Effect of calcineurin inhibition on cofilin dephosphorylation, Rac1 activation, and class II PAKs phosphorylation in thrombin-stimulated platelets. (A) Inhibition of cofilin dephosphorylation by the calcineurin inhibitory peptide CAID-11R and not by the control peptides CAID and NFAT-11R in thrombin-stimulated platelets. Platelet samples pretreated with CAID (-■-), CAID-11R (-□-), or NFAT-11R (-▴-; each 20 μM) and stimulated with 0.5 U/mL thrombin were immunoblotted with anti-cofilin and anti–phospho-cofilin antibodies. Top, representative immunoblots for cofilin phosphorylation. Bottom, graphic representation of the results. Values are the mean + SD or mean − SD of 3 independent experiments. (B) Inhibition of Rac1 activation by 20 μM CAID-11R. Rac1-GTP was pulled down from platelets untreated (−) or pretreated with CAID-11R (+) and then stimulated with 0.5 U/mL thrombin for 5 seconds. Representative immunoblots of pulled down Rac1-GTP and total Rac1. (C) Inhibition of class II PAKs phosphorylation by CAID-11R. Platelets stimulated with thrombin in the absence (-■-) or in the presence of CAID-11R (-□-) were immunoblotted with anti–phospho-PAK4/5/6 antibody. Top, representative immunoblots of PAK4/5/6 phosphorylation. Bottom, graphic representation of PAK4/5/6 phosphorylation. Values are the mean + SD or mean − SD for 3 independent experiments. * denotes statistical significance (P < .001) with respect to nontreated sample.