Partial purification and biological properties of thrombopoietin extracted from the urine of aplastic anemia patients.
Thrombopoietic (Tpo) and megakaryocyte-colony stimulating (Meg-CSF) activities were found in urinary extracts from patients with aplastic anemia. Preparative biochemical extractions were accomplished using Sephadex G-50 and DEAE-cellulose column chromatography. The biological activities of these extracts were assessed using not only an in vivo assay but were also examined in vitro employing the clonal development of megakaryocyte colonies. Both in vivo, as well as in vitro, biological activities were detected in the batch fraction which was stepwise eluted from DEAE-cellulose between 0.022 M NaCl in 0.016 M NaH2PO4 and 0.15 M NaCl in 0.05 M Na2HPO4 as a single fraction. When 0.4 mg of this fraction was injected daily into rats, a marked thrombopoiesis ensued producing an increase of 40% over initial platelet counts by 3 days after administration. This was followed by a decrease in platelets to a subnormal range by 21 days after the injection. Hemoglobin concentration gradually increased from 5% above initial value by day 7 to 20% above initial value by day 21. The effect of neuraminidase (NAse) on the properties of this extract was also examined. NAse-treated extracts, similar to the native extracts described above retained Tpo activity. Changes in megakaryocyte numbers in the spleen and bone marrow of rats were assayed with both the NAse-treated extract as well as with the native extract. A remarkable increase in megakaryocyte numbers, threefold above the normal count, was found in the spleens of rats given the native extract preparation; by contrast, however, no change was observed in splenic megakaryocyte numbers in rats given the NAse-treated extract. On the other hand, NAse-treated extract retained its ability to stimulate bone marrow megakaryocyte proliferation in the same rat. The urinary extract also revealed in vitro Meg-CSF activity with a specific activity of 31, 750 CFU-Meg colonies/mg of protein.
D000741:Anemia, Aplastic / Q000652:urine*; D000818:Animals; D002848:Chromatography, DEAE-Cellulose; D003114:Colony-Forming Units Assay; D004920:Erythropoiesis / Q000187:drug effects; D006023:Glycoproteins / Q000032:analysis*; D006410:Hematopoiesis / Q000187:drug effects; D006454:Hemoglobins / Q000032:analysis; D006801:Humans; D008297:Male; D008533:Megakaryocytes / Q000166:cytology; D051379:Mice; D008810:Mice, Inbred C57BL; D009439:Neuraminidase / Q000494:pharmacology; D010976:Platelet Count; D051381:Rats; D011919:Rats, Inbred Strains; D013926:Thrombopoietin / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D014556:Urine / Q000032:analysis*
D006023:Glycoproteins; D006454:Hemoglobins; D013926:Thrombopoietin; D009439:Neuraminidase