Characterisation of cyclic adenosine monophosphate/protein kinase A signalling networks in blood platelets

Platelet activation is a critical physiological event, whose main role is to prevent excessive blood loss and repair vessel wall injuries. However, platelet activation must be controlled to prevent unwanted and exaggerated responses leading to the occlusion of the blood vessel. The endothelial-derived inhibitors prostacyclin (PGI2) and nitric oxide (NO) are known to play a critical role in the control of platelet activity, although the mechanism underlying their actions remains unclear beyond the triggering of cyclic nucleotides signaling pathways. The aim of this study was to improve our understanding of platelet regulation by cAMP signaling networks.We observed differences in cAMP signaling depending on the agonists used. Using phosphorylation of PKA substrates as a marker of PKA activity, it was observed that PKA substrates were phosphorylated and dephosphorylated at different time points in a unique temporal pattern. Consistent with this observation we found that individual PKA isoforms, PKA I and II, were localized in distinct subcellular compartments, with PKA I being identified as a lipid raft protein. Our experimental data suggest that the localization of PKA I to lipid rafts is mediated by interaction with A-kinase anchoring proteins (AKAPs). Additionally, PKA signaling events were reversed when potential PKA type I interactions with AKAPs were disrupted with competitive peptides. Using this approach we found that the redistribution of PKA I to lipid rafts facilitated the phosphorylation of GPIbβ and the inhibition of von-Willebrand factor-mediated aggregation.Our data also demonstrated for the first time that the chemical disruption of lipid rafts increased platelet sensitivity to PGI₂, through increased cAMP production and PKA activity. The mechanism by which this occurs may involve sequestering a population of adenylyl cyclase 5/6 to a location remote from Gαs.In conclusion, data presented in this thesis suggest differential roles of PKA subtypes in the regulation of platelet activity. This involves, at least in part, the localisation of PKA I into specific subcellular compartments through an interaction with AKAPs. The potential presence of PKAII-AKAP interactions and the identification of specific AKAPs will be the main aim of future work.

Name dc:type.qualificationname

PhD

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

Hull York Medical School, the University of Hull and the University of York

Year dc:date.issued

2012

Author dc:creator

• Raslan, Zaher

• Medicine

Language dc:language

en

Identifier

oai:hull-repository.worktribe.com:4213632

OAI identifier oai:identifier

oai:hull-repository.worktribe.com:4213632