Protein Synthesis In Megakaryocytes Stimulated By Thrombo- Poietin In Vitro

Studies in our laboratories have been concerned with the responses of megakaryocytes to thrombopoietin in vitro. We have shown that preparations of thrombopoietin stimulate DNA synthesis in guinea pig megakaryocytes. The increase in ³H-thymidihe incorporation correlates with an increase in the labeling index of the megakaryocytes. After 2 and 3 days of incubation an increase in the ploidy levels of the megakaryocytes has been observed in thrombo- poietin-supplemented cultures compared to controls.

Recent studies have examined the incorporation of ³H-leucine in megakaryocyte cultures. Megakaryocytes were prepared on BSA or Percoll gradients to purities of 70-95%. ³H-leucine incorporation was measured after a 15 hr incubation of the megakaryocytes in medium containing 10% thrombopoietin or control preparations of normal plasma or phosphate-buffered saline. Utilization of isotope increased over a 24 hr period and was higher in the thrombopoietin-supplemented cultures. In addition, synthesis of specific proteins was analyzed by using SDS- polyacrylamide gel electrophoresis and quantitation was achieved by employing rocket immunoelectrophoresis. The results indicate that thrombopoietin stimulates endoredu- plication and protein synthesis in megakaryocytes in vitro and that this system may serve as a model for studying the mechanism of action of thrombopoietin in megakaryocytopoiesis.