Binding and regulation of thrombopoietin to human megakaryocytes

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-Mpk We have examined the binding of 125(I)-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 (K(d)) of K(d) = 223 pM, whereas primary human megakaryocytes exhibited 12140 receptors per cell and a dissociation constant of K(d)=749 pM. 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 3h 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. JIL-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.