The possible pathogenetic mechanisms responsible for the production of acquired amegakaryocytic thrombocytopenic purpura (AATP) were investigated in a group of patients with this disorder. Absence of megakaryocytes and small platelet glycoprotein-bearing mononuclear cells, as determined by immunochemical staining of patient marrows with an antisera to platelet glycoproteins, suggested that the defect in AATP occurs in an early progenitor cell of the megakaryocytic lineage. Using an in vitro clonal assay system for negakaryocytic progenitor cells or megakaryocyte colony-forming units (CFU-M), the proliferative capacity of AATP marrow cells was then assessed. Bone marrow cells from three of four patients formed virtually no megakaryocyte colonies, suggesting that in these individuals the AATP was due to an intrinsic defect in the CFU-M. Bone marrow cells from an additional patient, however, formed 12% of the normal numbers of colonies, providing evidence for at least partial integrity of the CFU-M compartment in this patient. Serum specimens from all six patients were screened for their capacity to alter in vitro megakaryocyte colony formation. Five of six sera enhanced colony formation in a stepwise fashion, demonstrating appropriately elevated levels of megakaryocyte colony- stimulating activity. The serum of the patient with partial integrity of the CFU-M compartment, however, stimulated colony formation only at low concentrations. At higher concentrations, this patient's serum actually inhibited the number of colonies cloned, suggesting the presence of a humoral inhibitor to CFU-M. Serum samples from all patients were further screened for such humoral inhibitors of megakaryocyte colony formation using a cytotoxicity assay. The patient whose serum was inhibitory to CFU-M at high concentrations was indeed found to have a complement-dependent serum IgG inhibitor that was cytotoxic to allogeneic and autologous marrow CFU-M but did not alter erythroid colony formation. These-studies suggest that AATP can be due to at least two mechanisms: either an intrinsic effect at the level of the CFU-M or a circulating cytotoxic autoantibody directed against the CFU-M.

The development of a satisfactory in vitro assay system for human megakaryocyte colony forming progenitor cells has been delayed by the lack of a suitable marker for cells of human megakaryocyte lineage. For this purpose we raised an antiserum directed against a purified human platelet glycoprotein preparation. In conjunction with indirect immunofluorescent staining of human bone marrow, this antiserum labeled only platelets, megakaryocytes, and an infrequent population of small mononuclear cells. These small mononuclear cells, not otherwise identifiable as members of the megakaryocyte series, constituted 22.9% of the total fluorescein positive nucleated bone marrow cells. This antiserum was also used to label colonies cultured from human peripheral blood mononuclear cells using a modified plasma clot technique. A mean of 123 fluorescein-labeled colonies were cloned per 10(6) mononuclear cells cultured. Granulocyte-macrophage and erythroid burst colonies did not label using this method. No augmentation of colony numbers was found with varying concentrations of erythropoietin, human embryonic kidney cell conditioned media (a source of thrombopoietin), or media conditioned by a human T lymphoblast cell line (a source of both colony stimulating and burst promoting activities). Immunofluorescent labeling for platelet glycoproteins is a convenient phenotypic marker for cells of human megakaryocyte lineage useful in the study of in vitro human megakaryocytopoiesis.