uPA is endocytosed by in vitro–grown MKs and stored in granules. (A) Schematics of the structures of scuPA (top) and uPAT (bottom). Full-length scuPA is composed of (1) an N-terminal growth factor-like domain (GFD, yellow) that binds uPAR; (2) a Kringle domain (K, green) that mediates LRP1-dependent intracellular uptake,³⁶ binding to integrins^37,38 and nuclear translocation³⁶; and (3) the protease domain (catalytic domain, gray).³⁹ Site of activation by plasmin is shown in red. uPAT is composed of the protease domain in which ¹⁵⁷F¹⁵⁸K has been deleted⁴⁰ to create a thrombin cleavage/activation site (shown in green). (B) Representative WB of lysates of in vitro–grown day-11 MKs in the presence (+) or absence (−) of scuPA (400 nM) added on day 10. WB at the top was with anti-human uPA mouse monoclonal antibody followed by horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody, and bottom shows HPR-conjugated anti–β-actin antibody as a loading control. Size marker is shown to the left of the blot. (C) Dose-dependent uptake of Alexa-568 scuPA by CD34⁺ MKs. y-axes denote mean fluorescence intensity (MFI) measured by flow cytometry. Mean ± 1 standard deviation (SD) of relative uptake of scuPA compared with the values in its absence. n = 4 independent studies. P values were determined by ordinary 1-way analysis of variance (ANOVA). (D) Same as in panel C but for time course of Alexa-568 scuPA (400 nM) uptake by the CD34⁺ MKs. (E) Visualization using confocal microscopy of Alexa-568 scuPA (top, red) or Alexa-488 uPAT (bottom, green) endocytosed by day-11 CD34⁺ MKs for 24 hours starting on day 10 initiation of culture. DAPI (4′,6-diamidino-2-phenylindole; blue) depicts the nuclei. Scale bar is shown.