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 α-granule secretion, and subsequent aggregation of thrombin-stimulated washed platelets.


OBJECTIVE
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 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%.


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.
Institute for Prevention of Cardiovascular Diseases, University of Munich, Munich, Germany.
Suman
S
Dwivedi
Dharmendra
D
Pandey
Anna L
AL
Khandoga
Richard
R
Brandl
Wolfgang
W
Siess
D000135:Acid Phosphatase / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000634:Aminoquinolines / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology; D003094:Collagen / Q000494:pharmacology; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D019007:P-Selectin / Q000378:metabolism; D058226:Plaque, Atherosclerotic / Q000473:pathology*; D010974:Platelet Aggregation / Q000187:drug effects*; D053657:Platelet-Rich Plasma / Q000378:metabolism; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism; D011743:Pyrimidines / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D000071681:Tartrate-Resistant Acid Phosphatase; D013927:Thrombosis / Q000473:pathology / Q000503:physiopathology; D020830:rac1 GTP-Binding Protein / Q000378:metabolism*
D000634:Aminoquinolines; D007527:Isoenzymes; C487513:NSC 23766; D019007:P-Selectin; D011743:Pyrimidines; C107953:RAC1 protein, human; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D003094:Collagen; D011451:Prostaglandin-Endoperoxide Synthases; D000135:Acid Phosphatase; D000071681:Tartrate-Resistant Acid Phosphatase; D020830:rac1 GTP-Binding Protein