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.
Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), Universidade de Santiago de Compostela, Santiago de Compostela, Spain.
Andrés F
AF
Parguiña
Jana
J
Alonso
Isaac
I
Rosa
Paula
P
Vélez
María J
MJ
González-López
Esteban
E
Guitián
Johannes A
JA
Eble
Johannes A
JA
Ebble
María I
MI
Loza
Ángel
Á
García
D001792:Blood Platelets / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D001798:Blood Proteins / Q000032:analysis; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000097:blood / Q000378:metabolism*; D037181:Lectins, C-Type / Q000378:metabolism* / Q000502:physiology*; D008562:Membrane Glycoproteins / Q000378:metabolism* / Q000502:physiology*; D010750:Phosphoproteins / Q000032:analysis / Q000097:blood / Q000378:metabolism; D015539:Platelet Activation / Q000187:drug effects*; D011485:Protein Binding / Q000187:drug effects; D020543:Proteome / Q000032:analysis* / Q000187:drug effects; D040901:Proteomics / Q000379:methods; D015398:Signal Transduction / Q000187:drug effects; D057825:Two-Dimensional Difference Gel Electrophoresis; D014443:Tyrosine / Q000378:metabolism; D054928:Validation Studies as Topic; D014757:Viper Venoms / Q000494:pharmacology*
D001798:Blood Proteins; C104778:CLEC2B protein, human; D047908:Intracellular Signaling Peptides and Proteins; D037181:Lectins, C-Type; D008562:Membrane Glycoproteins; D010750:Phosphoproteins; D020543:Proteome; D014757:Viper Venoms; C112292:rhodocytin protein, Calloselasma rhodostoma; D014443:Tyrosine