αIIbβ3 integrin activation and outside-in signaling in MYH9Δ platelets. (A) Flow-cytometry experiments showing integrin α_IIbβ₃ activation as revealed by Jon/A-PE (left panel) or fibrinogen (right panel) labeling following platelet activation by thrombin (thr, 1 U/mL) or collagen (coll, 100 μg/mL) in the absence of agitation. The amount of activated integrin is indicated by the geometric mean of the relative fluorescence intensity, in arbitrary units. Gray and black bars represent control and MYH9Δ platelets, respectively; mean (± SEM) n = 3 experiments. (B) Western blots showing phosphorylation of integrin β3 at Y773 (top panel) during stimulation of platelet suspension by thrombin (1 U/mL) for up to 3 minutes in the aggregometer. Identical protein loading was checked by reblotting with an anti-β3 antibody (bottom panel). Blots are representative of 2 experiments. (C) PtdIns(3,4)P₂ synthesis following thrombin (1 U/mL) or collagen (10 μg/mL) stimulation upon 2 minutes. Values have been normalized against total polyphosphoinositides and the results are presented as fold increase compared with nonstimulated platelets (± SEM, n = 3 independent experiments).