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Title: Targeting the nitric oxide signalling pathway to modulate platelet function
Author: Apostoli, Georgina Lorraine
ISNI:       0000 0004 6059 1252
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2015
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Nitric oxide (NO) negatively regulates platelets and impaired NO signalling can lead to arterial thrombosis. The source of platelet-derived NO is unclear with recent proposals of NO synthase (NOS) independent NO sources, such as S-nitrosothiols (RSNOs) and inorganic nitrate/nitrite. Sildenafil citrate, a phosphodiesterase 5 (PDE5) inhibitor, enhances NO/cGMP signals in cells expressing PDE5 such as platelets. The aims of this study were to investigate the antiplatelet properties of sildenafil, its mechanism of action and to determine the upstream sources of NO affecting platelet function. The functional effect of sildenafil was determined using a range of in vitro and in vivo platelet assays. The mechanism of action of sildenafil and upstream sources of NO/cGMP signals were assessed pharmacologically using established methods of in vitro and in vivo platelet aggregation. Bioconversion of nitrate to nitrite was determined using gas-phase chemiluminescence. The functional significance of NO/cGMP signalling events in platelets were investigated in vivo in W.T and eNOS-/- (a model of vascular dysfunction) mice. Sildenafil exerted an antiplatelet effect by enhancing transient NO/cGMP signals generated by platelets independent of NOS activity in vitro. Inhibition of proposed mechanisms of NO release from RSNOs did not modify the inhibitory effect of sildenafil suggesting that RSNOs did not mediate platelet NO/cGMP signals. Nitrite was able to drive inhibitory cGMP signalling events in platelets in vitro. Furthermore, nitrate inhibited platelet function in eNOS-/- mice in vivo following enhanced bioconversion to nitrite, potentially as a compensatory mechanism due to impaired NO signalling. In conclusion, inorganic nitrate/nitrite may critically regulate platelets following bioconversion to NO and dietary sources of nitrate/nitrite may generate compensatory NO during vascular disease. Furthermore, sildenafil may be beneficial in reducing the risk of platelet-driven cardiovascular disease by enhancing NO/cGMP signalling derived from both enzymic and inorganic sources and restoring impaired NO signalling during endothelial dysfunction.
Supervisor: Emerson, Michael Sponsor: Medical Research Council ; British Pharmacological Society
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral