Platelets are vital in haemostasis to prevent bleeding after injury. Uncontrolled platelet activation, however, leads to thrombosis. Platelet granule secretion is critical in thrombus formation and, due to the wide variety of granule contents released, also plays a role in many other physiological and pathological processes. The signalling molecule PKC is key in secretion of both dense and a-granules, but the downstream pathways that link it to secretion still need to be defined. A regulator of a-granule release, specifically, is ADP, which acts on the P2Y12 receptor after being secreted by dense granules. How it may control the release of functionally different cargoes is poorly understood. I first identified an extensive range of candidate platelet PKC substrates through proteomics screening using an anti-phosphopeptide antibody-based approach. Among these, I validated cytohesin-2, an Arf-GEF not previously characterised in platelets. This study demonstrated that, upon platelet activation, PKC phosphorylation of cytohesin-2 relieves the basal suppression of dense granule release, shedding new light on how PKC regulates secretion. I next examined the role of ADP in a-granule secretion with a specific focus on whether P2Y12 can induce differential cargo release, considering the implications for the clinical use of the P2Y12 blocker, clopidogrel. I investigated this using a P2Y12 antagonist and the Munc13-4-1 - mouse model where dense granule secretion is ablated and therefore autocrine ADP is not released. Results revealed that the secretion of certain a-granule contents is more dependent on ADP than others and that, in the absence of ADP signalling, granules may release contents without complete fusion with the plasma membrane. Taken together, this thesis provides new findings on the regulation of dense and a-granule secretion, which can be used in the design of improved therapies for thrombosis and other disorders where platelet granule contents play a role.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:685919 |
| Date | January 2015 |
| Creators | Van den Bosch, Marion T. J. |
| Publisher | University of Bristol |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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