Ablation of the αIIb C490-C545 disulfide bond extends residency time of αIIbβ3 integrin in focal adhesions due to a reduced rate of clathrin-mediated integrin internalization and recycling. (A) WT or cysteine mutant αIIβ3-expressing cells were seeded onto a fibrinogen-coated surface for 1 hour, fixed, stained for F-actin intensity, and the cell area measured. The surface area of spread cells is smaller when the αIIb C490-C545 disulfide bond is missing. The bars and errors are mean ± standard deviation (SD) of 8 biological replicates, and each data point represents the mean surface area of at least 500 cells. (B) WT or cysteine mutant αIIβ3-expressing cells were treated with 0.45 M sucrose to inhibit clathrin-mediated endocytosis (CME) before seeding on a fibrinogen-coated surface in the presence of β3 mAb VI-PL2 for 1 hour. After fixation, nuclei were stained with DAPI and spread area measured. Panel i is a representative image of WT cells before and after CME inhibition; panel ii is spread cell area for WT or cysteine mutant αIIβ3-expressing cells. The bars and errors are mean ± SD of 4 biological replicates, and each data point represents the mean surface area of at least 500 cells. (C) Cells stained with β3 mAb VI-PL2 were seeded onto a fibrinogen-coated surface and imaged every 30 seconds for 50 minutes. Integrin clusters were identified and observed. Panel i is the mean β3 cluster lifetime as a function of time for 3 independent experiments for at least 100 cells each; panel ii is the mean and SD of all measurements. (D) WT or cysteine mutant αIIβ3-expressing cells were seeded onto a fibrinogen-coated surface for 1 hour, αIIbβ3 immunoprecipitated from cell lysate, and blotted for αIIb and AP2. Panel i is a representative blot; panel ii includes AP2/αIIb band intensity ratios for 4 biological replicates. The bars and errors are mean ± SD. *P < .05, **P < .01, ****P < .0001 (1-way analysis of variance). ns, not significant.