GPVI and CLEC-2 regulation and signal transduction: from development through to adulthood

The platelet collagen receptor, GPVI, and podoplanin receptor, CLEC-2, have been shown to share a common tyrosine-kinase linked signalling pathway. GPVI contains a full ITAM (YxxLn6-12YxxL) within its intracellular tail, whilst CLEC-2 contains a hemITAM (YXXL). Both receptors rely on common, key signalling proteins including Src, Syk, LAT, Btk and PLCγ2, however the biological implications of GPVI and CLEC-2 stimulation are significantly different; GPVI is implicated in haemostasis and thrombosis responses whilst CLEC-2 appears to be involved in the newer roles of platelets, such as their roles in infection, immunity, and development. It has also been shown that the organisation and importance of the signalling proteins in the pathway differ between GPVI and CLEC-2. The aims of this thesis are to explore the regulation and function of the tyrosine kinase-linked signalling pathway downstream of the two (hem)ITAM receptors in response to different stimuli at different stages of development. In this thesis, I show that both GPVI and CLEC-2 require an intact, functional Syk kinase domain of Syk to support signalling, regardless of the maintained phosphorylation of certain adapter docking sites. I also show that both GPVI and CLEC-2 are hyporeactive and expressed at lower levels on the platelet surface throughout development, and that these defects appear to be tyrosine kinase-linked pathway specific. Lastly, using samples from homozygous GPVI deficient patients, I have shown that both fibrin and fibrinogen appear to be agonists for GPVI. Overall, I have shown that are likely subtle differences in the regulation and downstream signalling of GPVI and CLEC-2 throughout development and to several different stimuli – including the novel agonists fibrin and fibrinogen