ALBERT

Description: Megakaryopoiesis is a highly specialized cellular process which sustains platelet production. At the end of megakaryopoiesis, megakaryocyte (MKs) fragments into platelets via long and thin cytoplasmic extensions called proplatelets. Proplatelet formation (PPF) is associated essentially with cytoskeleton changes, including actin dynamics. The Rho/Rock pathway is a well characterized regulator of the actin reorganization. In the present study, we have tried to understand the precise role of the Rho/Rock pathway in PPF from human CD34+ derived MKs. Our results show that Rho is expressed in MKs and that its expression and activity remain stable during megakaryopoiesis. Overexpression of a RhoA dominant negatif (RhoA N19) in MKs leads to an increase in PPF. Conversely overexpression of a RhoA spontaneous active (RhoA V14) in MKs leads to a decrease in PPF. These results indicate that Rho activation could inhibit PPF in vitro. It is known that Rho/ROCK promotes actin cytoskeleton dynamics by regulating myosin light chain 2 (MLC2) phosphorylation. To demonstrate that Rho/Rock inhibits PPF through MLC2 phosphorylation, we added MLC kinase inhibitor (P18), Rho inhibitor (TatC3) and ROCK inhibitor (Y27362) in MKs culture just before PPF. Western blot analysis shows that MLC2 phosphorylation was inhibited by these 3 compounds, in contrast, PPF was significantly increased. Moreover, the platelet produced have an identical size and ultrastructure as control platelets and could be normally activated. These results suggest that Rho/ROCK could inhibit PPF through MLC phosphorylation during megakaryopoiesis.

Description: Recent studies suggest that multivesicular bodies are an intermediate stage in the formation of -granules. In contrast, the kinetics and mode of appearance of dense granules during megakaryocytic maturation has remained poorly understood. Immunoelectron microscopy was used to monitor the appearance of dense granular markers (granulophysin and serotonin) on cryosections of human megakaryocytes (MKs) cultured from CD34+ precursors. The monitoring was done on days 8 and 13 of culture. The data suggest that dense granules appear in immature MKs early during their maturation, concomitantly with -granule formation. In MKs of intermediary maturation stage, granulophysin was mainly localized within dense granules and multivesicular bodies (MVBs), which were also labeled for serotonin. This study provides evidence that granulophysin is a dense granule marker in human MKs and that MVBs are an intermediary stage of dense granule maturation and probably constitute a sorting compartment between -granules and dense granules.

Description: Erythroblastic synartesis is a rare form of acquired dyserythropoiesis, first described by Breton-Gorius et al in 1973. This syndrome is characterized by the presence of septate-like membrane junctions and “glove finger” invaginations between erythroblasts, which are very tightly linked together. This phenomenon, responsible for ineffective erythropoiesis, leads to an isolated severe anemia with reticulocytopenia. In the following report, we describe 3 new cases of erythroblastic synartesis associated with dysimmunity and monoclonal gammapathy. In all cases, the diagnosis was suggested by characteristic morphological appearance of bone marrow smears, and further confirmed by electron microscopy. Ultrastructural examination of abnormal erythroblast clusters showed that these cells were closely approximated with characteristic intercellular membrane junctions. The pathogenesis of the dyserythropoiesis was modeled in vitro using crossed erythroblast cultures and immunoelectron microscopy: when cultured in the presence of autologous serum, the erythroblasts from the patients displayed synartesis, whereas these disappeared when cultured in normal serum. Moreover, synartesis of normal erythroblasts were induced by the patient IgG fraction. Immunogold labeling showed that the monoclonal IgG were detected in, and restricted to, the synartesis. A discrete monoclonal plasmacytosis was also found in the patient bone marrow. The adhesion receptor CD36 appeared to be concentrated in the junctions, suggesting that it might be involved in the synartesis. These experiments indicated that a monoclonal serum immunoglobulin (IgG in the present cases) directed at erythroblast membrane antigen was responsible for the erythroblast abnormalities. Specific therapy of the underlying lymphoproliferation was followed by complete remission of the anemia in these cases.

