In Vitro Effects

TPO acts only through the c-mpl (TPO) receptor. Despite sharing considerable sequence and structural homology with EPO, TPO does not bind the EPO receptor and EPO does not bind the TPO receptor. Binding of TPO to its receptor causes receptor dimerization that has major effects on megakaryocyte precursors (megakaryocyte colony-forming cells [Meg-CFCs]) and megakaryocytes, including preventing apoptosis (37); increasing megakaryocyte number, size, and ploidy (4); and increasing the rate of cellular maturation. Increased megakaryocyte ploidy is associated with a gene dosage effect on the expression of lineage-specific proteins (38). All these events are mediated through signal transduction pathways involving Janus kinase (JAK) signal transducers and activators of transcription (STAT), mitogen-activated protein (MAP) kinase, and other intracellular mediators.

Addition of TPO to isolated human CD34+ cells can cause most cells to become megakaryocytes that shed platelets (39). This last step, the shedding of platelets from megakaryocytes, however, does not require, and actually may be inhibited by, the presence of TPO (40).

Although TPO affects late cellular maturation events only in megakaryocytes, it can stimulate in vitro early precursors of all lineages as well as pluripotential stem cells (41). Approximately 70% of CD34+ cells express the TPO receptor c-mpl (42). Indeed, in some infants born with amegakaryocytic thrombocytopenia and a defective c-mpl receptor, the initial thrombocytopenia at birth often evolves over a few years into anemia and leukopenia as the marrow precursor cells die off, presumably owing to lack of TPO effect on multilineage precursors (43,44). In general, TPO affects the growth of early progenitors of all lineages, including the pluripotential stem cells, but only affects the late maturation of megakaryocytes. Hence, it stimulates only the production of mature platelets in vitro and in vivo.

Binding of TPO to its receptor leads to internalization of the ligand-receptor complex that is the major clearance mechanism of TPO from the circulation (4,9,45). Platelets, and probably megakaryocytes, are the sites of this activity; once internalized, TPO is degraded (46-48) with no recycling of the TPO receptor (49).

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