A comparison of potency of and intracellular signaling events provoked by, small molecule and protein-based mpl agonists

Abstract

e14624 Background: Thrombopoietin (TPO) interacts with its receptor (mpl) to initiate signal transduction and increase platelet production. Other mpl agonists have been developed for clinical use: MDGF (a recombinant protein) and romiplostim (a peptibody) are protein-based mpl agonists; eltrombopag (a small molecule) is a nonpeptide mpl agonist. Methods: Mpl agonists were compared for their ability to induce cellular responses and activate relevant signaling pathways in mpl-expressing cells. Proliferation was assessed by ATP bioluminescence in Baf3/mpl cells treated with mpl agonists for 2 days. Differentiation was assessed by flow cytometric-detection of CD41 and CD61 expression on human CD34+ cells cultured with mpl agonists for 7 days. Activation of signaling pathways was determined by Western blot analysis of proteins from serum-starved Baf3/mpl cells treated with mpl agonists for 20 minutes. Experiments were repeated 3 or more times. Results: The TPO concentration that produced a half-maximal (EC50) proliferative response of Baf3/mpl cells was comparable with that of romiplostim (Table). In contrast, a 30-fold greater concentration of MGDF and a 2,000-fold greater concentration of small molecule were required to produce the same degree of proliferation. In addition, a 1,000,000-fold greater concentration of small molecule compared with romiplostim was required to produce an EC50 for differentiation of CD34+ cells. Mpl agonists activated identical signaling pathways in Baf3/mpl cells: a similar dose-response for phosphorylation of JAK2, Stat3, Stat5, Erk1/2, and AKT was observed at their respective EC10, EC50, and EC100 concentrations. Conclusions: In Baf3/mpl cells, the small molecule was between 1,000- and 1,000,000-fold less potent than protein-based mpl agonists when corrected for molecular mass differences. Small molecule and protein-based mpl agonists were indistinguishable when mpl intracellular signaling was compared at similarly effective concentrations. [Table: see text] [Table: see text]