Figure 4.

Shear stress exposure results in dramatically enhanced generation of MkMPs through activation of caspase-3. (A) Day 10 and 12 Mks were exposed to medium flow at a shear stress of 2.5 dyn/cm² for 0.5 hour. Whole cells were removed and the same amount of samples, as assessed by internal microbeads control, from slides exposed to shear flow and static control were analyzed by flow cytometry. PPTs were located on the high gate, PLPs in the middle gate, and MkMPs in the low gate. The number of particles in each gate is displayed below each gate. (B) MkMPs were smaller than microbeads of 1.34 µm diameter. (C) Expression of CD41 and CD42b on MkMPs from day 12 Mk cells. (D) CD62P expression and the corresponding IgG control of CD41⁺ MPs from day 12 Mk cells. (E) The relative number of CD41⁺ microparticles generated from day 10 or day 12 Mk cells either under static conditions or upon application of shear flow at 2.5 dyn/cm² for 0.5 hour (days 10 and 12) or 2 hours (day 10). (F) At days 10 and 12, DMSO (vehicle control) or z-VAD.fmk (pan-caspase inhibitor) or z-DEVD.fmk (caspase-3 inhibitor) treated Mks were exposed to shear flow at 2.5 dyn/cm² for 0.5 hour. After shear flow exposure, the number of isolated MkMPs was measured by flow cytometry. The number of MkMPs from 1 slide of Mks under shear flow was normalized by the number of MkMPs on a slide maintained under static culture conditions, and the resulting ratios were plotted. Error bars indicate SEM of 3 biological replicates. *P < .01 in panel (E) and *P < .05 in panel (F).