Humanized VB22B minibody for human Mpl stimulates human megakaryopoiesis but does not enhance platelet aggregation.

OBJECTIVE

Thrombopoietin stimulates megakaryopoiesis and platelet production by binding to its receptor, Mpl, on hematopoietic progenitor cells. Previously, a murine VB22B minibody for Mpl was shown to stimulate megakaryocyte colony formation in vitro and increase the platelet count in cynomolgus monkeys. In this study, we directly compared the effects of a humanized VB22B minibody (huVB22B) with those of thrombopoietin and eltrombopag under the hypothesis that Mpl agonists might have different biological effects on megakaryopoiesis, platelet production, intracellular signal transduction, and platelet function.

MATERIALS AND METHODS

Human bone marrow-derived CD34(+) cells were used for colony formation assays and proplatelet formation assays in vitro. The DNA ploidy in megakaryocytes was analyzed by flow cytometry. Phosphorylation of signal transducers and activators of transcription and mitogen-activated protein kinase was detected by Western blotting using specific antibodies. The effects of the Mpl agonists on platelet aggregation were analyzed by aggregometry using human platelets.

RESULTS

HuVB22B was as potent as thrombopoietin and eltrombopag in its ability to form mature megakaryocytes using human CD34(+) cells in vitro. It did not affect granulocyte-macrophage or erythroid colony formation. HuVB22B increased the number of proplatelet-forming megakaryocytes more efficiently than thrombopoietin or eltrombopag. Despite stronger phosphorylation of signal transducers and activators of transcription and mitogen-activated protein kinase compared with thrombopoietin in human platelets, huVB22B did not enhance adenosine diphosphate- or collagen-induced platelet aggregation. Eltrombopag did not enhance agonist-induced platelet aggregation.

CONCLUSIONS

We found that huVB22B, eltrombopag, and thrombopoietin have different effects on megakaryopoiesis, platelet function, and intracellular signaling. The precise mechanisms for these different biological effects regarding stimulation through the same receptor, Mpl, remain to be elucidated.