Human megakaryocyte colony-stimulating factor in sera from aplastic dogs: partial purification, characterization, and...

We have previously shown that human megakaryocyte colony-stimulating activity (Meg-CSA) is present in sera from patients with bone marrow megakaryocyte aplasia. In this report, we demonstrate that Meg-CSA is also present in sera from dogs rendered aplastic by 1000 rad total body irradiation. Canine serum Meg-CSA has activity comparable to human when assayed in plasma clot cultures containing human bone marrow mononuclear cells. Because of the uniform high potency and ready availability of aplastic canine sera, it was utilized initially for Meg-CSA purification. Sequential ammonium sulfate precipitation to approximately 80% saturation resulted in recovery of 59%-69% of the serum protein and of 75%-103% of the original serum Meg-CSA. The fraction precipitable between ammonium sulfate saturations of 0% and 44%-50% (fraction I) contained 53%-76% of the initial serum Meg-CSA and 25%-32% of the initial serum protein. This represents an enrichment of Meg-CSA specific activity by over 100%. The Meg-CSA eluted from Sephacryl S-300 in a single peak corresponding to a molecular weight of 175,000. This Meg-CSA peak also contained IgG, but the Meg-CSA did not bind to protein A-agarose. Meg-CSA was 90% inactivated by trypsin digestion for 4 h at 37 degrees C and by exposure to 5mM dithiothreitol for 2 h at room temperature. Exposure to either 6 M guanidine for 1 h at room temperature or 8 M urea for 1 h at 4 degrees C resulted in a 70% loss of Meg-CSA. At culture concentrations capable of stimulating maximal megakaryocyte colony formation, fraction I supported no colony growth by myeloid (CFU-GM) or late erythroid (CFU-E) human hematopoietic progenitor cells. Erythroid burst-promoting activity (BPA) was not detected in fraction I from two of three different aplastic canine sera tested. Therefore, Meg-CSA is functionally distinct from granulocyte-monocyte colony-stimulating factor (GM-CSF), erythropoietin, and BPA. The data indicate that serum Meg-CSA is a 175,000-dalton protein (megakaryocyte colony-stimulating factor, Meg-CSF) in which higher order structure and disulfide bonding are necessary for biologic activity. Partially purified Meg-CSF manifests functional specificity for the CFU-Meg hematopoietic progenitor cell.