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.

A convenient serum-free fibrin clot culture system for murine megakaryocyte progenitor cells was developed. The culture and counting of colonies is much easier in this system, when compared with previously reported serum-free culture methods. Recombinant murine interleukin-3 (rmIL-3) stimulated megakaryocyte colony formation in a dose-dependent manner in this system. While recombinant human granulocyte colony-stimulating factor (rhG-CSF) had no effect on megakaryocytopoiesis, recombinant human erythropoietin (rhEpo) and recombinant human interleukin-6 (rhIL-6) augmented megakaryocyte colony formation stimulated by rmIL-3. The depletion of adherent cells and T cells from the cultured bone marrow did not eliminate the synergistic effect of rhEpo and rhIL-6.