Model of Syk-mediated CLEC-2 signaling. (A) In WT platelets, Syk is phosphorylated after ligand binding initially on Y346 by the action of 1 or more Src kinases, followed by autophosphorylation of Y346. Phosphorylation of the CLEC-2 hemITAM leads to Syk recruitment and autophosphorylation at 519/20. This leads to activation of a downstream signaling pathway by Syk and Src (dotted line) that culminates in activation of PLCγ2. (B) In Syk^{R41Afl/fl;PF4-Cre} platelets, Syk is weakly phosphorylated on Y346 (dotted line) by Src, and Syk phosphorylates the CLEC-2 hemITAM in response to strong agonists (rhodocytin), but not weak agonists (antibody). Syk-R41A is not stably recruited to the phosphorylated hemITAM on CLEC-2 and is therefore unable to undergo autophosphorylation on Y519/20 and activate downstream signaling events. (C) In the presence of the Syk inhibitor PRT318, Syk-R41A is partially phosphorylated on Y346 by Src but is unable to phosphorylate the CLEC-2 hemITAM or undergo autophosphorylation. The increase in Src kinase phosphorylation of Y346 may reflect the loss of translocation of Syk to CLEC-2 through binding of its C-terminal SH2 domain.