Arf6 deletion does not alter the surface levels of total or activated integrin αIIbβ3 or the binding of Fg to platelets. (A) Comparison of surface levels of total integrin αIIbβ3 between WT (light gray) and KO (dark gray) platelets. Using FITC-anti-CD41/61 antibody, levels of total integrin αIIbβ3 were measured by flow cytometry, in resting (i) and thrombin-stimulated (0.1 U/mL; ii) platelets. Unlabeled platelets (black) were used as the background control. (iii) Quantification of flow cytometry data in panels Ai and Aii expressed as GMFI. The data shown are representative of at least 2 independent experiments with triplicates. (B) Comparison of surface levels of activated integrin αIIbβ3 between WT (light gray) and KO (dark gray) platelets. Using phycoerythrin (PE)–Jon/A antibody, levels of activated integrin αIIbβ3 were measured by flow cytometry, in resting (i) and thrombin-stimulated (0.1 U/mL; ii) platelets. Unlabeled platelets (black) were used as the background control. (iii) Quantification of flow cytometry data in panels Bi and Bii expressed as GMFI. Data shown are representative of at least 2 independent experiments with triplicates. (C) Comparison of Fg binding to platelet surface between WT and KO platelets. Washed platelets (5.0 × 108/mL) were incubated, on ice, with FITC-Fg at different concentrations for 20 minutes and then fixed with 2% paraformaldehyde overnight at 4°C. Platelets were analyzed by flow cytometry, and quantification is shown as GMFI. The data shown are representative of 2 independent experiments with triplicates.
