(A) Tyrosine phosphorylation of Vav in platelets stimulated by thrombopoietin (100 ng/mL). Platelets were lysed by the addition of an equal amount of a buffer containing 2% Triton X-100 before and after exposure to thrombopoietin (100 ng/mL) for various times as indicated. Vav was immunoprecipitated with specific anti-Vav antisera. Immune complexes were resuspended in SDS-sample buffer. Proteins were separated by 10% SDS-PAGE and transferred onto PVDF membranes. Immunoblots were probed with antiphosphotyrosine antibodies and bands were visualized by chemiluminescence (upper panel). The same PVDF membrane used in the upper panel was stripped and reprobed for Vav (lower panel). (B) Collagen- and thrombin-induced tyrosine phosphorylation of Vav in platelets. Platelet suspensions were incubated in the presence of RGDS peptide (200 μg/mL) for 5 minutes. Collagen (10 μg/mL) or thrombin (1 U/mL) was added to the suspension for various times as indicated. Platelets were lysed by the addition of an equal amount of a buffer containing 2% Triton X-100 before and after the addition of collagen or thrombin. Tyrosine phosphorylation of Vav was detected as in (A). (C) Tyrosine phosphorylation of Vav induced by various agonists. Platelet suspensions were incubated in the presence of RGDS peptide (200 μg/mL) for 5 minutes. Thrombin (1 U/mL), U46619 (1 μmol/L), ionomycin (20 nmol/L), PMA (100 nmol/L), or thrombopoietin (TPO, 100 ng/mL) was added to the suspension for 2 minutes. Platelets were lysed by the addition of an equal amount of a buffer containing 2% Triton X-100 and tyrosine phosphorylation of Vav was detected as in (A).