The objective of this project is to isolate, progressively purify and characterize facators derived from human urine, which can bring about increases in the number, maturity and size of single megakaryocytes and of megakaryocyte colonies in mouse marrow cell plasma clot cultures. Purification techniques involving size selective ultrafiltration, fractional precipitation conventional and high pressure liquid chromatography will be employed. It has been hypothesized that an increase in megakaryocytopoiesis is dependent on the action of at least two types of regulatory factors; one factor which primarily increases the number of cells capable of muturing into recognizable megakaryocytes and one factor which is needed to bring about megakaryocyte maturation. We have found that megakaryocyte colony stimulating factor (MEG-CSF) different from erythropoietin (EPO) is present in human urine. It will be attempted to discern whether a second """"""""potentiating"""""""" factor, similar to that described in conditioned media, is also present in urine. The dose dependence and machanism of action of these factors will be studied in experimental designs which will include preincubation of the mouse marrow target cells in suspension culture with a factor or with a combination of factors and subsequent challenge of these cells with appropriate factor(s) in clot culture. It will be determined whether an increase in numbers of megakaryocyte colony forming units (CFU-MEG) brought about by these factors is based on cell replication or on cell activation (sensitization). The nature of the target cells for the factors (direct versus indirect action, unipotent versus multipotent progenitor cells) will be investigated. The MEG-CSF content of urine from patients with abnormal platelet counts due to different causes and from normal individuals will be measured to detect possible correlations with other hematological parameters. An assay for the detection and quantitation of factors promoting megakaryocyte formation in culture based on the stimulation of the uptake of serotonin in mouse bone marrow cell suspension cultures will be developed to complement the plasma clot assay.
