Purification and Characterization of Thrombopoietin1

Abstract

A thrombopoietic factor, termed thrombopoietin (TPO), was highly purified directly from the plasma of sublethally irradiated 1,100 rats by measuring the production of megakaryocytes from a highly enriched population of rat megakaryocyte progenitor cells (CFU-MK). The rat plasma TPO is a glycoprotein and strongly hydrophobic. The total activity and purification yields obtained were about 29% and 1.49 x 10(8), respectively. The amino acid sequences of the two peptide fragments prepared from the purified 19 kDa TPO were analyzed, and used for the cloning of rat and human TPO cDNAs. It was found that the 19 kDa TPO was truncated but comprised at least 163 amino acids. The sequence of human TPO cDNA revealed that the TPO was identical to the c-Mpl ligand. Both rat and human TPOs expressed in COS-1 cells exhibited significant activity toward the CFU-MK in vitro, and were active in stimulating platelet production in mice. These results indicate that a thrombopoietic factor originally found in the irradiated rat plasma is a ligand for the rat c-Mpl.

CD34+/CD41a+ cells best predict platelet recovery after autologous peripheral blood stem cell transplantation

“…14,16 However, the CFU-MK assay has yielded variable results, in part, because best growth is obtained with a combination of growth stimulators, colony size can be quite small, and colony identification can present problems to the inexperienced observer. 25 In the present study, we used TPO and SCF as growth factors for CFU-MK; these produce more and larger colonies than previous methods, [18][19][20][21] and these CFU-MK characteristics were inversely correlated with the time to platelet recovery (r = Ϫ0.55). Although colony assays are reliable with TPO, the method is laborious and requires 2 weeks to obtain results, making it generally unsuitable for use as a routine method in a clinical laboratory.…”

“…13,14,16,17 Thrombopoietin (TPO), the ligand for the c-mpl receptor, has been shown to stimulate the growth of megakaryocyte colonies in vitro. [18][19][20] The number and size of CFU-MK induced by TPO was greater than that induced by any other combination of growth factors, suggesting that TPO may be useful for assaying CFU-MK. 21 Based on these observations, we analyzed the expression of different platelet glycoproteins on CD34 + cells by flow cytometry and the number of TPO-induced CFU-MK in PBSC grafts.…”

Thrombopoietin, c‐Mpl ligand, induces tyrosine phosphorylation of Tyk2, JAK2, and STAT3, and enhances agonists‐induced aggregation in platelets in vitro

“…Recently, several groups [2][3][4][5] cloned the ligand for the c-Mpl proto-oncogene [6] which is a member of the cytokine receptor superfamily. These studies and others have demonstrated that c-Mpl ligand plays a critical role in megakaryocytopoiesis and thrombopoiesis both in vitro and in vivo [2][3][4][5][7][8][9][10]. Therefore, c-Mpl ligand has been termed thrombopoietin (TPO) [3,7,8] or megakaryocyte growth and development factor (MGDF) [4].…”

Effects of mpl ligands on platelet production and function in nonhuman primates

“…Two Mpl ligands, full-length glycosylated recombinant human (rHu)TPO and poIyethyleneglycol (PEG)-derivitized 163-residue aminoterminus, known as PEG-rHu megakaryocyte growth and development factor (MGDF), are currently under clinical development. rHuTPO and PEG-rHuMGDF induce TPO receptor signaling of CD34' murine and human marrow cells to undergo hematopoietic stem cell differentiation into megakaryocyte progenitors [7, endoproliferative enlargement [9, lo], thereby amplifying the cytoplasmic substrate generating circulating platelets [4,5,[11][12][13][14][15][16]. Endogenous plasma TPO maintains peripheral platelet concentrations constant by modulating megakaryocytopoiesis to compensate for changing peripheral requirements [9,[17][18][19][20][21].…”