Alan M. Gewirtz

Inhibition of human megakaryocytopoiesis in vitro by platelet factor 4 (PF4) and a synthetic COOH-terminal PF4 peptide.

We report that highly purified human platelet factor 4 (PF4) inhibits human megakaryocytopoiesis in vitro. At greater than or equal to 25 micrograms/ml, PF4 inhibited megakaryocyte colony formation approximately 80% in unstimulated cultures, and approximately 58% in cultures containing recombinant human IL 3 and granulocyte-macrophage colony-stimulating factor. Because PF4 (25 micrograms/ml) had no effect on either myeloid or erythroid colony formation lineage specificity of this effect was suggested. A synthetic COOH-terminal PF4 peptide of 24, but not 13 residues, also inhibited megakaryocyte colony formation, whereas a synthetic 18-residue beta-thromboglobulin (beta-TG) peptide and native beta-TG had no such effect when assayed at similar concentrations. The mechanism of PF4-mediated inhibition was investigated. First, we enumerated total cell number, and examined cell maturation in control colonies (n = 200) and colonies (n = 100) that arose in PF4-containing cultures. Total cells per colony did not differ dramatically in the two groups (6.1 +/- 3.0 vs. 4.2 +/- 1.6, respectively), but the numbers of mature large cells per colony was significantly decreased in the presence of PF4 when compared with controls (1.6 +/- 1.5 vs. 3.9 +/- 2.3; P less than 0.001). Second, by using the human leukemia cell line HEL as a model for primitive megakaryocytic cells, we studied the effect of PF4 on cell doubling time, on the expression of both growth-regulated (H3, p53, c-myc,and c-myb), and non-growth-regulated (beta 2-microglobulin) genes. At high concentrations of native PF4 (50 micrograms/ml), no effect on cell doubling time, or H3 or p53 expression was discerned. In contrast, c-myc and c-myb were both upregulated. These results suggested the PF4 inhibited colony formation by impeding cell maturation, as opposed to cell proliferation, perhaps by inducing expression of c-myc and c-myb. The ability of PF4 to inhibit a normal cell maturation function was then tested. Megakaryocytes were incubated in synthetic PF4, or beta-TG peptides for 18 h and effect on Factor V steady-state mRNA levels was determined in 600 individual cells by in situ hybridization. beta-TG peptide had no effect on FV mRNA levels, whereas a approximately 60% decrease in expression of Factor V mRNA was found in megakaryocytes exposed to greater than or equal 100 ng/ml synthetic COOH-terminal PF4 peptide. Accordingly, PF4 modulates megakaryocyte maturation in vitro, and may function as a negative autocrine regulator of human megakaryocytopoiesis.

Stage-related proliferative activity determines c-myb functional requirements during normal human hematopoiesis.

To determine if MYB protein is preferentially required during specific stages of normal human hematopoiesis we incubated normal marrow mononuclear cells (MNC) with c-myb antisense oligodeoxynucleotides. Treated cells were cultured in semisolid medium under conditions designed to favor the growth of specific progenitor cell types. Compared with untreated controls, granulocyte-macrophage (GM) CFU-derived colonies decreased 77% when driven by recombinant human (rH) IL-3, and 85% when stimulated by rH GM colony-stimulating factor (CSF); erythroid burst-forming unit (BFU-E)- and CFU-E-derived colonies decreased 48 and 78%, respectively. In contrast, numbers of G-CSF-stimulated granulocyte colonies derived from antisense treated MNC were unchanged from controls, though the numbers of cells composing these colonies decreased approximately 90%. Similar results were obtained when MY10+ cells were exposed to c-myb antisense oligomers. When compared with untreated controls, numbers of CFU-GM and BFU-E colonies derived from MY10+ cells were unchanged, but the numbers of cells composing these colonies were reduced approximately 75 and greater than 90%, respectively, in comparison with controls. c-myc sense and antisense oligomers were without significant effect in these assays. Using the reverse transcription-polymerase chain reaction, c-myb mRNA was detected in developing hematopoietic cells on days 0-8. At day 14 c-myb expression was no longer detectable using this technique. These results suggest that c-myb is required for proliferation of intermediate-late myeloid and erythroid progenitors, but is less important for lineage commitment and early progenitor cell amplification.

