title: Actin reorganization and proplatelet formation in murine megakaryocytes: the role of protein kinase calpha.
abstract: With the recent cloning and characterization of thrombopoietin, appreciation of the molecular events surrounding megakaryocyte (MK) development is growing. However, the final stages of platelet formation are less well understood. Platelet production occurs after the formation of MK proplatelet processes. In a study to explore the molecular mechanisms underlying this process, mature MKs isolated from suspension murine bone marrow cell cultures were induced to form proplatelets by exposure to plasma, and the role of various cell-signaling pathways was assessed. The results showed that (1) bis-indolylmaleimide I, which blocks protein kinase C (PKC) activation; (2) down-modulation of conventional or novel classes of PKC by phorbol myristate acetate; and (3) ribozymes specific for PKCalpha each inhibited proplatelet formation. Inhibition of several MAP kinases, PI3 kinase, or protein kinase A failed to affect MK proplatelet formation. To gain further insights into the function of PKCalpha in proplatelet formation, its subcellular localization was investigated. In cultures containing active proplatelet formation, cytoplasmic polymerized actin was highly aggregated, its subcellular distribution was reorganized, and PKCalpha colocalized with the cellular actin aggregates. A number of MK manipulations, including blockade of integrin signaling with a disintegrin or inhibition of actin polymerization with cytochalasin D, interrupted actin reorganization, PKC relocalization, and proplatelet formation. These findings suggest an important role for PKCalpha in proplatelet development and suggest that it acts by altering actin dynamics in proplatelet-forming MKs. Identification of the upstream and downstream pathways involved in proplatelet formation should provide greater insights into thrombopoiesis, potentially allowing pharmacologic manipulation of the process.
authors.affiliation: Division of Hematology, University of Washington, Seattle, WA 98195, USA.
authors.forename: P
authors.initials: P
authors.lastname: Rojnuckarin
authors.forename: K
authors.initials: K
authors.lastname: Kaushansky
mesh_terms: D008841:Actin Cytoskeleton / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D000199:Actins / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000648:ultrastructure; D000818:Animals; D001792:Blood Platelets / Q000166:cytology* / Q000187:drug effects; D015638:Cytochalasin D / Q000494:pharmacology; D003593:Cytoplasm; D019483:Disintegrins / Q000494:pharmacology; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000494:pharmacology / Q000502:physiology; D008533:Megakaryocytes / Q000166:cytology* / Q000187:drug effects; D051379:Mice; D008815:Mice, Inbred Strains; D008856:Microscopy, Fluorescence; D010949:Plasma; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000494:pharmacology / Q000502:physiology; D051571:Protein Kinase C-alpha; D016337:RNA, Catalytic / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D000199:Actins; D019483:Disintegrins; D007527:Isoenzymes; D016337:RNA, Catalytic; D015638:Cytochalasin D; C496630:Prkca protein, mouse; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D013755:Tetradecanoylphorbol Acetate