Shear force promotes proplatelet fission and platelet release. (A–H) Proplatelets released from fetal liver–derived megakaryocytes were cultured for 2 h in the presence or absence of shear (∼0.5 Pa). Every 20 min, samples were removed and probed with a rabbit polyclonal antibody against β1-tubulin and analyzed by immunofluorescence microscopy for different-sized objects (A–D) or labeled with a CD42a-specifc, FITC-conjugated antibody and analyzed by flow cytometry (G and H). (A–C) Representative micrographs of the culture after 0 (A), 60 (B), and 120 (C) min of shear. (insets) High magnification views of the composite images are shown. (D) Compared with no shear controls, platelet numbers increased over time, whereas proplatelet numbers decreased (n = 4). (E and F) Representative quantification of culture intermediates under shear with time. Platelet and PP progeny (cells of area = 1–137 µm2; E) and proplatelet progenitors (138–227 µm2; F) were binned by size (area), and their relative counts over time were spread across multiple bins to resolve the mechanism of platelet release. The data describe an inverse relationship between smaller and larger object counts that reveal dynamic proplatelet fission and continuous platelet release under shear with time. (G) Flow cytometric analysis of cultured proplatelet intermediates under shear with time (n = 3). (H) Representative quantification of cultured proplatelet intermediates by flow cytometry. Samples support immunofluorescence microscopy data and reveal a significant decrease in the number of large proplatelets and PP intermediates (small ProPLTs) after 2 h relative to no shear control. Error bars indicate mean ± standard deviation.
