Adducin in platelets: activation-induced phosphorylation by PKC and proteolysis by calpain.
Adducins are a family of cytoskeletal proteins encoded by 3 genes (alpha, beta, and gamma). Platelets express alpha and gamma adducins, in contrast to red blood cells that express alpha and beta adducins. During platelet activation with thrombin, calcium ionophore A23187, or phorbol 12-myristate 13-acetate, alpha and gamma adducins were phosphorylated by protein kinase C (PKC) as detected by an antibody specific for a phosphopeptide sequence in the highly conserved carboxy terminus. Platelet activation also led to adducin proteolysis; inhibition by calpeptin suggests that the protease was calpain. The kinase inhibitor staurosporine inhibited PKC phosphorylation of adducin and also inhibited proteolysis of adducin. Experiments with recombinant alpha adducin demonstrated that the PKC-phosphorylated form was proteolyzed at a significantly faster rate than the unphosphorylated form. The concentration of adducin in platelets was estimated at 6 microM, similar to the concentration of capping protein. Fractionation of platelets into high-speed supernatant (cytosol) and pellet (membrane and cytoskeleton) revealed a shift of PKC-phosphorylated adducin to the cytosol during platelet activation. Platelet aggregation detected turbidometrically was decreased in the presence of staurosporine and was completely inhibited by calpeptin. Thrombin-induced changes in morphology were assessed by confocal microscopy with fluorescein phalloidin and were not prevented by staurosporine or calpeptin. Our results suggest that regulation of adducin function by PKC and calpain may play a role in platelet aggregation.
Gilligan
Diana M
DM
Department of Internal Medicine (Hematology), Yale University School of Medicine, New Haven, CT, USA. dianag@psbc.org
Sarid
Rami
R
Weese
Joleen
J
D000328:Adult; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000097:blood*; D002154:Calpain / Q000097:blood*; D003598:Cytoskeletal Proteins / Q000097:blood; D004151:Dipeptides / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D010766:Phosphorylation; D015539:Platelet Activation; D010974:Platelet Aggregation; D011493:Protein Kinase C / Q000097:blood*; D011994:Recombinant Proteins / Q000378:metabolism; D012044:Regression Analysis; D019311:Staurosporine / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology; D000099291:Adducins
D002148:Calmodulin-Binding Proteins; D002154:Calpain; D003598:Cytoskeletal Proteins; D004151:Dipeptides; D011493:Protein Kinase C; D011994:Recombinant Proteins; D019311:Staurosporine; D013917:Thrombin; D000099291:Adducins; C071482:calpeptin