Proteomic identification of pleckstrin-associated proteins in platelets: Possible interactions with actin

Abstract

Pleckstrin (plek)-null platelets from a knockout mouse have been shown to be defective in granule secretion, aggregation and actin polymerization. However, the mechanism of plek signaling is currently unknown. Therefore, we sought to identify plek-binding proteins in platelets by using GST pulldown assays and immunoprecipitation to isolate proteins from extracts of protein kinase C-activated or inhibited human platelets. Co-purified plek-binding proteins were resolved by SDS-PAGE and identified via nanospray quadruple TOF MS. Identified proteins may be involved in various cellular processes including cytoskeletal reorganization (moesin, radixin and alpha-actinin) and signal transduction (serum deprivation response protein, 17 beta-hydroxysteroid dehydrogenase 4 and factor XIIIA). Both platelet aggregation and/or secretion require actin polymerization. However, studies have shown no direct association between plek and actin. Based on our findings we propose indirect associations between plek and actin through 17 beta-hydroxysteroid dehydrogenase 4, alpha-actinin, moesin, radixin and factor XIIIA, which in turn suggest new roles for plek in platelet biology.