Izquierdo Berjano, IreneNúñez Barrachina, MaríaHermida Nogueira, LidiaCasas, VanessaMorán Lara, Luis ArturoLacerenza, SerenaPinto-Llorente, RobertoEble, Johannes A.Ríos, Vivian de losDomínguez Medina, EduardoLoza García, María IsabelCasal, José IgnacioCarrascal, MontserratAbián, JoaquínGarcía Alonso, Ángel2020-02-052021-01-092020Thromb Haemost 2020; 120(02): 262-276. DOI: 10.1055/s-0039-34002950340-6245http://hdl.handle.net/10347/20681This is an Accepted Manuscript of an article published by Thieme Publishing Group in Thrombosis and Haemostasis on 04 January 2020, available online at https://www.thieme-connect.de/products/ejournals/abstract/10.1055/s-0039-3400295C-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 signalosomeeng© 2020 Georg Thieme Verlag KG.PlateletsCLEC-2 signalingTyrosine phosphoproteomejournal article10.1055/s-0039-3400295open access