cAMP signaling reverses platelet spreading via inhibition of RhoA

Yusuf, Muhammad Zuhair

October 2016

Thesis or dissertation

© 2016 Muhammad Zuhair Yusuf. All rights reserved. No part of this publication may be reproduced without the written permission of the copyright holder.

Prostacyclin (PGI₂) is a key regulator of platelet function. There is a significant field of research which outlines the role of PGI₂ in inhibiting platelets in circulation, and therefore playing a major role in the prevention of excessive thrombus formation. PGI₂ signalling is countered by strong activatory stimuli leading to platelet spreading and thrombus formation. However little is known if PGI₂ plays a role after platelet activation. Therefore, the aim of this study was to identify the effect of PGI₂ on already activated platelets. Therefore, thrombi were formed on fibrinogen and/or collagen, and then PGI₂ was flowed over the top. This revealed a significant reduction in thrombus surface area on fibrinogen, and both surface area coverage, and thrombus height on collagen, demonstrating the ability of PGI₂ to modulate a pre-formed thrombus. To understand the mechanism behind this event it was postulated that PGI₂ was modulating the actin cytoskeleton of an activated platelet, leading to thrombus instability.

To identify if cytoskeletal changes were induced by PGI₂, platelet spreading was monitored. Platelet spreading follows a defined sequence of events, with the formation of filopodia, actin nodules, lamellipodia and finally stress fibres. By understanding the percentage of platelets containing each structure, it would be possible to identify the role of PGI₂ within platelet spreading. Therefore, platelets were allowed to spread, before stimulation with PGI₂. This identified that PGI₂ induced a significant reduction in stress fibre formation whilst inducing actin nodule formation. Alongside this there was a reduction in the surface area of the platelet. Further to this PGI₂ was shown to induce a strong elevation of cAMP within fully spread platelets. This reversal of stress fibre formation occurred in a PKA dependent manner as inhibition of PKA induced an inhibition of the stress fibre reversal induced by PGI₂.

Stress fibre formation is linked to RhoA activity. Inorder to understand if PKA was modulating RhoA, the phosphorylation and activity status of RhoA was monitored. We showed clearly that PKA both induced the phosphorylation, but also significantly reduced the activity of RhoA in spread platelets. Further to this, there was a reduction in the phosphorylation of the light chains of Myosin II.

Thus, PGI₂ modulates the actin cytoskeleton via a PKA dependent inactivation of RhoA, leading to stress fibre reversal and thrombus instability.

Hull York Medical School, The University of Hull and University of York
Calaminus, Simon
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