Interaction of CD154 with CD40 on the cell surface of activated platelets leads to cleavage of CD154.

(A) Platelets were either left unstimulated (○) or were activated with 0.2 U/mL thrombin for the indicated times, and the concentration of sCD154 in the supernatants was determined using a specific ELISA. The data are shown as mean values ± SD of duplicates and are representative of 4 independent experiments. The low background levels of sCD154 observed in unstimulated platelets probably reflect a minimal activation of platelets through the isolation and incubation procedures. ●, activated; ▪, activated plus mAb MOPC-21; ▴, activated plus mAb G28-5. (B) CD154 was immunoprecipitated from lysates and supernatants of unstimulated or activated (0.2 U/mL thrombin for 1 hour) platelets with mAb TRAP2 and detected by Western blotting with a CD154-specific antiserum. In parallel, platelet activation was performed in the presence of anti-CD40 mAb G28-5 (10 μg/mL). Unstim indicates unstimulated; Lys, lysate; Sup, supernatant. (C) Platelets were either left unstimulated or activated for the indicated time periods, and sCD154 was immunoprecipitated with a chimeric CD40-Ig reagent. The immunoprecipitate was subjected to SDS-PAGE and detected by Western blotting with a CD154-specific antiserum. Unstim indicates unstimulated.