Thrombin activation of human platelets dissociates a complex containing gelsolin and actin from phosphatidylinositide-specific phospholipase Cγ1

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Abstract
We have examined the association of two cytoskeleton proteins, gelsolin and actin, with phosphatidylinositide-specific phospholipase Cγ1 (PLCγ1) in resting and thrombin-stimulated human platelets. In unstimulated platelets, gelsolin, actin and PLCγ1 were immunoprecipitated as a complex by a polyclonal antibody to PLCγ1. The association of gelsolin and actin was specific for PLCγ1 because immunoprecipitates of PLCs β2, β3, γ2 and δ1, which are also expressed in human platelets, did not contain detectable gelsolin or actin. Activation with thrombin resulted in platelet aggregation and the dissociation of gelsolin and actin from PLCγ1. Inhibition of thrombin-induced platelet aggregation blocked the dissociation of gelsolin and actin from PLCγ1. After stimulation with thrombin, PLCγ1 activity in immunoprecipitates was increased 2–3-fold. This elevation in PLCγ1 activity in response to thrombin activation was not observed when platelet aggregation was blocked. Although PLCγ1 is tyrosine phosphorylated in response to many agonists, we could not detect, by Western analysis with anti-phosphotyrosine antibodies, tyrosine phosphorylation of PLCγ1 immunoprecipitated from thrombin-stimulated platelets. These results demonstrate that PLCγ1 is associated with gelsolin and actin in resting platelets, and that thrombin-induced platelet aggregation results in the dissociation of PLCγ1 from gelsolin and actin, and the stimulation of PLCγ1 activity.