Erythroid accelerating factor detected in serum from rats with drug induced hemolysis

We have previously observed that an erythroid enhancing activity presents in rat serum in the early stage of drug induced hemolytic anemia. The further studies on biological and physicochemical aspects of this erythroid accelerating factor (EAF) is described in this paper. Hemolytic anemia was induced in rats by single intraperitoneal injection of acetylphenylhydrazine (APH) and serum was obtained from the rats on day 1 after APH injection. It was first fractionated by ultrafiltration on Amicon Diaflo membranes to give a series of fractions lying in the following ranges of molecular weight: 10-30 kDa, 30-50 kDa, 50-100 kDa, and >100 kDa. Among those fractions, largest increase in the number of colony forming unit erythroid CFU-E) colonies was shown in the fraction of >100 kDa that was subsequently fractionated by fast protein liquid chromatography (FPLC) system. EAF activity for CFU-E proliferation was detected in a FPLC fraction corresponding to a molecular weight of about 160 kDa. An addition of EAF significantly increased with dose dependent manner in the number of CFU-E colonies from rat bone marrow mononuclear cells. EAF alone had no burst promoting activity and exhibited no distinct activity to proliferate burst forming unit-erythroid even when interleukin-3 (IL-3) and high concentration (2 U/ml) of erythropoietin (Epo) were added together to the culture. The stimulating effect of EAF on CFU-E was markedly dependent on the presence of adherent cells in the culture. Partially purified protein was relatively heat-unstable (60% at 75 degrees C, 30 minutes) and sensitive to treatment with trypsin and alpha-galactosidase. These results suggest that EAF is a novel factor, possible glycoprotein to reinforce Epo function and is different from various cytokines previously documented because of differences of approximate molecular weight.