Partial purification and characterization of human megakaryocyte colony-stimulating factor (Meg-CSF)

Abstract

Megakaryocyte colony-stimulating factor (Meg-CSF) in urinary extracts from patients with aplastic anemia was partially characterized and purified. Using Meg-CSF-enriched fractions, we established that the moiety has the following characteristics: 1) portions of the molecules having Meg-CSF activity have sialic acid, probably with a biantennary structure, and beta-galactose residues as the terminal and penultimate sugars; 2) disulfide residues are an essential chemical group of the molecule and are located on its surface; and 3) Meg-CSF activity is stable in n-propanol, but not in acetonitrile with trifluoroacetic acid. Partial purification of Meg-CSF by a four-step procedure of ethanol precipitation, CM Affi-Gel Blue chromatography, wheat germ agglutinin-sepharose chromatography, and high-resolution hydroxyapatite chromatography, yielded a concentrate with a 430- to 630-fold increase in specific activity. The partially purified Meg-CSF fractions stimulated both human and murine megakaryocytopoiesis in vitro (CFU-meg). When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreduced conditions, Meg-CSF activity was recovered in the 29-34 kDa molecular weight fractions. We have also shown that Meg-CSF, purified from the urine of aplastic anemia patients, stimulated murine megakaryocytopoiesis and platelet production in vivo. Final purification of human urinary Meg-CSF is currently in progress.

Megakaryocyte growth and development factor. Analyses of in vitro effects on human megakaryopoiesis and endogenous serum levels during chemotherapy-induced thrombocytopenia.

“…Measurable levels of MGDF in serum collected from patients undergoing myeloablative therapy and transplantation demonstrate the physiological relevance of this molecule. These data support the concept that all of the megakaryopoietic and thrombopoietic activities present in plasma, sera, or urine from models of bone marrow aplasia or thrombocytopenia (2,11,(17)(18)(19)(20) can be substantially, if not wholly, accounted for by the cytokine MGDF.…”

High Concentrations of Thrombopoietin Activate Platelets In Vitro

“…Other factors that regulated the growth and maturation of hematopoietic cells were identified and cloned. Some of these factors increased the growth of megakaryocytes in vitro, including c-kit ligand (or stem cell factor), interleukin (IL)-3, IL-6, IL-11, leukemia inhibitory factor (LIF), and granulocyte-macrophage colony stimulating factor (GM-CSF) (10)(11)(12)(13)(14)(15)(16)(17)(18). However, none of these factors appeared to be TPO itself.…”

The physiologic role and therapeutic potential of the Mpl‐ligand in thrombopoiesis

“…The early growth and the later differentiation phases of megakaryopoiesis have traditionally been considered as separate events under distinct regulatory controls [2-51. The primary factor guiding early megakaryopoiesis, megakaryocyte colony-stimdating factor, had been thought to induce growth of megakaryocyte precursor cells (colony formation) but not megakaryocyte maturation [6-91. The later-acting factor, thrombopoietin (TPO), had been thought to regulate terminal megakaryocyte differentiation and platelet shedding, but not progenitor cell colony formation [ 10-141. Both of these activities had been identified in the urine, serum or plasma from aplastic and/or thrombocytopenic humans [9,[15][16][17], rats [12], rabbits [13, 14, 181 and dogs [19]. However, the low concentrations of these activities as well as the complexity of these sources had for many years prevented the unambiguous identification or characterization of either putative cytokine.…”