PB0817 - Visualising the dynamics of movement of GPVI and CLEC-2 upon ligand engagement

Background: The platelet receptors, Glycoprotein VI (GPVI) and C-type lectin-like receptor 2 (CLEC-2) have emerged as targets for new anti-platelet therapy due to their major role in thrombosis and minor role in haemostasis. Receptor activation is mediated by clustering but its regulation and how it influences activation and signalling isn’t fully understood. Valuable kinetic information in the membrane such as dimer lifetimes and association/dissociation rates are missing. This information will aid our understanding of receptor organisation and regulation which is important in the development of better therapeutics.

Aims: Measure the kinetics of GPVI and CLEC-2 dimerisation, including dimer lifetimes in the membrane using state-of-the-art advanced microscopy.

Methods: We used two-colour single particle tracking (SPT) using total internal reflection fluorescence (TIRF) microscopy to image Snaptag and Halotag versions of GPVI and CLEC-2 in resting and stimulated transfected live CHO cells. Advanced computational analyses were performed to generate receptor trajectories and determine kinetic outputs.

Results: Using fluorescence correlation spectroscopy (FCS) and photobleaching we have shown that GPVI and CLEC-2 are a mixture of monomers and dimers. For SPT, productive interactions for GPVI and CLEC-2 were not observed at basal level suggesting the receptors are monomers at low expression levels. Upon addition of novel trivalent nanobodies (100 nM), GPVI and CLEC-2 formed immobile clusters with an increase in the immobile fraction from 26.9% to 41.9% and 18.9% to 40.7% respectively. Studies are on-going to study the relationship between cluster size and ligand valency, and to determine the kinetics of cluster formation.

Conclusion(s): We have monitored single receptor movement in the membrane to measure interactions and their associated kinetic information. The results show that GPVI and CLEC-2 are monomeric at low receptor levels highlighting the importance of expression levels. Ligand addition causes receptors to become immobile which could be due to variables such as ligand valency and cluster size.