Expression, activation, and subcellular localization of the Rap1 GTPase in cord blood-derived human megakaryocytes

• Published in: Experimental cell research 2004-10, Vol.300 (1), p.84-93
• Main Authors: Balduini, Alessandra, Pecci, Alessandro, Lova, Paolo, Arezzi, Nicoletta, Marseglia, Carmela, Bellora, Federica, Perotti, Cesare, Balduini, Cesare, Balduini, Carlo L., Torti, Mauro
• Format: Article
• Language: English
• Subjects: Activation; Cell Compartmentation - drug effects; Cell Compartmentation - physiology; Cell Differentiation - drug effects; Cell Differentiation - physiology; Cell Membrane - drug effects; Cell Membrane - metabolism; Cells, Cultured; Cytoplasmic Granules - drug effects; Cytoplasmic Granules - metabolism; Differentiation; Dose-Response Relationship, Drug; Fetal Blood - cytology; Fetal Blood - drug effects; Fetal Blood - enzymology; Fetal Blood - metabolism; Humans; Infant, Newborn; Megakaryocytes; Megakaryocytes - cytology; Megakaryocytes - drug effects; Megakaryocytes - enzymology; Megakaryocytes - metabolism; Platelet Glycoprotein GPIb-IX Complex - drug effects; Platelet Glycoprotein GPIb-IX Complex - metabolism; Platelet Membrane Glycoprotein IIb - drug effects; Platelet Membrane Glycoprotein IIb - metabolism; Protein Transport - drug effects; Protein Transport - physiology; Rap1; rap1 GTP-Binding Proteins - drug effects; rap1 GTP-Binding Proteins - metabolism; rhoA GTP-Binding Protein - drug effects; rhoA GTP-Binding Protein - metabolism; Thrombin; Thrombin - metabolism; Thrombin - pharmacology
• ISSN: 0014-4827; 1090-2422
• DOI: 10.1016/j.yexcr.2004.07.011

The expression of the small GTPase Rap1 in human megakaryocytes (MKs) differentiated from cord blood (CB)-derived progenitors was investigated. High levels of Rap1 were detected in the majority of mature megakaryocytes independently of days of culture, while a very low percentage of immature megakaryocytes was found to express a small amount of the protein. Rap1 was predominantly detected on internal α-granule but not on the plasma membrane. By contrast, CD41 was clearly present on the peripheral plasma membrane, although it also displayed an intracellular localization similar to that of Rap1. Upon thrombin stimulation, both Rap1 and CD41 translocated to the periphery of the cell. At the opposite, RhoA GTPase and glycoprotein Ibα were predominantly located at the plasma membrane and did not undergo relocation upon thrombin stimulation. Thrombin induced a dose- and time-dependent activation of Rap1 in mature megakaryocytes. By using a confocal microscopy approach with a specific probe, active Rap1 was detected exclusively at the peripheral plasma membrane. These results demonstrate that expression of Rap1 occurs during maturation rather than differentiation of megakaryocytes from cord blood progenitor cells. Moreover, we demonstrate that thrombin-activated Rap1 is exclusively localized at the peripheral plasma membrane.