Plasminogen activator expression in normal stem cells and during normal myeloid differentiation
It is unclear whether expression of a u-PA in myeloid blasts reflects their stage of maturation and is therefore a feature of normal myeloid precursors, or whether it is a feature unique to malignancy. Clarification will provide insight into the properties of normal developing myeloid cells and increase our understanding of the bleeding mechanisms in leukaemia. In addition, u-PA may have an extravascular proteolytic role in bone marrow, participating in cell differentiation/maturation and mobilisation that may be relevant to both malignant and non-malignant myelopoiesis. This thesis describes the expression of plasminogen activator (PA) activity in normal peripheral blood stem cells (PBSCS) and during their subsequent normal myeloid differentiation and maturation. Fresh mononuclear cells obtained from G-CSF-stimulated autologous or normal allogeneic donors undergoing peripheral blood harvests were cultured in semi-solid medium with recombinant cytokines (IL3, SCF and GM-CSF) to stimulate differentiation and maturation of PBSCs along the myeloid pathway. At intervals, cells were harvested for examination uPA antigen and activity were detected using immunoblotting, SDS-PAGE / zymography and clot lysis assay and uPA mRNA using RT-PCR. The results showed that normal PBSCs did not show detectable u-PA activity, antigen or mRNA but acquired the ability to express PA in the form of u-PA during the process of myeloid differentiation in culture in vitro. The u-PA was found to be active and capable of lysing plasminogen-containing fibrin clot in vitro. These results suggest that the fibrinolytic bleeding syndromes seen in some forms of acute myeloid leukaemia seem likely to reflect accumulation of immature cells at stages during which u-PA generation is a prominent feature and not dependent on the malignant process itself. Inhibition of u-PA activity did not directly influence myeloid differentiation in vitro but it remains possible that u-PA may interact with marrow stromal elements that influence the process or participate in the release of myeloid cells into the circulation.