S-EPMC3018435 - Rac1-mediated signaling plays a central role in secretion-dependent platelet aggregation in human blood stimulated by atherosclerotic plaque. - OmicsDI

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Dataset Information

Rac1-mediated signaling plays a central role in secretion-dependent platelet aggregation in human blood stimulated by atherosclerotic plaque.

ABSTRACT:

Background

Platelet activation requires rapid remodeling of the actin cytoskeleton which is regulated by small GTP-binding proteins. By using the Rac1-specific inhibitor NSC23766, we have recently found that Rac1 is a central component of a signaling pathway that regulates dephosphorylation and activation of the actin-dynamising protein cofilin, dense and ?-granule secretion, and subsequent aggregation of thrombin-stimulated washed platelets.

Objectives

To study whether NSC23766 inhibits stimulus-induced platelet secretion and aggregation in blood.

Methods

Human platelet aggregation and ATP-secretion were measured in hirudin-anticoagulated blood and platelet-rich plasma (PRP) by using multiple electrode aggregometry and the Lumi-aggregometer. Platelet P-selectin expression was quantified by flow cytometry.

Results

NSC23766 (300 ?M) inhibited TRAP-, collagen-, atherosclerotic plaque-, and ADP-induced platelet aggregation in blood by 95.1%, 93.4%, 92.6%, and 70%, respectively. The IC50 values for inhibition of TRAP-, collagen-, and atherosclerotic plaque-, were 50 ± 18 ?M, 64 ± 35 ?M, and 50 ± 30 ?M NSC23766 (mean ± SD, n = 3-7), respectively. In blood containing RGDS to block integrin ?IIb?3-mediated platelet aggregation, NSC23766 (300 ?M) completely inhibited P-selectin expression and reduced ATP-secretion after TRAP and collagen stimulation by 73% and 85%, respectively. In ADP-stimulated PRP, NSC23766 almost completely inhibited P-selectin expression, in contrast to aspirin, which was ineffective. Moreover, NSC23766 (300 ?M) decreased plaque-stimulated platelet adhesion/aggregate formation under arterial flow conditions (1500s-1) by 72%.

Conclusions

Rac1-mediated signaling plays a central role in secretion-dependent platelet aggregation in blood stimulated by a wide array of platelet agonists including atherosclerotic plaque. By specifically inhibiting platelet secretion, the pharmacological targeting of Rac1 could be an interesting approach in the development of future antiplatelet drugs.

SUBMITTER: Dwivedi S

PROVIDER: S-EPMC3018435 | biostudies-literature | 2010 Dec

REPOSITORIES: biostudies-literature

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Publications

Rac1-mediated signaling plays a central role in secretion-dependent platelet aggregation in human blood stimulated by atherosclerotic plaque.

Dwivedi Suman S Pandey Dharmendra D Khandoga Anna L AL Brandl Richard R Siess Wolfgang W

Journal of translational medicine 20101206

<h4>Background</h4>Platelet activation requires rapid remodeling of the actin cytoskeleton which is regulated by small GTP-binding proteins. By using the Rac1-specific inhibitor NSC23766, we have recently found that Rac1 is a central component of a signaling pathway that regulates dephosphorylation and activation of the actin-dynamising protein cofilin, dense and α-granule secretion, and subsequent aggregation of thrombin-stimulated washed platelets.<h4>Objectives</h4>To study whether NSC23766 i ...[more]

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Project description:ObjectiveCalcification of atherosclerotic plaque is traditionally associated with increased cardiovascular event risk; however, recent studies have found increased calcium density to be associated with more stable disease. 3-hydroxy-3-methylglutaryl coenzymeA reductase inhibitors or statins reduce cardiovascular events. Invasive clinical studies have found that statins alter both the lipid and calcium composition of plaque but the molecular mechanisms of statin-mediated effects on plaque calcium composition remain unclear. We recently defined a macrophage Rac (Ras-related C3 botulinum toxin substrate)-IL-1β (interleukin-1 beta) signaling axis to be a key mechanism in promoting atherosclerotic calcification and sought to define the impact of statin therapy on this pathway. Approach and Results: Here, we demonstrate that statin therapy is independently associated with elevated coronary calcification in a high-risk patient population and that statins disrupt the complex between Rac1 and its inhibitor RhoGDI (Rho GDP-dissociation inhibitor), leading to increased active (GTP bound) Rac1 in primary monocytes/macrophages. Rac1 activation is prevented by rescue with the isoprenyl precursor geranylgeranyl diphosphate. Statin-treated macrophages exhibit increased activation of NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells), increased IL-1β mRNA, and increased Rac1-dependent IL-1β protein secretion in response to inflammasome stimulation. Using an animal model of calcific atherosclerosis, inclusion of statin in the atherogenic diet led to a myeloid Rac1-dependent increase in atherosclerotic calcification, which was associated with increased serum IL-1β expression, increased plaque Rac1 activation, and increased plaque expression of the osteogenic markers, alkaline phosphatase and RUNX2 (Runt-related transcription factor 2).ConclusionsStatins are capable of increasing atherosclerotic calcification through disinhibition of a macrophage Rac1-IL-1β signaling axis.