Spain
,
Joaquín Abián
2  CSIC/UAB Proteomics Laboratory, Institute of Biomedical Research of Barcelona, Spanish National Research Council (IIBB-CSIC/IDIBAPS), Barcelona, Spain
,
Angel García
1  Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), Universidad de Santiago de Compostela, and Instituto de Investigación Sanitaria (IDIS), Santiago de Compostela, Spain
› Author Affiliations
Funding This study was supported by the Spanish Ministry of Economy and Competitiveness (MINECO) [grant No. SAF2016-79662-R], co-funded by the European Regional Development Fund (ERDF); and the Consellería de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia [ED431C 2018/21; predoctoral grant Plan I2C 2014; and Centro Singular de investigación de Galicia accreditation 2016-2019, ED431G/05], co-funded by the European Regional Development Fund (ERDF). The study also received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 766118. J.A.E. is supported by Deutsche Forschungsgemeinschaft [DFG grant: EB177/13-1].
Further Information

Publication History

01 July 2019

08 October 2019

Publication Date:
04 January 2020 (online)

Abstract

C-type lectin-like receptor 2 (CLEC-2) plays a crucial role in different platelet-related physiological and pathological processes. It signals through a tyrosine kinase-mediated pathway that is highly dependent on the positive feedback exerted by the platelet-derived secondary mediators, adenosine diphosphate (ADP) and thromboxane A2 (TXA2). Here, we aimed to analyze the tyrosine phosphoproteome of platelets activated with the CLEC-2 agonist rhodocytin to identify relevant phosphorylated tyrosine residues (p-Tyr) and proteins involved in platelet activation downstream of this receptor. We identified 363 differentially p-Tyr residues, corresponding to the majority of proteins previously known to participate in CLEC-2 signaling and also novel ones, including adaptors (e.g., DAPP1, Dok1/3, CASS4, Nck1/2), kinases/phosphatases (e.g., FAK1, FES, FGR, JAK2, SHIP2), and membrane proteins (e.g., G6F, JAM-A, PECAM-1, TLT-1). To elucidate the contribution of ADP and TXA2 at different points of the CLEC-2 signaling cascade, we evaluated p-Tyr levels of residues identified in the analysis and known to be essential for the catalytic activity of kinases Syk(p-Tyr525+526) and Src(p-Tyr419), and for PLCγ2 activity (p-Tyr759). We demonstrated that Syk phosphorylation at Tyr525+526 also happens in the presence of ADP and TXA2 inhibitors, which is not the case for Src-pTyr419 and PLCγ2-pTyr759. Kinetics studies for the three phosphoproteins show some differences in the phosphorylation profile. Ca2+ mobilization assays confirmed the relevance of ADP and TXA2 for full CLEC-2-mediated platelet activation. The present study provides significant insights into the intracellular events that take place following CLEC-2 activation in platelets, contributing to elucidate in detail the CLEC-2 signalosome.

Authors' Contributions

I.I.: performed research, analyzed data, and wrote the paper. M.N.B., L.H.-N., V.C., L.A.M., S.L., R.P.-L., and V.d.l.R,: performed research. J.I.C., M.C., and J.A.: contributed with key analytical tools and analyzed and integrated mass spec data. J.A.E.: provided vital reagents. E.D. and M.I.L.: provided vital reagents and analytical tools for the calcium release experiments. A.G.: designed research, analyzed data, provided vital reagents and analytical tools, and wrote the paper.