Phosphorylation of PSP. (A) Phosphorylation of affinity-purified platelet PSP. Immunoaffinity purified PSP (2 μg) was phosphorylated in vitro with purified PKC (6.3 ng) and [γ³²P]ATP for 20 minutes at 30°C and subjected to SDS-PAGE. Phosphoproteins were detected by phosphorimaging. Relative migration of molecular standards (in kilodaltons) is indicated at left. (B) Phosphorylation of PSP in thrombin-activated and unstimulated platelets. Washed human platelets (1.3 × 10⁹ cells/100 μL) were permeabilized with saponin in the presence of [γ³²P]ATP and incubated with or without thrombin (0.15 U/mL) at 30°C for 1 to 10 minutes. The cells were lysed, boiled for 3 minutes, and diluted in radioimmunoassay precipitation buffer. After immunoprecipitation with anti-PSP antibody or a control antibody, the phosphoproteins were subjected to SDS-PAGE on 7.5% gels and phosphorimaging. The presence or absence of thrombin, the precipitating antibody, and the time of incubation are shown for each lane. Arrow indicates PSP. (C) The role of PKC in phosphorylation of PSP. As described above, washed, saponin-permeabilized platelets were incubated with prostaglandin E₁ (PGE1; 10 μmol/L), thrombin (0.15 U/mL), or PMA (100 nmol/L) in the presence or absence of the PKC inhibitor Ro-31-8220 (10 μmol/L) for 3 minutes at 30°C. The cells were lysed, boiled, and diluted in radioimmunoprecipitation buffer. After immunoprecipitation with the anti-PSP antibody, the pellet was subjected to SDS-PAGE and phosphorimaging.