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

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.