Description: Multimerin is a massive, soluble, homomultimeric, factor V-binding protein found in platelet -granules and in vascular endothelium. Unlike platelets, endothelial cells contain multimerin within granules that lack the secretory granule membrane protein P-selectin, and in culture, they constitutively secrete most of their synthesized multimerin. To further evaluate multimerin’s posttranslational processing and storage, we expressed human endothelial cell prepromultimerin in a variety of cell lines, with and without pathways for regulated secretion. The recombinant multimerin produced by these different cells showed variations in its glycosylation, proteolytic processing, and multimer profile, and human embryonic kidney 293 cells recapitulated multimerin’s normal processing for constitutive secretion by human endothelial cells. When multimerin was expressed in a neuroendocrine cell line capable of regulated protein secretion, it was efficiently targeted for regulated secretion. However, the multimerin stored in these cells was proteolyzed more extensively than normally occurs in platelets, suggesting that endoproteases similar to those expressed by megakaryocytes are required to produce platelet-type multimerin. The impact of the tissue-specific differences in multimerin’s posttranslational processing on its functions is not yet known. Multimerin’s sorting and targeting for regulated secretion may be important for its functions and its association with factor V in secretion granules.

Description: The late stages of megakaryocytopoiesis, consisting of the terminal processes of cytoplasmic maturation and platelet shedding, remain poorly understood. A simple liquid culture system using CD34+ cells in serum-free medium has been developed to study the regulation of platelet production in vitro. Platelets produced in vitro were enumerated by flow cytometry. A truncated form of human Mpl-Ligand conjugated to polyethylene glycol (PEG-rHuMGDF) played a crucial role in both proplatelet formation and platelet production. A combination of stem cell factor (SCF), interleukin-3 (IL-3), and IL-6 was as potent as PEG-rHuMGDF for the growth of megakaryocytes (MKs). However, the number of proplatelet-displaying MKs and platelets was increased 10-fold when PEG-rHuMGDF was used. Peripheral blood mobilized CD34+ cells gave rise to a threefold augmentation of platelets compared with marrow CD34+ cells. This finding was related to the higher proliferative capacity of the former population because the proportion of proplatelet-displaying MKs was similar for both types of CD34+ cells. The production of platelets per MK from CD34+ cells was low, perhaps because of the low ploidy of the cultured MKs. This defect in polyploidization correlated with the degree of proliferation of MK progenitors induced by cytokines. In contrast, ploidy development closer to that observed in marrow MKs was observed in MKs derived from the low proliferative CD34+CD41+ progenitors and was associated with a twofold to threefold increment in platelet production per MK. As shown using this CD34+ CD41+ cell population, PEG-rHuMGDF was required throughout the culture period to potently promote platelet production, but was not involved directly in the process of platelet shedding. IL-3, SCF, and IL-6 alone had a very weak effect on proplatelet formation and platelet shedding. Surprisingly, when used in combination, these cytokines elicited a degree of platelet production which was decreased only 2.4-fold in comparison with PEG-rHuMGDF. This suggests that proplatelet formation may be inhibited by non-MK cells which contaminate the cultures when the entire CD34+ cell population is used. Cultured platelets derived from PEG-rHuMGDF– or cytokine combination-stimulated cultures had similar ultrastructural features and a nearly similar response to activation by thrombin. The data show that this culture system may be useful to study the effects of cytokines and the role of polyploidization on platelet production and function.

Description: Vascular endothelial cells are thought to be the main source of plasma tissue-type plasminogen activator (t-PA) and von Willebrand factor (VWF). Previous studies have suggested that both t-PA and VWF are acutely released in response to the same stimuli, both in cultured endothelial cells and in vivo. However, the subcellular storage compartment in endothelial cells has not been definitively established. We tested the hypothesis that t-PA is localized in Weibel-Palade (WP) bodies, the specialized endothelial storage granules for VWF. In cultured human umbilical vein endothelial cells (HUVECs), t-PA was expressed in a minority of cells and found in WP bodies by immunofluorescence. After up-regulation of t-PA synthesis either by vascular endothelial growth factor (VEGF) and retinoic acid or by sodium butyrate, there was a large increase in t-PA–positive cells. t-PA was exclusively located to WP bodies, an observation confirmed by immunoelectron microscopy. Incubation with histamine, forskolin, and epinephrine induced the rapid, coordinate release of both t-PA and VWF, consistent with a single storage compartment. In native human skeletal muscle, t-PA was expressed in endothelial cells from arterioles and venules, along with VWF. The 2 proteins were found to be colocalized in WP bodies by immunoelectron microscopy. These data indicate that t-PA and VWF are colocalized in WP bodies, both in HUVECs and in vivo. Release of both t-PA and VWF from the same storage pool likely accounts for the coordinate increase in the plasma level of the 2 proteins in response to numerous stimuli, such as physical activity, β-adrenergic agents, and 1-deamino-8d-arginine vasopressin (DDAVP) among others.

