Statins are 3-hydroxy, 3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, which block the cholesterol biosynthetic pathway to lower total serum levels and LDL-cholesterol. The cholesterol pathway also provides a supply of isoprenoids (farnesyl and geranylgeranyl) for the prenylation of signaling molecules, which include the families of Ras and Rho small GTPases. Prenyl groups provide a membrane anchor that is essential for the correct membrane localisation and function of these proteins. Statins deplete cells of lipid geranylgeranyl diphosphate (GGPP) thereby inhibiting progression of the mevalonate pathway and prenylation of proteins. Two such proteins are Rab27b and Rap1, small GTPase proteins that are involved in the secretion of platelet granule and integrin activation. We hypothesise that statins can impair prenylation of Rab27b and Rap1a in platelets and thereby attenuate platelet function. The specific aims of the project were to analyse the impact of statins on the prenylation status of Rab27b and Rap1a in platelets. As Rab27b and Rap1a are known to be involved in secretion of platelet granules a secondary aim was to analyse the downstream effects of statins on this process following activation. Finally, we assessed the impact of treatment of platelets with statins on thrombus formation, stability and resistance to fibrinolysis. Platelets incubated with statins overnight were separated into cytosolic (aqueous) and membrane (detergent) components and visualised by Western blot. An accumulation of Rab27b and Rap1a was observed in the cytosolic compartments of statins treated platelets compared to untreated platelets, thus indicating indirect evidence that statins attenuate prenylation of Rab27b and Rap1a in platelets. The most effective statin in attenuating prenylation of Rab27b and Rap1a was atorvastatin (ATV). The inhibitory effect of statins on prenylation was recovered by GGPP, indicating that the mechanism of inhibition involved the mevalonate pathway. Release of ADP from platelet dense granules was significantly impeded following overnight treatment with ATV. In line with the inhibition of prenylation of Rab27b and Rap1a by ATV, addition of GGPP rescued the release of ADP from platelet dense granules. This suggests that attenuation of dense granules release by ATV occurs via interference in the mevalonate pathway and the inhibition of Rab27b prenylation. Furthermore, ATV significantly attenuates α-granules release in thrombin stimulated platelets, which was visualised as impaired accumulation of endogenous P-selectin, PAI-1 and fibrinogen on the activated membrane. Changes in the activation of α₁₁bβ₃ integrin on the stimulated platelet surface, observed as defective binding of exogenous fibrinogen and PAC-1, were also evident following treatment of platelets with ATV. In addition, ATV treatment of platelets reduced binding of CD41a, indicating that the copy number and activation of α₁₁bβ₃ integrin on stimulated platelets was significantly reduced. Statins were also found to significantly inhibit thrombin-induced platelet aggregation following incubation of platelets overnight with therapeutic concentrations of statins. Surprisingly GGPP did not rescue platelet aggregation indicating that different mechanisms are involved in inhibition of platelet responses by statins. Incubation of whole blood with ATV overnight significantly altered several haemostatic parameters. Using thromboelastography we demonstrated a delay in the coagulation time and clot formation time. Maximum clot firmness was also significantly reduced in the presence of statins compared to the control. The effect on clot firmness generally arises from platelet dysfunction and/or a change in fibrinogen concentration and function; the latter was ruled out using a Fibtem test, which shows no difference between treated and untreated whole blood. Similarly, formation of platelet-rich plasma clots was significantly delayed following pre-treatment with ATV overnight. These clots also exhibited lower maximal absorbances, which could represent differences in the fibrin network structure. In line with the reduction in fibrinogen binding defective clot retraction was also observed in platelet-rich plasma pre-treated with ATV overnight. Similar clot retraction results were observed with tirofiban and CytoD, suggesting that the inhibitory effect of ATV may involve modulation of α₁₁bβ₃ integrin activation. Platelet-rich plasma clots formed post-treatment with statins were visualised by confocal microscopy and revealed significant alterations in clot structure; observed as thinner fibrin fibres and fewer platelet aggregates. Additionally, we demonstrated that statins modulate clot stability and shorten time to lysis. Clots formed from platelet rich plasma that was subjected to incubation with ATV overnight revealed faster lysis by tPA compared to the absence of statin. These findings are also in agreement with the lysis of Chandler model thrombi formed from overnight incubated whole blood with ATV, which demonstrated faster lysis rate mediated by tPA. Furthermore, statins were shown to change the clot thrombodynamics as assessed by HemaCore analyser, which shows that stains implicate both clot growth in response to TF-coated comb and spontaneous clot lysis by tPA. In conclusion, statins directly inhibit Rab27b and Rap1a prenylation in platelets and down-regulated dense granules release. Inhibition of Rab27b and Rap1a prenylation, and dense granules release was recovered by GGPP, indicating that these effects are mediated through the mevalonate pathway. Impairment of platelet aggregation by statins resulted via multiple mechanisms as GGPP did not recovered the inhibition of aggregation by ATV. Statins also modulate fibrinogen binding, α-granules release, clot retraction and clot formation and stability in vitro. Together these results suggest that statins may directly attenuate the platelet response in vivo. The pleotropic effect of statins on platelets may contribute to the protective function of these class of drugs in cardiovascular diseases.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:731642 |
| Date | January 2017 |
| Creators | Jalal, Mohammed Mansour |
| Publisher | University of Aberdeen |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=235575 |
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