The effect of D3 and ADP on the composition of the cytoskeleton of platelet aggregates.

After 6 minutes in the aggregometer, platelet samples were lysed, and the low-speed cytoskeletal pellet was isolated. Pellets were solubilized with reducing sample buffer and electrophoresed through a 5% to 20% exponential gradient polyacrylamide gel. (A) Coomassie blue-stained gel. The gel shown is representative of 3 experiments. Lane 1 contains spontaneous aggregation; lane 2, D3 aggregated platelets; lane 3, ADP aggregated platelets; lane 4, D3/ADP added simultaneously; lane 5, D3 2 minutes preincubation and ADP; lane 6, D3 and ADP preincubation 2 minutes; lane 7, whole platelets; lane 8, molecular weight standards. (B) Quantitative Western blot. Cytoskeletal cores isolated from D3 aggregated platelets (lanes 1, 4, 7, and 10), ADP aggregated platelets (lanes 2, 5, 8, and 11) and D3 2 minutes of preincubation and ADP (lanes 3, 6, 9 and 12) were electrophoresed at protein gel loads of 100%, 75%, 50%, and 25% and transferred to nitrocellulose. The blots were probed with a goat antihuman talin antibody and binding was detected using an HRP conjugated secondary antibody and chemiluminescence. Shown is a representative blot of 3 independent experiments. WP indicates whole platelets.