The study of platelet messenger and micro-RNAs is of increasing interest owing to the fact that platelets contain the machinery to splice and translate mRNA into proteins in response to inhibitory or activating signals. However, the relatively small size (roughly 4000-5000 transcripts) and short half-life of the platelet transcriptome makes this a technically challenging aspect of platelet biology to investigate. The aims of these thesis investigations were therefore to optimise protocols for the isolation of platelets for downstream RNA analyses and function testing, to investigate the functional capabilities of platelet subpopulations rich in RNA, and to understand the functional and transcriptomic impact of gene mutations predicted to influence platelet function. I found that the optimal method for isolating platelets from whole blood is to use simple single step centrifugation to obtain platelet rich plasma. This method is as effective as more involved methods at reducing white blood cell contamination whilst causing minimal platelet activation. Using this method in combination with flow cytometric cell sorting techniques I was able to isolate the newly formed reticulated platelet sub-population and to confirm the link between reticulation status and increased RNA content. Furthermore, using a range of platelet function assays I demonstrated that reticulated platelets are more reactive than non-reticulated platelets. By obtaining blood samples from a patient with a PLA2G4A mutation I was able to show that loss of cPLA2α enzymatic activity alters both platelet function and the expression of certain mRNA transcripts. My investigations using samples from a range of patients with bleeding tendencies show the benefit of combining deep platelet phenotyping with next generation sequencing to understand the causation of bleeding disorders. Together these investigations highlight the utility of genomic DNA and platelet specific mRNA studies in providing novel insights in to pathways regulating platelet reactivity.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:766125 |
| Date | January 2018 |
| Creators | Hayman, Melissa Anne |
| Publisher | Queen Mary, University of London |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://qmro.qmul.ac.uk/xmlui/handle/123456789/36221 |
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