Cytochalasin D inhibits cytoskeletal translocation of Src and GPIb and increases Src activity in VWF-stimulated platelets.

Platelets were preincubated for 10 minutes at 37°C with 0.25% dimethyl sulfoxide (DMSO) as control or 1 μM cytochalasin D (Cyto D), and stimulated with 6 μg/mL botrocetin plus 10 μg/mL VWF for the indicated time periods (seconds). The platelets were solubilized with lysis buffer containing Complete. (A) The platelet lysates were immunoprecipitated with anti-Src MoAb, and the precipitated proteins were analyzed by an in vitro kinase assay using enolase as the exogenous substrate. The arrow represents the band presumably derived from IgG heavy chain. (B) Triton X–insoluble fractions were harvested by centrifugation at 15 000g for 5 minutes. The pellets were solubilized with Laemmli buffer. Cytoskeletal proteins were analyzed by immunoblotting with anti-Src and anti-GPIb MoAbs. (C) Supernatant after removal of the Triton X–insoluble fraction (Triton X-100-soluble fraction) was mixed with Laemmli sample buffer. Fractions were separated by SDS-PAGE and analyzed by immunoblotting with anti-Src and anti-GPIb MoAbs.