The effect of β3 substitutions on αIIbβ3 and αvβ3 outside-in signaling–related functions and complex stability. (A) Cell adhesion to fibrinogen-coated wells was evaluated after 30-minute incubation. The cells were fixed and stained by May-Grünwald eosin–methylene blue solution. Surface coverage was measured using Impact R analyzer. Results present as percentage surface coverage relative to WT cells ± SD (N ≥ 3). (B) Spreading was evaluated in cells harboring WT αIIbβ3 or αIIbβ3-261Lys and by staining with anti-actin FITC-conjugated antibody after 30-minute incubation. The actin-stained cells of 10 fields were counted and percentage of cells fully spread was calculated out of total adhered cells (presented in the right corner ± SD). (C) Band intensity of immunoblotting of anti-Fak and anti-Fak phosphorylated (FAK-PY) on WT αIIbβ3 and αIIbβ3-261Lys cell lysates. Measurements of band intensity were done using the EzQuant densitometry program. Index phosphorylation was calculated as the ratio between FAK-PY and FAK band intensity (presented on the top of the bars). (D) Clot retraction of cells resuspended in DMEM in the presence of fibrinogen and thrombin after incubation for 18 hours at 37°C. (E) Surface expression of integrin complexes was measured by flow cytometry using P2-FITC antibody against αIIbβ3 and LM609-FITC against αvβ3. Cells were incubated with or without 5mM EDTA for 30 minutes at room temperature. The results are presented as the proportion of MFI in the presence and absence of EDTA (n ≥ 3).