Effect of whole-platelet lysate on TPO mRNA expression in BM stromal cells.
Equal amounts of lysate from freeze-thawed platelets were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and the presence of PF4 and TSP was confirmed by Western blot analysis using anti-PF4 (2 μg/mL) (A) and anti-TSP (2 μg/mL) antibodies (B). Serial dilutions of lysate from 1 × 109platelets/mL were added to the stromal cell cultures, resulting in a dose-dependent inhibition of TPO mRNA expression measured with the QISH assay (C). The concentration of lysate added is expressed as the amount of platelet granular constituents in increasing numbers of platelets per milliliter. GAPDH mRNA expression remained unchanged. The lysate inhibition of TPO mRNA was partly relieved with the incubation of an antibody specific to PF4, whereas the nonimmune IgG control had no effect; GAPDH mRNA expression remained unchanged in cells exposed to lysate or antibody (D). The results shown are the mean ± SD values from 3 separate experiments performed in triplicate. ▪, TPO; □, GAPDH. * P < 0.05 compared with the control.
