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

Suman Dwivedi,Anna L Khandoga,Richard Brandl,Wolfgang Siess,Dharmendra Pandey

doi:10.1186/1479-5876-8-128

Abstract

BackgroundPlatelet 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.ObjectivesTo study whether NSC23766 inhibits stimulus-induced platelet secretion and aggregation in blood.MethodsHuman 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.ResultsNSC23766 (300 μM) 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 μM, 64 ± 35 μM, and 50 ± 30 μM NSC23766 (mean ± SD, n = 3-7), respectively. In blood containing RGDS to block integrin αIIbβ3-mediated platelet aggregation, NSC23766 (300 μM) 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 μM) decreased plaque-stimulated platelet adhesion/aggregate formation under arterial flow conditions (1500s-1) by 72%.ConclusionsRac1-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.

Highlights

Platelet activation requires rapid remodeling of the actin cytoskeleton which is regulated by small GTP-binding proteins
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
We report here that NSC23766 blocks secretion and secretion-dependent aggregation in platelet-rich plasma (PRP) and blood induced by Adenosine 3’-phosphate 5’-phosphate (ADP), TRAP, collagen and human atherosclerotic plaque, and notably plaque-stimulated platelet thrombi formation under arterial flow conditions

Summary

Introduction

Platelet activation requires rapid remodeling of the actin cytoskeleton which is regulated by small GTP-binding proteins. After rupture of atherosclerotic plaques thrombogenic matrix components and lipids are locally exposed to circulating platelets [1,2,3,4,5] By adhering to these sites, platelets rapidly become activated, leading to secretion of their granule contents such as ADP that recruits circulating platelets into large aggregates culminating in the formation of platelet thrombi [5,6]. The major molecules involved in actin dynamics are the small GTP-binding proteins Rho, Rac, and Cdc42 These proteins differentially regulate the reorganization of the actin cytoskeleton, leading to the formation of different cellular structures. In the two other studies, thrombin-induced secretion and aggregation were not affected; Rac was found to be involved only in collagen/glycoprotein VI-mediated platelet activation [9,14]

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