GPVI receptor spatial organisation and signalling in platelets

Platelets are crucial players in many (patho)physiological processes including haemostasis, thrombosis and inflammation. The immunoreceptor tyrosine-based activation motif (ITAM)-containing receptor glycoprotein VI (GPVI) is the major platelet signalling receptor for collagen, which is exposed upon vascular injury. Once bound to collagen, multiple GPVI dimers cluster at the platelet surface increasing the avidity for collagen and regulating the signalling. In suspension, GPVI is cleaved by the metalloproteinases ADAM10/17 upon collagen activation. This thesis aimed to elucidate the signalling dynamics underlying GPVI clustering in platelets spread on collagen. Super-resolution microscopy and two-layer cluster analysis revealed that immobilised collagen induces long-term GPVI clustering and signalling in tandem with minimal shedding and low levels of GPVI/ADAM10 colocalisation. Conversely, GPVI is massively shed and the signalling rapidly downregulated in platelet suspensions stimulated with collagen. Syk, but not Src, kinase-dependent signalling is required to maintain clustering of the collagen integrin receptor α2β1, whilst neither is required for GPVI. This thesis proposes that GPVI clustering on immobilised collagen protects GPVI from shedding, thereby enabling longer-lasting Src and Syk-dependent signalling, integrin α2β1 activation and stable adhesion to collagen. Nanobodies targeting human GPVI have potential uses in many different biological applications from anti-platelet approaches to super-resolution imaging.