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Markham-Lee, Zoe Jade (2024) Structural studies of Von Willebrand Factor regulators for Thrombotic Thrombocytopenic Purpura. PhD thesis, University of Nottingham.

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Abstract

Blood clotting requires an urgent and efficient response with von Willebrand factor (VWF) playing a major role through recruitment of platelets forming a haemostatic plug. VWF is a large multi-domain glycoprotein, with the A1 domain for platelet binding via Gp1bα and the A2 domain containing the cleavage site to reduce multimer size. VWF is regulated by A Disintegrin-like And Metalloprotease with Thrombospondin type I repeats, member 13 (ADAMTS13) to maintain the delicate haemostatic thrombotic balance. ADAMTS13 is a highly specific protease, only cleaving VWF at the scissile bond under specific flow conditions. The process by which this regulation is maintained is still unclear, with details on ADAMTS13 latency and subsequent interaction and cleavage of VWF still to be elucidated.

Thrombotic thrombocytopenic purpura TTP is the clinical deficiency of ADAMTS13 and is a life-threatening thrombotic disorder. The disease is characterised by excessive clotting in the microvasculature and has ~90% mortality if untreated. Caplacizumab is a nanobody recently approved for treatment of TTP, and the structure was resolved in complex with the VWF A1 domain. The VWF A1 domain consists of the main domain region and two flanking autoinhibitory modules termed the NAIM and CAIM, which inhibit A1 activation through disruption of Gp1bα binding. Although mortality rates have subsequently improved, the use of caplacizumab is associated with an increased risk of bleeding so alternative treatment options must be explored to improve patient outcomes. This thesis aims to investigate the structures of ADAMTS13 and VWF A2 domain, as well as VWF A1 domain and the nanobody ND6 to better understand VWF regulation and subsequently inform treatment of thrombotic diseases.

Analysis of TTP patient mutations identified a lack of associations between genotype and phenotype information highlighting the need for a novel ADAMTS13-VWF structure to interrogate the activation cycle of the proteins. Computational studies utilising molecular modelling and docking enabled design of novel VWF constructs, with expression and purification of SUMO-tagged VWF A2 domain fragments as well as a fusion of 2GKG tag to VWF A2 fragments. Initial characterisation of these constructs revealed that, in preliminary binding analysis, the VWF Y1605C mutant showed a stronger binding affinity to ADAMTS13 than wildtype VWF (KD of 42nm and 450nm respectively), as well as successful complex formation visible utilising size exclusion analysis. This characterisation will help progress understanding of VWF regulation as well as ADAMTS13 structure in normal and disease states following resolution of the crystal structure. Furthermore, the successfully resolved structure of VWF A1 domain in complex with a novel nanobody ND6, gives detailed interface information and a unique conformation of VWF not elucidated in previous structures. The O-glycosylation of T1468 and S1263 is visualised in this novel structure, as well as the interface between both the NAIM and CAIM of VWF A1 occurring from the main body of the domain. Together this information will help deepen the understanding on the fundamental biology of TTP and explores ways to harness this knowledge for improvement in thrombosis disease treatments.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Emsley, Jonas Dreveny, Ingrid Morgan, Neil
Keywords:	blood clotting, TTP, ADAMTS13, VWF, Nanobody, X-ray Crystallography
Subjects:	Q Science > QP Physiology > QP1 Physiology (General) including influence of the environment R Medicine > RC Internal medicine
Faculties/Schools:	UK Campuses > Faculty of Science > School of Pharmacy
Item ID:	79450
Depositing User:	Markham-Lee, Zoe
Date Deposited:	13 Dec 2024 04:40
Last Modified:	13 Dec 2024 04:40
URI:	https://eprints.nottingham.ac.uk/id/eprint/79450

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