Rac1-mediated signaling plays a central role in secretion-dependent platelet aggregation in human blood stimulated by at
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RESEARCH
Open Access
Rac1-mediated signaling plays a central role in secretion-dependent platelet aggregation in human blood stimulated by atherosclerotic plaque Suman Dwivedi1, Dharmendra Pandey1,3, Anna L Khandoga1, Richard Brandl2, Wolfgang Siess1*
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 a-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 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 aIIbb3-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%. 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.
Background After rupture of atherosclerotic plaques thrombogenic matrix components and lipids are locally exposed to circulating platelets [1-5]. 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 latter are * Correspondence: [email protected] 1 Institute for Prevention of Cardiovascular Diseases, University of Munich, Munich, Germany Full list of author information is available at the end of the article
potentially life-threatening by occluding coronary and cerebral arteries. The step-wise activation of platelets (adhesion, shape change, secretion and aggregation) in
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