Association of Vav and GPIIb α with the Triton X-100 insoluble residue (A) and its cleavage in platelets (B and C). (A) Platelets were lysed with Triton X-100-EGTA buffer before or after stimulation with thrombin (1 U/mL) with or without stirring. Lysates were separated into soluble and insoluble residue. Proteins from each fractions were separated by 10% SDS-PAGE and immunoblotted with Vav antisera (lower panel) or an anti-GPIIbα monoclonal antibody (AP4) (upper panel). Lane 1, Triton X-100 soluble residue of resting cells (7.5 × 10⁶ cells); lane 2, Triton X-100 insoluble residue of resting cells (3.0 × 10⁷ cells); lanes 3 to 6, Triton X-100 insoluble residue from 3.0 × 10⁷ cells, 15 seconds, 1 minute, 5 minutes, and 10 minutes after exposure to thrombin (1 U/mL) with stirring; lane 7, Triton X-100 insoluble residue of cells (3.0 × 10⁷ cells) stimulated for 10 minutes with thrombin without stirring. (B) Cleavage of calpain (upper panel) and Vav (lower panel). Cleavage of the 80-kD subunit of μ-calpain in human platelets. Platelets were incubated with either calpeptin or DMSO (vehicle of calpeptin, final 0.1%) for 5 minutes. Platelets were then treated with 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. Calpain (upper panel) or Vav (lower panel) was detected by immunoblotting with specific monoclonal antibodies. The arrow indicates the relative position of the intact 80-kD subunit of μ-calpain (upper panel) or Vav (lower panel). Lane 1, control, DMSO (final 0.2%) + 1 mmol/L CaCl₂; lane 2, A23187 (1 μmol/L, for 5 minutes) + 1 mmol/L CaCl₂; lane 3, A23187 (1 μmol/L, for 5 minutes) + 5 mmol/L EGTA; lane 4, A23187 (1 μmol/L, for 5 minutes) + 1 mmol/L CaCl₂ + calpeptin (20 μmol/L); lane 5, dibucaine (1 mmol/L, for 15 minutes) + 1 mmol/L CaCl₂; lane 6, dibucaine (1 mmol/L, for 15 minutes) + 5 mmol/L EGTA; lane 7, dibucaine (1 mmol/L, for 15 minutes) + 1 mmol/L CaCl₂ + calpeptin (20 μmol/L). (C) Cleavage of Vav during platelet aggregation. Platelets were stimulated with thrombin (1 U/mL) for 30 minutes with or with stirring. After the addition of EGTA (final, 5 mmol/L) and EDTA (final, 5 mmol/L), platelets were lysed by boiling in SDS sample buffer. Vav was detected by immunoblotting as described as above. Lane 1, resting; lane 2, thrombin stimulation for 30 minutes with stirring; lane 3, thrombin stimulation for 30 minutes without stirring.