Exploring zinc homeostasis and utilising quantitative proteomics to understand platelet (patho)physiology

Platelets are central to haemostasis and play an increasingly recognised role in immune modulation. Their dysregulation contributes to thrombotic events and cardiovascular complications, especially in type 1 diabetes (T1D). Elucidating the molecular mechanisms underlying these processes is an active research area. This thesis explores four distinct, yet interconnected, aspects of platelet biology in both physiological and clinical contexts. Firstly, an examination of zinc transporters expression in megakaryoblast cells and platelets was undertaken to determine the mechanisms that govern zinc homeostasis in hematopoietic cells. A diverse array of zinc transporters were identified in both platelets and megakaryocytes, aided through the development of a low-contamination platelet proteomic protocol. Secondly, this work builds on preliminary findings suggesting the presence of the mitsugumin 23 (MG23) channel in platelets. MG23 is hypothesised to mediate crosstalk between Zn²⁺ and Ca²⁺ signalling and was identified here for the first time in megakaryoblast cells. However, functional assays, a phenome-wide association study and quantitative proteomics did not show a major contribution of MG23 to platelet function. Thirdly, a comparative quantitative proteomic study examining platelets from T1D individuals and controls was performed. This revealed significant alterations in protein expression, providing molecular insight into the hyperactive phenotype of T1D platelets. Finally, the immunopeptidome of platelet-derived extracellular vesicles (PEVs) was investigated. It was demonstrated that PEVs possess an immunopeptidome and that the repertoire of peptides presented by human leukocyte antigens (HLAs) varies depending on the activation state of the platelet. Collectively, the work presented in this thesis underscores the multi-faceted roles of platelets in haemostasis, immune functioning and T1D pathogenesis.