SIRT1; A Novel Antithrombotic Target in Cardiovascular Disease?

Blanco Lopez, Maria Rosario (2025) SIRT1; A Novel Antithrombotic Target in Cardiovascular Disease? Doctoral thesis (PhD), Manchester Metropolitan University.

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Abstract

Platelets play a pivotal role in atherothrombosis, the primary cause of heart attacks and ischaemic strokes. As a consequence, antiplatelet drugs provide an important reduction in cardiovascular events. However, adverse bleeding events and variable patient responses limit the safety and efficacy of current antithrombotic treatments, which generates the clinical need to develop novel therapeutical approaches. Sirtuin 1 (SIRT1) is an NAD+ dependent deacetylase protective against oxidative damage. Individuals at high risk of atherothrombosis, such as diabetic and obese patients, display reduced SIRT1 levels, which were associated with enhanced thrombus formation in a murine model of arterial thrombosis. However, the importance of SIRT1 in regulating haemostasis and thrombosis has not been evaluated. The aim of this study was to assess the potential use of the SIRT1 selective agonist SRT1720 as a novel antithrombotic therapy by evaluating the effect of SIRT1 activation on platelet and endothelial function. First, the expression of all sirtuins was confirmed in platelets. Then, different platelet functional assays with SRT1720-treated (10 μM) platelet-rich plasma were performed. Using plate-based aggregometry, it was demonstrated that SIRT1 activation attenuates platelet aggregation induced with TRAP-6, collagen and ADP. Further evaluation of the role of SIRT1 in platelet activation showed that SRT1720 decreases fibrinogen and PAC-1 binding, which indicates that this drug reduces integrin αIIbβ3 activation. Moreover, SIRT1 activation decreased the percentage of platelets that release dense granules, while no effects were observed in α-granule secretion. In vitro arterial thrombus formation assays demonstrated that SRT1720 reduces the area covered by the thrombi on collagen. Considering the decrease in thrombus formation caused by SRT1720, the effect of SIRT1 activation in platelet functions dependent on integrin αIIbβ3 “outside-in” signalling and cytoskeletal reorganisation was explored. Activation of SIRT1 inhibited platelet adhesion and spreading on collagen and fibrinogen, but it did not alter the tubulin ring. Incubation with SRT1720 disrupted the actin cytoskeleton of basal and activated platelets and significantly reduced actin polymerization after stimulation. The results from this study also demonstrate that SRT1720 reduces the late stage of clot retraction, which suggests that SIRT1 activation could potentiate the bioavailability of fibrinolytics inside of the thrombi by reducing its density. To investigate the signalling behind the effects of SRT1720 in integrin αIIbβ3 activity and cytoskeletal rearrangement, the phosphorylation status of key regulators, including Y773-β3, S3-cofilin-1, Y397-FAK, and S19-MLC2, was assessed in resting and activated platelets following SIRT1 activation. Although the phosphorylation levels of these proteins changed upon platelet agonist stimulation, SRT1720 had no effect. Thus, the precise mechanism by which SIRT1 exerts its effects in platelets remains unclear. To evaluate the global effect of SIRT1 activation in thrombosis and haemostasis, the impact of SRT1720 in healthy and dysfunctional human coronary artery endothelial cells (HCAECs) was also investigated. This study confirmed that HCAECs express SIRT1 and revealed that SRT1720 is not toxic at a dose of 3 µM or lower. SRT1720 (0.1 - 3 μM) failed to reverse TNF-α-induced NF-κB nuclear translocation in HCAEC. In a gap closure assay, a high but non-toxic dose of SRT1720 (3 μM) significantly reduced HCAEC migration. Investigation of the mechanism behind these effects revealed that HCAEC F-actin levels remained unaltered upon TNF-α and SRT1720 treatment (0.1 - 3 µM). Using a FITC-dextran permeability assay, it was identified that SIRT1 activation is protective against LPS-induced hyperpermeability in HCAEC. To assess the role of SIRT1 in the context of atherosclerosis and thromboinflammation in vitro, the effect of a panel of endothelial dysfunction inducers, including IL-6 (50 ng/mL), LPS (100 ng/mL), TNF-α (10 ng/mL) and H2O2 (100 μM), was tested in HCAEC, revealing that LPS was the best molecule to induce dysfunction. The effect of SIRT1 activation on thrombosis was tested in vitro using an endothelialised arterial flow model, revealing that the area covered by the thrombi on the HCAEC treated with SRT1720 in the presence of LPS was significantly reduced versus the vehicle control. To elucidate the mechanism behind these protective effects, the impact of SIRT1 activation on the mRNA expression of endothelial regulators of haemostasis in healthy and dysfunctional HCAEC was evaluated using RT-qPCR. SRT1720 (1 µM) increased NOS3 mRNA levels only in healthy HCAEC, while it was unable to recover NOS3 LPS-induced downregulation. By contrast, DDAH1 mRNA levels were increased by SRT1720 in the presence of LPS. Moreover, SIRT1 activation increased THBD mRNA expression in LPS-treated and healthy HCAEC. In conclusion, the investigation performed for this thesis indicates that SIRT1 activation could be a novel alternative to current antiplatelet agents and a potential adjuvant drug during fibrinolytic therapy. Moreover, the protective effects of SIRT1 activation observed in HCAEC suggest that SIRT1 agonists could be useful to prevent endothelial dysfunction.to prevent endothelial dysfunction.