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

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 alpha 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 mu 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 mu M, 64 +/- 35 mu M, and 50 +/- 30 mu M
NSC23766 (mean +/- SD, n = 3-7), respectively. In blood containing
RGDS to block integrin alpha(IIb)beta(3)-mediated platelet
aggregation, NSC23766 (300 mu 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
mu 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.