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

We have previously observed that an erythroid enhancing activity presents i n rat serum in the early stage of drug induced hemolytic anemia. The furthe r studies on biological and physicochemical aspects of this erythroid accel erating factor (EAF) is described in this paper. Hemolytic anemia was induc ed in rats by single intraperitoneal injection of acetylphenylhydrazine (AP H) and serum was obtained from the rats on day 1 after APH injection. It wa s 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, large st increase in the number of colony forming unit erythroid CFU-E) colonies was shown in the fraction of > 100 kDa that was subsequently fractionated b y 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 EAP significantly increased with do se dependent manner in the number of CFU-E colonies from rat bone marrow mo nonuclear cells. EAF alone had no burst promoting activity and exhibited no distinct activity to proliferate burst forming unit-erythroid even when in terleukin-3 (IL-3) and high concentration (2 U/ml) of erythropoietin (Epo) mere added together to the culture. The stmiulating effect of EAF on CPU-E was markedly dependent on the presence of adherent cells in the culture. Pa rtialy purified protein was relatively heat-unstable (60% at 75 degrees C, 30 minutes) and sensitive to treatment with trypsin and cy-galactosidase. T hese results suggest that EAF is a novel factor, possible glycoprotein to r einforce Epo function and is different from various cytokines previously do cumented because of differences of approximate molecular weight. (C) 1998 T ohoku University Medical Press.