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Title: Protein kinase-dependent regulation of platelet function
Author: Savage, Joshua S.
ISNI:       0000 0004 2723 5133
Awarding Body: University of Bristol
Current Institution: University of Bristol
Date of Award: 2012
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Platelets are essential in the initiation, growth and stabilisation of thrombi in normal haemostasis upon injury to the vasculature, or in cardiovascular disease upon rupture of an atherosclerotic plaque. Protein kinases are a crucial component in platelet signalling pathways, playing a combination of positive and negative regulatory roles. The AGC kinase family contains several known key regulatory kinases in platelet signalling, most notably PKC. PKC is largely considered a positive regulator of platelet function, although recent data suggests that some PKC isoforms play minor negative regulatory roles. Secretion of pro-aggregatory and pro-inflammatory mediators from platelet granules, a process previously shown to be PKC-dependent, is necessary for propagation of activation and for thrombus growth and stability. For this reason, in this study a clinically relevant PKCP inhibitor ruboxistaurin, used in the treatment of diabetic retinopathy, has been demonstrated to be an effective anti-platelet compound acting primarily through the inhibition of secretion. Ruboxistaurin ablated platelet aggregation which could be restored by exogenous ADP, unlike secretion which remained strongly inhibited. Additionally, thrombus formation under flow was significantly reduced in the presence of ruboxistaurin. This promising data suggests there may be potential clinical uses of ruboxistaurin as an anti-thrombotic agent. The mechanism by which PKC regulates granule secretion is yet to be elucidated. Unc13d'inx mice lack Munc13-4, a protein responsible for coordinating components of the secretory complex, possibly directly regulated by or downstream of PKC. Munc13-4 was shown to be absolutely essential for dense granule secretion, and knockdown of the gene caused a reduction in aggregation and thrombus formation, both of which were rescued by exogenous ADP. However, this study also reveals the existence of a Munc13-4 independent pathway, and a novel positive feedback mechanism for u granule release via ADP and P2Y12. The role of another AGC kinase, PKN, had not previously been studied in platelets. It was , hypothesised that, like PKC, PKNl and PKN3, the isoforms identified in platelets, would act as regulatory proteins via secretion and cytoskeletal reorganisation. Using PKNl/3-I- mice, PKN was revealed to be a negative regulator of aggregation, integrin UlIbP3 activation and spreading. The work presented here demonstrates the varied positive and negative roles that protein kinases play in platelet function, including, but by no means exclusively, in secretion. It also reveals a novel ADP-dependent pathway in platelet secretion and demonstrates that a clinically tested PKC inhibitor has potential as a novel anti-thrombotic therapy.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available