Transitory hypomegakaryocytic thrombocytopenia: aetiological association with ethanol abuse and implications regarding regulation of human megakaryocytopoiesis

Summary. We studied a patient with a long history of ethanol abuse who presented to the hospital with profound weakness, anaemia and thrombocytopenia. Evaluation of these problems revealed the patients bone marrow to be hypercellular but severely iron depleted and almost totally devoid of morphologically recognizable megakaryocytes. However, we were able to detect the presence of non‐morphologically recognizable, immature megakaryocytes in the same sample using an immunochemical detection technique. This circumstance allowed us to study the relative importance of both megakaryocyte maturation and peripheral blood platelet count on the production of megakaryocyte colony stimulating activity (Meg‐CSA), a putative regulator of the megakaryocyte colony forming unit (CFU‐M). The results of our investigations disclosed a rapid decline in serum Meg‐CSA levels which preceded recovery of the platelet count and appeared to coincide with the maturation of megakaryocytes into the morphologically recognizable pool. The effect of ETOH on the patients CFU‐M cloning efficiency was also studied. ETOH in amounts up to 454 mg/dl did not inhibit cloning of the patients peripheral blood CFU‐M in plasma clot cultures. Our results suggest that regulation of Meg‐CSA production is a complex function which appears to be dependent on a number of factors including the level of megakaryocyte maturation in the marrow. We also speculate that ethanol associated thrombocytopenia may occasionally be brought about by a disruption in the process of megakaryocyte maturation at the level of a progenitor more mature than the CFU‐M.

Human platelet factor V is crosslinked to actin by FXIIIa during platelet activation by thrombin

Although it has been established that factor V (FV) becomes associated irreversibly with the platelet cytoskeleton after stimulation with thrombin, the chemical nature of this complex is not known. Factor V has recently been demonstrated to be a substrate for factor XIIIa and to form factor V oligomers. We now show that thrombin-activated 125I-FV specifically links to a single protein (43 kDa) of the solubilized platelet membrane in a reaction which requires Ca++ and factor XIIIa. In a purified system, FV, activated by thrombin, forms covalently linked high molecular complexes with 125I-actin catalyzed by factor XIIIa. The site of crosslinking of actin was the factor V fragments, 150 kDa (connecting peptide, C1) and its parent molecule 200 kDa (B). Using radiolabeled actin and unlabeled FV, factor XIIIa catalysed the formation of both homopolymers and heteropolymers. Unlabeled actin was found to compete with radiolabeled FV as a substrate for FXIIIa. To evaluate the biological significance of the crosslinking of factor V to actin, intact platelets were treated with B10 (monoclonal antibody to C1), or monospecific polyclonal antibodies to actin or FXIII. After stimulation with thrombin, the cytoskeleton (material insoluble in Triton X-100) showed markedly decreased 125I-FV in the crosslinked complexes. FV coagulant activity associated with platelet cytoskeleton was also diminished following incubation with an antibody to actin, factor XIII, or B10. These data suggest that FV, through the C1 domain, is crosslinked to actin in the cytoskeleton of thrombin-treated platelets. Activated factor XIII may play a role in plasma FV-platelet interaction as well as the expression of FV derived from the alpha-granules on the cytoskeleton during platelet stimulation.

Current considerations of the etiology of aplastic anemia.

Aplastic anemia is a disorder characterized by marrow aplasia and pancytopenia. The pathogenetic mechanisms that lead to bone marrow aplasia have been intensively studied. Data obtained from these studies suggest that aplastic anemia is a heterogeneous disorder with regards to pathogenesis. Bone marrow aplasia may result from a number of abnormalities including qualitative or quantitative abnormalities of hematopoietic stem cells, abnormal interaction between bone marrow accessory cells (lymphocytes and macrophages) and hematopoietic stem cells, cytotoxic humoral inhibitors of hematopoiesis, and abnormalities of the bone marrow microenvironment. A number of new therapeutic options have improved the survival of patients with aplastic anemia. Allogeneic bone marrow transplantation has actually resulted in the cure of patients. Unfortunately, only a minority of patients have a suitable bone marrow donor and alternate modes of therapy have been sought. Encouraging results have been reported from several centers concerning the use of antilymphocyte serum in patients with aplastic anemia. Certainty of the ultimate long-term benefit of this type of immunosuppressive therapy is not possible until careful, randomized, prospective studies of its use are completed.

Exogenous and Endogenous Regulations of Human Megakaryocytopoiesis

Megakaryocytopoiesis may be viewed as a developmental continuum which begins when an undifferentiated hematopoietic stem cell commits to maturation within this lineage (for recent review see ref.#l) The process of commitment results in the generation of megakaryocyte progenitor cells (CFU-Meg). When appropriately stimulated, CFU-Meg are capable of intense proliferative activity which determines the numbers of megakaryocytes which will ultimately populate the bone marrow. As proliferative activity declines, CFU-Meg progeny mature into precursor cells which terminally differentiate into polyploid megakaryocytes capable of releasing several thousand platelets each into the circulation.