Figure 3.

Inhibition of metalloproteinases in culture restores GPIbα expression in ESPs. (A) On day 10 of ESC culture, 1% DMSO alone (vehicle) or 100 μM GM6001 dissolved with 1% DMSO was added to the culture medium. On day 12 of culture, representative flow cytometry dot plots of mature megakaryocytes derived from ESCs (ESC-MKs) in the absence of GM6001 (left) and ESPs in the absence or presence of GM6001 are shown. (B) The graph shows the percentage of GPIbα⁺ of all αIIb⁺ ESPs on day 12 in the absence or presence of 1–10 μM TAPI-1 or 100 μM GM6001. Results are shown as mean ± SEM from three independent experiments. (C) The graph summarizes total numbers of αIIb⁺ (αIIb⁺GPIbα⁺ plus αIIb⁺GPIbα⁻) ESPs per well of a 6-well plate on day 12. Results are the mean ± SEM from four independent experiments. (D) The graph shows the effects of the metalloproteinase inhibitor GM6001 on the surface expression of GPV or GPVI in αIIb⁺ ESPs on day12. Results are mean ± SEM from four independent experiments. (E) Expression of GPIbα or GC in lysates from ESC-MKs (i), in lysates from pellets containing ESPs depleted of MKs (ii), or in supernatant (iii). In panel (i) or (ii), lysates were analyzed by Western blot (7.5% SDS-PAGE) with an anti-GPIbα antibody. In panel (iii), supernatant derived from culture media of ESPs pretreated with or without GM6001 were subjected to immunoprecipitation followed by immunoblotting with anti-GPIbα. In vitro–aged platelets from an adult mouse were used as a positive control in panel (iii). Similar results were obtained from three independent experiments. (F) The graphs show the percentage of positive cells of annexin V or P-selectin from the total αIIb⁺ ESPs on day 12 in the absence or presence of GM6001. Results are the mean ± SEM from three independent experiments.