Filamin A binds to activated integrin αIIbβ3 in stimulated human platelets. (A) Washed human platelets were stimulated with 0.5 mM Mn²⁺ and stirred in the presence of 25 μg/mL fibrinogen in suspension and lysed at indicated time points. Platelet lysates were immunoprecipitated with antibodies against integrin αIIbβ3 and then immunoblotted with antibodies against filamin A, talin-1, and integrin β3, respectively. (B) Representative confocal microscopic images colocalizing integrin αIIbβ3 with filamin A or talin-1 in human platelets spread on fibrinogen at the indicated time points. Experiments were repeated independently at least 3 times with similar results. Colocalization of integrin αIIbβ3 with filamin A (C) or talin-1 (D) was quantified using Pearson correlation coefficient calculation, using National Institutes of Health Image J JACoP plug-in⁵⁷ with Costes automatic threshold. Twenty images from each time point were analyzed. A representative result from 2 independent experiments.