Binding and regulation of thrombopoietin to human megakaryocytes

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Abstract
Thrombopoietin (TPO, c‐Mpl ligand) is considered to play an important role in the regulation of megakaryocytopoiesis and platelet production by activating the cytokine receptor c‐Mpl. We have examined the binding of 125I‐TPO to the human megakaryocytic cell line, CMK, and to primary human megakaryocytes. Scatchard analysis of TPO binding to its cognate receptor in megakaryocytic cells suggested the existence of a single class of c‐Mpl receptors. CMK cells exhibited 1223 receptors per cell with a dissociation constant (Kd) of Kd = 223 p M, whereas primary human megakaryocytes exhibited 12 140 receptors per cell and a dissociation constant of Kd = 749 p M. The pretreatment of CMK cells and primary bone marrow megakaryocytes with TPO resulted in a decreased binding of TPO to the c‐Mpl receptors. This down‐regulation was observed within 3 h and was not inhibited by cycloheximide. Phorbol ester, an activator of protein kinase C, also inhibited TPO binding to the c‐Mpl receptors by reducing the number of these receptors. The pretreatment of CMK cells with IL‐3, IL‐6 and DMSO, all of which induced the differentiation of CMK cells, did not affect the binding of TPO to the c‐Mpl receptors. These results suggest an additional mechanism, where protein kinase C may help to regulate the binding of TPO to these cells.