BINDING AND REGULATION OF THROMBOPOIETIN TO HUMAN MEGAKARYOCYTES

Thrombopoietin (TPO, c-Mpl ligand) is considered to play an important role in the regulation of megakaryo-cytopoiesis and platelet productio n by activating the cytokine receptor c-Mpl. We have examined the bind ing of I-125-TPO to the human megakaryocytic cell line, CMK, and to pr imary human megakaryocytes. Scatchard analysis of TPO binding to its c ognate receptor in megakaryocytic cells suggested the existence of a s ingle class of c-Mpl receptors. CMK cells exhibited 1223 receptors per cell with a dissociation constant (K-d) Of K-d = 223 pm whereas prima ry human megakaryocytes exhibited 12140 receptors per cell and a disso ciation constant of K-d = 749 pm. The pretreatment of CMK cells and pr imary bone marrow megakaryocytes with TPO resulted in a decreased bind ing of TPO to the c-Mpl receptors. This down-regulation was observed w ithin 3h and was not inhibited by cycloheximide. Phorbol ester, an act ivator of protein kinase C, also inhibited TPO binding to the c-Mpl re ceptors by reducing the number of these receptors. The pretreatment of Chill cells with IL-3, IL-6 and DMSO, all of which induced the differ entiation of CMK cells, did not affect the binding of TPO to the c-Mpl receptors. These results suggest an additional mechanism, where prote in kinase C may help to regulate the binding of TPO to these cells.