The role of dimerisation in the activation of platelets by the collagen receptor GPVI

Clark, Joanne Carol ORCID: 0000-0002-1912-7816 (2021). The role of dimerisation in the activation of platelets by the collagen receptor GPVI. University of Birmingham. Ph.D.

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Abstract

Cardiovascular disease is the leading cause of death globally and platelets have emerged as key markers for disease pathophysiology. Cardiovascular disorders caused by pathological thrombosis are divided into two underlying causes’ namely, arterial thrombosis and thrombo-inflammation. Current antiplatelet treatments however have a high risk of causing excessive bleeding and are largely ineffective against thrombo-inflammation. Therefore there is a need to develop new antiplatelet treatments including targeting the receptor tyrosine kinase, glycoprotein VI (GPVI). GPVI has emerged as a promising therapeutic target due to it having a key role in arterial thrombosis (at site of plaque rupture) and a number of thrombo-inflammatory disorders (including ischaemic stroke) but a minimal role in haemostasis. This glycoprotein is a critical signalling receptor responsible for collagen-induced platelet responses and a receptor for additional endogenous ligands including fibrin(ogen). GPVI is also a receptor for snake venom toxins and charged surfaces. GPVI has two immunoglobulin (Ig) domains (D1 & D2), a single transmembrane helix and a mucin rich stalk. It has been proposed that GPVI is expressed as a dimer through association via its D2 domains, that collagen binds to a unique epitope in dimeric GPVI, that the number of GPVI dimers increases upon platelet activation and that dimerisation of GPVI is inhibited by elevation of cyclic adenosine monophosphate (cAMP). In this thesis, I confirm that elevation of cAMP reduces dimerisation but show this has a minimal effect on collagen signalling. Furthermore, through functional studies on transfected cells expressing GPVI mutants I provide evidence that dimerisation is not critical for activation of the collagen receptor. In addition, I show using super-resolution single molecule microscopy, fluorescence correlation spectroscopy (FCS) and bioluminescence resonance energy transfer (BRET) that GPVI is expressed as a mixture of monomers and dimers in transfected cell lines but is predominately monomeric, and that collagen increases the degree of dimerisation/oligomerisation. The results suggest that a dimer-specific conformation is not critical for collagen binding and receptor activation but supports collagen signalling through an increase in avidity. The results are discussed in the context of GPVI as a novel target in thrombosis.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):