Cleavage of WASP. (A) Platelets were incubated with either calpeptin (20 μmol/L) or DMSO (vehicle of calpeptin; final concentration, 0.1%) for 5 minutes. Platelets were then treated with calcium ionophore A23187 (1 μmol/L for 5 minutes) or dibucaine (1 mmol/L for 15 minutes) in the presence of 1 mmol/L CaCl₂ or 5 mmol/L EGTA. Whole platelet lysates (1.5 × 10⁷ cells/lane) were analyzed by 10% SDS-PAGE. Separated proteins were electrophoretically transferred from the gel onto nitrocellulose membranes. WASP was detected by immunoblotting with polyclonal antibody 503. The top arrow indicates the relative position of the intact 64-kD subunit of WASP. The lower arrows indicate the positions of apparently cleaved products of WASP. Lane 1, control, DMSO + CaCl₂; lane 2, A23187 + CaCl₂; lane 3, A23187 + EGTA; lane 4, A23187 + CaCl₂ + calpeptin ; lane 5, dibucaine + CaCl₂; lane 6, dibucaine + EGTA; lane 7, dibucaine + CaCl₂ + calpeptin. (B) Cleavage of WASP during platelet aggregation. Platelets were stimulated with thrombin (1 U/mL) for 30 minutes with or without stirring. After the addition of EGTA (5 mmol/L) and EDTA (5 mmol/L), platelets were lysed by boiling in SDS sample buffer. WASP was detected by immunoblotting as described in Fig 1. Lane 1, resting platelets; lane 2, thrombin stimulation of platelets for 30 minutes with stirring; lane 3, thrombin stimulation for 30 minutes without stirring. The arrows indicate the same cleaved fragments of WASP as in (A).