A sensitive sandwich enzyme-linked immunosorbent assay (ELISA) has been established to estimate serum thrombopoietin (TPO) concentrations in healthy volunteers and patients with haemopoietic disorders. The ELISA uses a mouse monoclonal antibody (Ab) as the capture Ab and a biotinylated rabbit polyclonal Ab as the detector. The ELISA was reproducible, highly sensitive and specific for human TPO. The coefficients of intra-and inter assay variation were from 3.0% to 4.9% and from 5.9% to 6.1%, respectively. The quantitative limit of the ELISA was 0.09 fmol/ml in serum. The quantitative limit was lower than the normal level. The dose-response curves of serum samples from healthy volunteers and patients with haemopoietic disorders were parallel to the standard curves. The ELISA did not cross-react with a variety of blood components and cytokines to produce false-positive results. The serum TPO concentrations from 29 normal males and 21 females were 0.79 +/- 0.35 and 0.70 +/- 0.26 fmol/ml, respectively. Serum TPO levels in patients with aplastic anaemia (AA), acute lymphocytic leukemia (ALL) and essential thrombocythaemia (ET) were measured using the ELISA. The serum TPO levels in the patients with ET (n = 6, 2.80 +/- 1.55 fmol/ml) were higher than the normal level. The patients with AA (n = 7, 18.53 +/- 12.37 fmol/ml) and ALL (n = 5, 10.36 +/- 5.57 fmol/ml) had significantly higher serum TPO levels than normal individuals. These results indicate that the ELISA specific to TPO should prove useful in measuring the TPO concentration in serum samples.

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.

The present study shows that recombinant human megakaryocyte growth and development factor (r-HuMGDF) behaves both as a megakaryocyte colony stimulating factor and as a differentiation factor in human progenitor cell cultures. Megakaryocyte colony formation induced with rHuMGDF is synergistically affected by stem cell factor but not by interleukin 3. Megakaryocytes stimulated with rHuMGDF demonstrate progressive cytoplasmic and nuclear maturation. Measurable levels of megakaryocyte growth and development factor in serum from patients undergoing myeloablative therapy and transplantation are shown to be elaborated in response to thrombocytopenic stress. These data support the concept that megakaryocyte growth and development factor is a physiologically regulated cytokine that is capable of supporting several aspects of megakaryopoiesis. (J. Clin. Invest. 1995Invest. . 95:2973Invest. -2978

We demonstrated that local expression of a dominant-negative type II TGF-beta receptor prevents live fibrogenesis and dysfunction in dimethylnitrosamine-treated rats. Using the same model, we have now tested whether a soluble TGF-beta receptor expressed in skeletal muscle can effectively suppress TGF-beta signaling in a remote organ (the liver). We constructed an adenovirus expressing an entire ectodomain of human TGF-beta type II receptor fused to the Fc portion of human IgG (AdTbeta-ExR). This soluble receptor secreted from AdTbeta-ExR-infected cells bound TGF-beta and blocked TGF-beta-signaling in vitro. After intramuscular injection of AdTbeta-ExR in rats, the soluble receptor protein was detectable in the blood for at least 3 weeks. When such rats were treated with dimethylnitrosamine, liver fibrosis was markedly attenuated without apparent systemic or local side effects. The hepatic hydroxyproline content was reduced to a level indistinguishable from that achieved by local expression of the dominant-negative TGF-beta receptor. Since a qualitatively and quantitatively similar suppression was achieved by the two methods, it may be concluded that the new strategy can achieve a complete inhibition of TGF-beta signaling under pathophysiological conditions in vivo. This strategy should facilitate clarification of the role of TGF-beta in vivo in various organs where direct gene transfer seems to be difficult.

Thrombopoietin is known to be essential for megakaryocytopoiesis and thrombopoiesis. Recently, we and others have shown that thrombopoietin induces rapid tyrosine phosphorylation of Jak2 and other proteins in human platelets and BaF3 cells, genetically engineered to express c- Mpl, a receptor for thrombopoietin. The Jak family of tyrosine kinases are known to mediate some of the effects of cytokines or hematopoietic growth factors by recruitment and tyrosine phosphorylation of a variety of Stat (signal transducers and activators of transcription) proteins. Hence, we have investigated whether Stat proteins are present in platelets and, if so, whether they become tyrosine phosphorylated in response to thrombopoietin. We immunologically identified Stat1, Stat2, Stat3, and Stat5 in human platelet lysates. Thrombopoietin induced tyrosine phosphorylation of Stat3 and Stat5 in these cells. Thrombopoietin also induced tyrosine phosphorylation of Stat3 and Stat5 in FDCP-2 cells genetically engineered to constitutively express human c-Mpl. Thus, our data indicate that Stat3 and Stat5 may be involved in signal transduction after ligand binding to c-Mpl and that this event may have a role in megakaryopoiesis/thrombopoiesis or possibly a mature platelet function such as aggregation.