mTOR is expressed in CD34⁺ stem cell–derived megakaryocytes, proplatelets, and circulating human platelets. Megakaryocytes were cultured from CD34⁺ stem cells and induced to extend proplatelets as described.⁴  (A) mTOR was identified by immunostaining (red fluorescence) in megakaryocytes and megakaryocytes with proplatelet extensions. In parallel, the cells were labeled with wheat germ agglutinin (WGA; green immunofluorescence). Arrows indicate proplatelet tips, the sites of platelet budding, in developing and fully extended proplatelets (middle and right panels). Scale bar, 25 μm. (B) mTOR (red) and polymerized actin (green) were imaged in quiescent and activated, aggregated platelets. Immunostaining of mTOR was done with the same antibody as in panel A. Platelets were activated with thrombin for 30 minutes. The bottom panels are enlargements of the areas outlined in the 2 middle panels. Images in panels A and B are each representative of 5 independent experiments. (C) Western analysis of mTOR was done using lysates of freshly isolated human platelets under unstimulated conditions (control) and after activation with thrombin (0.5 U/mL) for the indicated times.