Unraveling a novel Rac1-mediated signaling pathway that regulates cofilin dephosphorylation and secretion in thrombin-stimulated platelets.
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 Ca(2+)-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 seconds) 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 Ca(2+)/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 Ca(2+)-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 protein kinase C (PKC) pathway mediating secretion.
Institute for Prevention of Cardiovascular Diseases, University of Munich, Munich, Germany.
Dharmendra
D
Pandey
Pankaj
P
Goyal
Suman
S
Dwivedi
Wolfgang
W
Siess
D051339:Actin Depolymerizing Factors / Q000378:metabolism*; D000634:Aminoquinolines / Q000494:pharmacology; D001792:Blood Platelets / Q000378:metabolism*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007527:Isoenzymes / Q000145:classification / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D015539:Platelet Activation / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism; D011743:Pyrimidines / Q000494:pharmacology; D015398:Signal Transduction; D013917:Thrombin / Q000378:metabolism*; D054462:p21-Activated Kinases / Q000145:classification / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000037:antagonists & inhibitors / Q000378:metabolism*
D051339:Actin Depolymerizing Factors; D000634:Aminoquinolines; D004791:Enzyme Inhibitors; D007527:Isoenzymes; C487513:NSC 23766; D011743:Pyrimidines; D019869:Phosphatidylinositol 3-Kinases; D054462:p21-Activated Kinases; D011493:Protein Kinase C; D013917:Thrombin; D020830:rac1 GTP-Binding Protein