Clustering of GPVI dimers upon adhesion to collagen as a mechanism to regulate GPVI signalling in platelets

Background: Platelet GPVI binding to subendothelial collagen exposed upon blood vessel injury initiates thrombus formation. Dimeric GPVI has high affinity for collagen and occurs constitutively on resting platelets. Objective: To identify higher order oligomerisation (clustering) of pre-existing GPVI-dimers upon interaction with collagen as a mechanism to initiate GPVI-mediated signalling. Methods: GPVI was located using fluorophore conjugated GPVI-dimer-specific Fab (antigen-binding fragment. Tested substrates include Horm collagen I fibres, soluble collagen III, GPVI-specific collagen peptides and fibrinogen. GPVI-dimer clusters on the platelet surface interacting with these substrates were visualised\ud using complementary imaging techniques: Total Internal Reflection Fluorescence Microscopy (TIRFM) to monitor real time interactions and direct Stochastic Optical Reconstruction Microscopy (dSTORM), providing relative quantification of GPVI cluster size and density. Confocal microscopy was used to locate GPVI-dimer clusters, GPIb, integrin α2β1, and phosphotyrosine. Results: Upon platelet adhesion to all collagenous substrates, GPVI-dimers coalesced to form clusters; notably clusters formed along the fibres of Horm collagen. dSTORM revealed that GPVI density within clusters depended on the substrate,\ud collagen III being most effective. Clusters on fibrinogen-adherred platelets were much smaller and more numerous; whether these are pre-existing oligomers of GPVI-dimers or\ud fibrinogen-induced is not conclusive. Some GPVI-dimer clusters colocalized with areas of phosphotyrosine, indicative of signalling activity. Integrin α2β1 localized to collagen fibres close to GPVI-dimer clusters. GPVI-clustering depends on a dynamic actin cytoskeleton.\ud Conclusions: Platelet adhesion to collagen induces GPVI-dimer clustering. GPVI-clustering increases both avidity for collagen and proximity of GPVI-associated signalling molecules, which may be crucial for initiation and persistence of signalling.