A detailed proteomic analysis of rhodocytin-activated platelets reveals novel clues on the CLEC-2 signalosome: implications for CLEC-2 signaling regulation

C-type lectin-like receptor 2 (CLEC-2) is an essential platelet-activating receptor in hemostasis and thrombosis that is activated by the snake venom rhodocytin. We present here a differential proteomic analysis of basal and rhodocytin-activated platelets with the aim of providing novel clues on CLEC-2 signaling regulation. Proteome analysis was based on 2D-DIGE, phosphotyrosine immunoprecipitations followed by 1D SDS-PAGE and mass spectrometry. Protein-protein interactions were studied by coimmunoprecipitations and a systems biology approach. Overall, we identified 132 proteins differentially regulated after CLEC-2 platelet activation, including most of the major players reported so far in the signaling cascade. In addition, we identified various proteins not previously known to participate in CLEC-2 signaling, such as the adapters Dok-2 and ADAP, tyrosine kinase Fer, and tyrosine phosphatase SHIP-1. We also report an increased association between Dok-2 and SHIP-1 in rhodocytin-stimulated platelets, which might negatively regulate CLEC-2 signaling. Moreover, we also present a comparative analysis of proteomic data for CLEC-2 and glycoprotein VI signaling. We think that our data provide thrombosis-relevant information on CLEC-2 signaling regulation, contributing to a better understanding of this important signaling cascade.

This work was supported by the Spanish Ministry of Economy and Competitiveness (MINECO; grant SAF2010-22151), the European regional development fund, and the Galician Government (Consellería de Educación, Xunta de Galicia, Spain; grant INCITE09PXIB203145PR). A.G. is a Ramón y Cajal Research fellow (MINECO). A.F.P. is an FPI predoctoral fellow (MINECO). J.A.E. received financial support from the Deutsche Forschungsgemeinschaft (SFB/TR23, project A8).

Peer reviewed