Figure 2.

Shear stress promotes PS externalization and caspase-3 activation. Caspase-3 is involved in shear-stress enhanced PPTs and PLPs formation. (A) Percent of Annexin V⁺ Mks at days 8 and 10 after shear-flow application at 1 dyn/cm² for 2 hours vs static control. Adherent and nonadherent Mks were analyzed separately. (B) Percent of Annexin V⁺ day 10 Mks exposed to 2.5 dyn/cm² for 0 (static control), 15, 30, 60, or 120 minutes. (C) Correlation between caspase-3 activation and PPT formation of Mks under static culture conditions. X-axis: caspase-3 activation level defined as the ratio of MFI of active caspase-3 over IgG control. Y-axis: percent of Mks bearing PPTs. (D) MFI of active caspase-3 per µm² of adherent day 10 and 12 Mks from different donors (don) after shear-flow exposure at 1.0 dyn/cm² for 2 hours vs static control. The ratio of MFI of caspase-3 of Mks under shear-flow conditions over static control is indicated above the bars. Under shear flow, MFI values for caspase-3 activation were well above the MFI for isotype control, so there was no need to correct for isotype control. (E-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, PPTs and PLPs were harvested and counted. The number of PPTs (E) or PLPs (F) from 1 slide of Mks exposed to shear flow was normalized by number of PPTs or PLPs on a slide under static conditions, and the resulting ratios are plotted. Error bars indicate SEM of 3∼4 biological replicates in panel (A-B,E-F) and 6∼10 different images in panel (D). *P < .05; **P < .01. ns, not significant.