Pleckstrin, the platelet and leukocyte C kinase substrate, is a prominent substrate of PKC in platelets, monocytes, macrophages, lymphocytes, and granulo - cytes. Pleckstrin accounts for 1% of the total protein in these cells, but it is best known for containing the 2 prototypic Pleckstrin homology, or PH, domains. Overexpressedpleckstrin can affectpoly - phosphoinositide second messenger - based signaling events; however, its true in vivo role has been unknown. Here, we describe mice containing a null mutation within the pleckstrin gene. Platelets lacking pleckstrin exhibit a marked defect in exocytosis of δ and α granules, αllbβ3 activation, actin assembly, and aggregation after exposure to the PKC stimulant, PMA. Pleckstrin-null platelets aggregate normally in response to thrombin, but they fail to aggregate in response to thrombin in the presence of Pi3K inhibitors, suggesting that a Pi3K-dependent signaling pathway compensates for the loss of pleckstrin. Although pleckstrin - null platelets merged their granules in response to stimulation of PKC, they failed to empty their contents into the open canalicular system. This might be attributable to impaired actin assembly present in cells lacking pleckstrin. These data show that pleckstrin regulates the fusion of granules to the cell membrane and is an essential component of PKC-mediated exocytosis. © 2009 by The American Society of Hematology.