Receptor homodimerisation significantly prolongs the lifetime of ligand-induced crosslinking of CLEC-2 but not GPVI

The platelet ITAM receptors, GPVI and CLEC-2, are activated by a diversity of ligands which bind to distinct epitopes indicating that competitive antagonists will not block activation by all stimuli. The present study used two-colour single-particle tracking to investigate the dynamic nanoscale organisation of the two receptors in the cell membrane to identify new strategies for inhibition. The studies were performed in CHO-K1 cells which lack the tyrosine kinase Syk. The results show that when expressed at low level, GPVI and CLEC-2 diffuse over the cell surface as monomers with the presence of dimers due to random collisions. The addition of divalent and trivalent nanobody ligands induces homodimerisation and cessation of movement of both receptors proportionate to ligand valency. Dimers of CLEC-2 are longer-lived than those of GPVI despite a lower affinity of the monomeric nanobody that forms the ligand backbone. Dimerisation of recombinant monomeric CLEC-2 but not GPVI was detected by surface plasmon resonance with a KD of 18.5 μM. The results suggest that the prolonged lifetime of the CLEC-2 interactions is due to synergy between ligand-induced crosslinking and receptor homodimerisation. Blocking dimerisation may be an effective way to inhibit activation of CLEC-2 by its diverse range of ligands.