Clustering extent-dependent differential signaling by CLEC-2 receptors in platelets.

Background: C-type lectin receptor family members play a role in many cells including platelets, where they are crucial in the separation of lymphatic and blood vessels during development. The C-type lectin-like receptor 2 (CLEC-2) receptor contains the canonical intracellular hemITAM motif through which it signals to activate Syk. Objectives: One proposed hypothesis for signaling cascade is that Syk bridges two receptors through phosphorylated hemITAM motifs. We demonstrated that the phosphorylated hemITAM stimulates PI3 kinase/Btk pathways to activate Syk. To address this controversy, we used a CLEC-2 selective agonist and studied the role of Btk in platelet activation. Results and Conclusions: Platelet activation and downstream signaling were abolished in murine and human platelets in the presence of the Btk inhibitors ibrutinib or acalabrutinib when a low concentration of a CLEC-2 antibody was used to crosslink CLEC-2 receptors. This inhibition was overcome by increasing concentrations of the CLEC-2 antibody. Similar results were obtained in X-linked immunodeficient mouse platelets, with an inactivating mutation in Btk or in Lyn null platelets. We conclude that at low crosslinking conditions of CLEC-2, Btk plays an important role in the activation of Syk, but at higher crosslinking conditions their role becomes less important and other mechanisms take over to activate Syk.