mTOR is required for platelet aggregate stability under flow. (A) Purified human platelets (2 × 107/mL) were spread on glass coverslips coated with 100 μg/mL fibrinogen. After 30 minutes, unbound platelets were removed and activated platelets were treated with vehicle (DMSO), WYE-354 (10μM), or EHT 1864 (50μM) for an additional 30 minutes. Platelet spreading and lamellipodial withdrawal were evaluated by DIC microscopy. Scale bar = 10 μm. (B) PPACK-anticoagulated blood was perfused over collagen for 4 minutes to produce platelet aggregates that were subsequently perfused with buffer containing fibrinogen for an additional 4 minutes before perfusion with buffer, fibrinogen, and vehicle (DMSO), WYE-354 (10μM), or EHT 1864 (50μM). Representative DIC images of platelet aggregates are shown. Arrow indicates direction of flow. Scale bar = 50 μm. (C) Quantification of platelet disaggregation promoted by WYE-354 or EHT 1864 under flow (n = 3). Data are represented as mean ± SEM.
