Unraveling a novel Rac1-mediated signaling pathway that regulates cofilin dephosphorylation and secretion in thrombin-stimulated platelets

Abstract

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 thrombinstimulated 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؉ /calmodulindependent 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 thrombinstimulated platelets. Our study unravels a Ca 2؉ -dependent pathway of secretion in stimulated platelets as a signaling pathway linking Rac1 activation to actin dynamics: calcineurin3Rac13class II PAKs3cofilin activation. We further demonstrate that this pathway is separate and independent of the protein kinase C (PKC) pathway mediating secretion. (Blood. 2009;114:415-424)

Wiskott-Aldrich Syndrome Protein (WASp) Controls the Delivery of Platelet Transforming Growth Factor-β1

“…Both GTPases can modulate platelet secretion (47)(48)(49), and several reports have described impaired secretion in Rac1-null platelets (49) or in NSC23766-treated platelets (48). In our study, TGF-␤1 secretion diminished when platelets were treated with the Rac1 inhibitor NSC23766, as was cytokine release from OG-permeabilized platelets reconstituted with N17Rac1.…”

In vivo imaging visualizes discoid platelet aggregations without endothelium disruption and implicates contribution of inflammatory cytokine and integrin signaling

“…16,17 In addition, Rac1 is reportedly required for thrombus stabilization in conventional in vivo thrombosis models entailing thrombin-stimulated actin reorganization, 16 and in thrombin-stimulated granule secretion. 41 When we assessed thrombus formation in platelet-specific Rac1 KO mice (platelet factor 4 promoter-dependent deletion of Rac1: Pf4-Rac1 KO), we found that only the late-phase thrombus stabilization was impaired, but the effect of Pf4-Rac1 KO was modest in comparison to Y747A KI ( Figure 7D-F, and 7H-J). This finding indicates that both thrombin-PAR signaling-mediated integrin activation and Rac1 activation may be involved in thrombus stabilization, but not in initial platelet adhesion, and further confirm that tyrosine phosphorylation at Y747 of ␤3 integrin is likely required for stable adhesion of thrombi to the endothelium.…”

βArrestin1 Regulates the Guanine Nucleotide Exchange Factor RasGRF2 Expression and the Small GTPase Rac-mediated Formation of Membrane Protrusion and Cell Motility

“…Cofilin is known to be activated by, among others, Rac (22). Higher activities for both Rac and cofilin in ␤Arr1 Ϫ/Ϫ MEFs suggest that they function in the same pathway following ␤Arr1 Rac GTP FIGURE 3.…”