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

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.

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.