Regulation of Thrombopoietin Levels by c-mpl-Mediated Binding to Platelets

The involvement of platelets and the c-mpl receptor in the regulation of thrombopoietin (TPO) plasma concentrations and tissue mRNA levels was investigated in both normal mice and mice defective in c-mpl (c-mpl-’-). Although cmpl-‘- mice have fewer platelets and higher plasma TPO activity than normal mice, there was no increase in TPO mRNA levels as measured by an S1 nuclease protection assay. After the intravenous injection of ’“I-TPO, specific uptake of radioactivity by the spleen and blood cells was present in the normal mice, but absent in the c-mpl-’- mice. Platelet-rich plasma (PRP) from normal mice was able to bind and internalize ’251-TP0, whereas PRP from c-mpl-/mice lacked this ability. Analysis of ‘251-TP0 binding to norLATELETS ARE small anucleated cells that play an important role in wound healing and blood clotting. Normal hemostasis requires the maintenance of an adequate number of functional platelets in the blood. Production of circulating blood cells by bone marrow cells is mediated by humoral hematopoietic growth factors. The primary regulator of platelet production has recently been identified as the ligand for the c-mpl protooncogene’.2 and has been termed thrombopoietin (TPO)3-s or megakaryocyte growth and development factor (MGDF). In vitro, TPO supports both megakaryocyte colony formation6-@ and the formation of fully mature megakaryocytes and functional platelets in liquid culture^.^"^ Injection of recombinant TPO into mice and nonhuman primates increases platelet counts, spleen and bone marrow megakaryocytes, colony-forming units-megakaryocyte (CFU-MK),3-63” and burst-forming unit erythrocyte.I2*” Mice defective in the c-mpl or the TPO gene show a greater than 80% reduction in their megakaryocyte and platelet numbers, without affecting the other hematopoietic lineages.I4*I5 These results indicate that TPO is the primary physiologic regulator of both proliferation and differentiation of hematopoietic progenitor cells into mature megakaryocytes. Circulating levels of TPO present in the serum of thrombocytopenic animals are elevated,4.’.I6”@ suggesting that TPO is the physiologic regulator of platelet production during a platelet emergency situation. However, the mechanism by P