P2Y12 and PI3K signaling is needed for persistent αIIbβ3 activation under flow. (A) Human blood was flowed over VWF/collagen at 1000 s–1 for 4 minutes. Thrombi on coverslips were perfused again for 10 minutes with PPACK-anticoagulated plasma, and stained with FITC-PAC1 (1:20). Indicated antagonists were present during secondary perfusion: vehicle (i), AR-C69931MX (30 μM) (ii-iii), and wortmannin (1 μM) (iv). As a positive control, single platelets on coverslips were allowed to spread on collagen for 20 minutes before staining (ii). Shown are representative fluorescent images (180 × 180 μm) after secondary perfusion. (B) Mean surface area coverage of FITC-PAC1 fluorescence after secondary perfusion (n = 3); iloprost (20 nM) was present as indicated. Mean ± SEM; *P = .05 vs vehicle.
