Characterisation of platelet membrane receptors in their native environment using SMALP nanodiscs

Wang, Xueqing ORCID: 0000-0002-3391-770X (2023). Characterisation of platelet membrane receptors in their native environment using SMALP nanodiscs. University of Birmingham. Ph.D.

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Abstract

Platelets are small, anucleate blood cells, and their major role is in haemostasis, whereby they prevent excessive blood loss by adhesion, activation and aggregation at sites of blood vessel injury. However, they can also give rise to thrombosis, leading to myocardial infarction or ischaemic stroke, when they are activated in diseased arteries following the rupture of atherosclerotic plaques. These behaviours are dependent on the interaction between platelet membrane protein receptors and their subendothelial matrix or soluble ligands. Traditionally, membrane protein studies relied on detergent-based solubilisation methods, which remove most of the native plasma membrane lipid species and can affect protein structure and function. Recently, a novel methodology, namely styrene-maleic acid lipid particles (SMALPs), has emerged for the extraction of membrane proteins. This method utilises styrene-maleic acid co-polymers (SMA), which are amphiphilic polymer chains, to directly cut patches out of the native membrane continuum, generating SMALPs encapsulating intact membrane proteins with their surrounding lipids and partner proteins, belted by SMA, without the addition of detergent molecules in any steps. The aim of this thesis was to adopt SMALPs for the characterisation of platelet membrane protein structure and interaction partners, with a main focus on the platelet-activating collagen receptor GPVI. In transfected HEK-293T cells, GPVI was successfully purified in SMALPs together with its associated FcRγ signalling chain. Gel filtration followed by a native polyacrylamide gel electrophoresis (PAGE) method, SMA-PAGE, revealed multiple sizes of the purified GPVI/FcRγ SMALPs, suggesting the potential existence of GPVI oligomers. Importantly, GPVI/FcRγ SMALPs were functional in binding to collagen, and contained single-dispersed protein particles that could be observed under negative stain electron microscopy (EM), suggesting great potential for future investigation of GPVI stoichiometry and structure. In human platelets, the SMALP generation protocol was optimised and simplified for bench-top biochemical characterisations. This demonstrated that membrane protein complexes could be successfully extracted into the same SMALPs, including GPVI/FcRγ and the ‘molecular scissor’ ADAM10 with its regulatory tetraspanins Tspan15. In addition, SMA-PAGE has been proved as a convenient method to investigate a wide range of platelet membrane protein interactions, by revealing the molecular weights of native protein complexes in SMALPs. Taken together, the work in this thesis suggests that SMALPs are a promising tool for the characterisation of platelet receptor structures in their native lipid bilayer and for the identification of novel membrane protein interaction partners. Such information will be valuable for the future characterisation of new antiplatelet therapeutic targets designed to prevent thrombosis with minimal effects on haemostasis.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):