Submitted October 9, 2008
Accepted April 11, 2009
Unraveling a novel Rac1-mediated signaling pathway that regulates cofilin dephosphorylation and secretion in thrombin-stimulated platelets
Dharmendra Pandey, Pankaj Goyal, Suman Dwivedi, and Wolfgang Siess*
Institute for Prevention of Cardiovascular Diseases, University of Munich, Munich, Germany
* Corresponding author; email: wsiess{at}med.uni-muenchen.de.
In platelets stimulated by thrombin to secrete and aggregate, cofilin is rapidly dephosphorylated leading to its activation. Cofilin by severing existing actin filaments and stimulating F-actin polymerization on newly created barbed ends dynamizes the actin cytoskeleton. We previously found that cofilin dephosphorylation is Ca2+-dependent and occurs upstream of degranulation in stimulated platelets. We report now in thrombin-stimulated platelets that Rac1 and class-II PAKs (PAK4/5/6) were rapidly (within 5 sec) activated, whereas PAK1/2 (class-I PAKs) phosphorylation was slower. The Rac1 specific inhibitor NSC23766 blocked phosphorylation of class-II PAKs, but not PAK1/2. Moreover, inhibition of the Ca2+/calmodulin-dependent phosphatase calcineurin inhibited Rac1 activation and class-II PAKs phosphorylation. Prevention of Rac1 activation by calcineurin inhibition or NSC23766 also blocked cofilin dephosphorylation and platelet granule secretion indicating that a calcineurin/Rac1/class-II PAKs pathway regulates cofilin dephosphorylation leading to secretion. We further found that PI3-kinases were activated downstream of Rac1, but were not involved in regulating cofilin dephosphorylation and secretion in thrombin-stimulated platelets. Our study unravels a Ca2+-dependent pathway of secretion in stimulated platelets as a signaling pathway linking Rac1 activation to actin dynamics: Calcineurin 
Rac1 class-II PAKs cofilin activation. We further demonstrate that this pathway is separate and independent of the PKC pathway mediating secretion.
