An investigation into the effect of oxLDL and cyclic nucleotides on platelet cytoskeletal function

Ahmed, Yusra

Abstract

Atherosclerosis is a major underlying cause in cardiovascular disease and a significant health concern in developed countries. Part of the mechanism by which platelets are linked to this process is via their activation by oxidised Low-Density Lipoproteins (oxLDL), which drives both atherogenesis and thrombosis. However, the mechanism by which oxLDL drives platelet
activation and thrombosis is unclear.
The combinations of matrix protein within the vascular wall and within the thrombus, especially if oxLDL is present can affect the size and strength of the thrombus formed. We have previously shown that platelets bind effectively to fibrinogen, but that this can be reversed by prostacyclin (PGI2). We now demonstrate that the combination of fibrinogen and oxLDL as a matrix protein induced a significant increase in platelet adhesion and spreading in a dose-dependent manner. Furthermore, we identified that oxLDL partially blocked the reversal of spread platelets by PGI2, as the surface area of the platelet was unaffected in contrast to those platelets spread only on fibrinogen. This suggested that oxLDL modulated the reversal of platelet spreading by PGI2. However, oxLDL mediated inhibition of surface area reduction by PGI2 was independent of the secondary mediators ADP and TxA2.
To understand how oxLDL might cause this change in PGI2 mediated reversal of platelet function, we first identified the cAMP production in spread cells. This indicated that cAMP production was inhibited by oxLDL but was still significantly higher than control spread platelets. This indicated that the reversal of stress fibres could be induced by limited increases in cAMP whilst higher increases then affected lamellipodia and reduced overall platelet surface area. These two pieces of data suggested that the RhoGTPases, Rac and RhoA might be differentially controlled within spread platelets. This was confirmed as platelets spread on oxLDL and fibrinogen in the presence of PGI2 showed elevated levels of Rac activity, whilst RhoA activity was significantly inhibited. Furthermore, we found that pMLC was elevated in platelets spread on fibrinogen, fibrinogen and nLDL as well as oxLDL, in line with stress fibre formation and increase in Rho activity in the absence of PGI2.
To understand the effect of this reversal of stress fibres on thrombus formation, we post perfused cAMP elevating agents over platelet aggregates preformed on fibrinogen +/- oxLDL or nLDL. Our data suggest that at high shear PGI2 or the AC (adenylyl cyclase) activator, forskolin cannot induce reversal of stress fibres, whilst the phosphodiesterase 3 inhibitor, milrinone can. In contrast at low shear, PGI2 and forskolin can induce a complete reversal of stress fibres. This data indicates that shear stress can alter PGI2 effects on spread platelets, potentially due to changes in PDE3 activity, and therefore can help to provide a prothrombotic environment.
In summary, our data shows that oxLDL and fibrinogen play an important role in promoting a prothrombotic and proinflammatory phenotype, contributing to atherogenicity and atherothrombosis. Investigating the contribution of oxLDL and fibrinogen and their involvement in atherothrombosis, in the context of these results will provide novel insights and identify new pathways of immunothrombosis.

Citation

Ahmed, Y. An investigation into the effect of oxLDL and cyclic nucleotides on platelet cytoskeletal function. (Thesis). Hull York Medical School, The University of Hull and The University of York. https://hull-repository.worktribe.com/output/4200904

• Thesis Type: Thesis
• Deposit Date: Feb 15, 2023
• Publicly Available Date: Feb 15, 2023
• Keywords: Medicine ; Medical sciences
• Public URL: https://hull-repository.worktribe.com/output/4200904
• Award Date: 2020-09