Description: We recently described a Quebec family with an autosomal dominant bleeding disorder characterized by mildly reduced-low normal platelet counts, an epinephrine aggregation defect, multimerin deficiency, and proteolytic degradation of several, soluble α-granular proteins. Similar clinical features led us to investigate a second family with an unexplained, autosomal dominant bleeding disorder. The affected individuals had reduced to normal platelet counts, absent platelet aggregation with epinephrine, and multimerin deficiency. Their platelet α-granular proteins factor V, thrombospondin, von Willebrand factor, fibrinogen, fibronectin, osteonectin, and P-selectin were proteolyzed and comigrated with the degradation products found in patients from the other family. However, their platelet albumin, IgG, external membrane glycoproteins, CD63 (a lysosomal and dense granular protein), calpain, and plasma von Willebrand factor were normal, indicating restriction in the proteins proteolyzed. Electron microscopy studies indicated preserved α-granular ultrastructure, despite degradation of soluble and membrane α-granular proteins. Immunoelectron microscopy studies of the patients' platelets indicated that fibrinogen, von Willebrand factor, P-selectin, multimerin, and factor V were within α-granules, with normal to reduced labeling for these proteins. Pathologic proteolysis of α-granular contents, rather than a defect in targeting proteins to α-granules, may be the cause of the protein degradation in the Quebec platelet disorder.

Description: Multimerin is a novel, massive, soluble protein that resembles von Willebrand factor in its repeating, homomultimeric structure. Both proteins are expressed by megakaryocytes and endothelial cells and are stored in the region of platelet α-granules resembling Weibel-Palade bodies. These findings led us to study the distribution of multimerin within human endothelial cells. Multimerin was identified in vascular endothelium in situ. In cultured endothelial cells, multimerin was identified within round to rod-shaped, dense-core granules, some of which contained intragranular, longitudinally arranged tubules and resembled Weibel-Palade bodies. However, multimerin was found primarily in different structures than the Weibel-Palade body proteins von Willebrand factor and P-selectin. After stimulation with secretagogues, multimerin was observed to redistribute from intracellular structures to the external cellular membrane, without detectable accompanied secretion of multimerin into the culture media. In early passage endothelial cell cultures, multimerin was associated with extensive, fibrillary, extracellular matrix structures, in a different distribution than fibronectin. Although multimerin and von Willebrand factor are stored together in platelets, they are mainly found within different structures in endothelial cells, indicating that there are tissue-specific differences in the sorting of these soluble, multimeric proteins.

Description: To evaluate the effects of long-term, high-dose exposure to thrombopoietin (TPO), lethally irradiated mice were grafted with bone marrow cells infected with a retrovirus carrying the murine TPO cDNA. Mice were studied for 10 months after transplantation. In plasma, TPO levels were highly elevated (104 U/mL) throughout the course of the study. All mice developed a lethal myeloproliferative disorder evolving in two successive phases. During the first phase (7-9 weeks posttransplant), platelet and white blood cell (WBC) counts rose four- and ten-fold, respectively, whereas hematocrits decreased slightly to 29% ± 3%. The WBC were mainly mature granulocytes, but myeloid precursor cells were invariably observed as well as giant platelets with an irregular granule distribution. The striking features were a massive hyperplasia of megakaryocytes and granulocytes in the spleen and bone marrow and a hypoplasia of erythroblasts in bone marrow. Total numbers of megakaryocyte colony-forming cell, burst-forming unit-erythroid, and granulocytemacrophage colony-forming cells were increased but colony-forming unit-erythroid numbers decreased. From 10 weeks posttransplant and thereafter, WBC, platelets, and red blood cell numbers declined dramatically. The absolute numbers of progenitor cells were very low in the spleen and bone marrow, but sharply increased in the blood and peritoneal cavity. Extramedullary hematopoiesis was observed in several organs. Histologic sections of the spleen and bones revealed severe fibrosis and osteosclerosis. The mean survival time was 7 months posttransplant and mice died with severe pancytopenia. Notably, two mice died between 3 and 4 months posttransplant with a leukemic transformation. This disorder was transplantable into secondary recipients who developed an attenuated form of the disease similar to the one previously described (Yan et al, Blood 86:4025, 1995). Taken together, our data show that high and persistent TPO production by transduced hematopoietic cells in mice results in a fatal myeloproliferative disorder that has a number of features in common with human idiopathic myelofibrosis.