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.

Increased numbers of bone marrow megakaryocytes and thrombocytosis are frequently observed in patients with myeloproliferative disorders (MPD). Increased marrow megakaryocytes and thrombocytosis are also noted in a variety of inflammatory and neoplastic disease leading to the phenomenon of reactive thrombocytosis (RT). The pathogenesis of this finding remains incompletely understood. Using methodology developed in our laboratory, we investigated the causative role of megakaryocyte colony-stimulating activity (Meg-CSA) in generating this phenomenon. We also examined the cloning efficiency of colony-forming units-megakaryocyte (CFU-M) and their responsiveness to an exogenous source of Meg-CSA in patients with these diseases. The results of our investigations suggest that: (1) increased production of Meg-CSA is not responsible for the megakaryocyte hyperplasia and thrombocytosis noted in these patients; (2) the intrinsic stem cell defect described in MPD appears to affect the CFU-M of these patients as well, resulting in an effective expansion of the CFU-M pool with consequent megakaryocyte hyperplasia and thrombocytosis; (3) the CFU-M of patients with MPD remain responsive to an exogenous source of Meg-CSA, suggesting that this megakaryocyte hyperplasia may not be entirely autonomous of its effects; and (4) the CFU-M pool in RT is normal both in size and responsiveness to Meg-CSA, suggesting that in these disorders, the stimulus leading to megakaryocyte hyperplasia and thrombocytosis is active at the post-CFU-M level of megakaryocyte differentiation.