Physiological Regulation of Early and Late Stages of Megakaryocytopoiesis by Thrombopoietin

Thrombopoietin (TPO) has recently been cloned and shown to regulate megakaryocyte and platelet production by activating the cytokine receptor c-mpl. To determine whether TPO is the only ligand for c-mpl and the major regulator of megakaryocytopoiesis, TPO deficient mice were generated by gene targeting. TPO-/-mice have a >80% decrease in their platelets and megakaryocytes but have normal levels of all the other hematopoietic cell types. A gene dosage effect observed in heterozygous mice suggests that the TPO gene is constitutively expressed and that the circulating TPO level is directly regulated by the platelet mass. Bone marrow from TPO-/-mice have decreased numbers of megakaryocyte-committed progenitors as well as lower ploidy in the megakaryocytes that are present. These results demonstrate that TPO alone is the major physiological regulator of both proliferation and differentiation ofhe-matopoietic progenitor cells into mature megakaryocytes but that TPO is not critical to the final step ofplatelet production. p latelets are small anucleated cells produced from mega-karyocytes. Normal hemostasis requires the maintenance of an adequate number of circulating platelets in the peripheral blood. Multiple cytokines (IL-1, IL-3, IL-6, IL-11, GM-CSF, and erythropoietin) stimulate megakaryocyto-poiesis both in vitro and in vivo (1, 2), but their effect is only modest compared with the recently identified ligand for the protooncogene c-mpl (3-9). This ligand, called thrombopoietin (TPO), is a novel cytokine with a unique structure; the NH2-terminal domain is homologous to erythropoietin, and the COOH-terminal glycosylated domain is unrelated to any known protein (6-8). In vitro and in vivo experiments with recombinant TPO indicate that it stimulates both megakaryocyte colony formation and megakaryocyte maturation (6, 8-10). TPO stimulates the formation ofCFU-megakaryocytes (CFU-megs) both alone and in combination with early acting factors (9, 11). It stimulates the production ofmegakaryocytes and functional platelets from enriched murine or human stem cell populations (12, 13). Injection of TPO into mice increases platelet counts 4-6-fold and stimulates a 20-fold increase in bone marrow megakaryocytes (7, 9). The importance of c-mpl in the physiological regulation of platelet production in vivo was demonstrated by the generation of mice deficient in c-mpl (14). These mice exhibit an 85% reduction in peripheral platelet counts and in marrow and spleen mega-karyocytes. Although these results demonstrate that c-mpl is critical in thrombopoiesis, they do not exclude the possibility that another receptor could be activated by TPO and could therefore be responsible for the remaining platelets in the c-mpl-/-mice. To investigate this, and the …