PB0395 - Avidity-dependent differential signaling by CLEC-2 receptors

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 CLEC-2 receptor contains the canonical intracellular hemITAM motif through which it signals to activate Syk. One proposed hypothesis for the signaling cascade is that Syk bridges two receptors through phosphorylated hemITAM motifs. We demonstrated that the phosphorylated hemITAM activates PI3 kinase/Tec kinase pathways to activate Syk.

Aims: To address this controversy, we used a CLEC-2 selective agonist and studied the role of Tec kinases in platelet activation.

Methods: We activated platelets with different concentrations of CLEC-2 monoclonal antibodies in the presence and absence of different inhibitors.

Results: Platelet activation and downstream signaling were abolished in murine and human platelets in the presence of the Tec kinase inhibitor Ibrutinib 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 Lyn null platelets or X-linked immunodeficient (Xid) mouse platelets, with an inactivating mutation in Btk.

Conclusion(s): We conclude that at low crosslinking (avidity) conditions of CLEC-2, Tec kinases play an important role in the activation of Syk but, at higher crosslinking (avidity) conditions, their role becomes less important and other mechanisms take over to activate Syk.