[Thrombopenia. Diagnostic orientation].
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Humans↗
Biomedical subjects
E Dupuy
Publications and source records attributed to E Dupuy.
[Surveillance of treatment with K antivitamins].
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Anticoagulants↗
[Gray platelet syndrome, an example of myelofibrosis of megakaryocytic origin].
In this study, the clinical history of two patients with the gray platelet syndrome, a rare congenital disorder associating thrombopathia and myelofibrosis is recalled. Complementary studies on platelets and megakaryocytes were performed, mainly with an immunocytochemical approach. In gray platelets, a general decrease of alpha-granule proteins, including PF4, beta tg and PDGF was observed. The decrease in platelet mitogenic activity (PDGF) was confirmed by biological and radio-immunological measurements. An abnormally high level of these compounds was also found in the plasma. In megakaryocytes cultured from the bone marrow of these patients, alpha-granule proteins were normally expressed in early maturation stages, whereas they were found to be absent in the mature megakaryocytes. An alpha-granule membrane glycoprotein, GMP 140 has been studied in resting and thrombin stimulated gray platelets and was found to be normally expressed at the surface of stimulated platelets. GMP140 was studied in resting platelets by immunoelectron microscopy and found to be present in vacuole probably corresponding to empty granules. This observation allows to conclude that alpha-granule membrane is formed in the gray platelet syndrome, but that there is a storage defect of alpha-granule soluble proteins, possibly due to an abnormal targetting of these proteins to the alpha-granule. Synthesis and subsequent release of these proteins, namely of the mitogenic factors, which can induce myelofibrosis and lung fibrosis by abnormal fibroblast stimulation, is discussed.
Adult↗
Basic fibroblast growth factor expression in human omental microvascular endothelial cells and the effect of phorbol ester.
The human omentum contains a potent, not yet identified angiogenic activity. The omentum is very vascularized. Therefore, we investigated whether human omental microvascular endothelial cells (HOME cells) express the angiogenic peptide basic fibroblast growth factor (bFGF). Cytosol prepared from HOME cells stimulated DNA synthesis in bovine epithelial lens cells (BEL cells). The mitogenic activity could be neutralized by an anti-bFGF antibody. Basic FGF-like material from the HOME cell cytosol was bound onto a heparin-Sepharose column at 0.6 M and was eluted at 3 M NaCl. The 3 M NaCl eluted material reacted with the specific anti-bFGF antibody in an ELISA and stimulated DNA synthesis. It did not react with a specific anti-acidic fibroblast growth factor (aFGF) antibody. Western blotting experiments using the same bFGF antibody showed the presence of a major band of 17 Kd and a doublet of 20-22 Kd. Northern blotting of non-stimulated HOME cells using a specific 1.4 kb bFGF probe showed the presence of 5 molecular species of 6.6, 3.7, 2.2, 2.0, and 1.0 kb. No aFGF mRNA was detected with a specific previously characterized 4.04 kb probe. 12-O-tetradecanoylphorbol 13-acetate (TPA) did not influence significantly the expression of bFGF at the protein and mRNA level in HOME cells. Thus, protein kinase C activation by TPA did not appear to modulate significantly the expression of bFGF for that cell type. Contrastingly, human umbilical vein endothelial cells (HUVE cells), which expressed no bFGF and aFGF mRNA at a basal level, were induced to express bFGF but not aFGF mRNA when stimulated by TPA. These results suggest that the described angiogenic activity could be the bFGF-like mitogen contained in HOME cells and that these cells are different from endothelial cells derived from large vessels (HUVE cells) regarding the expression of bFGF.
Biological Assay↗
Binding, internalization, and degradation of basic fibroblast growth factor in human microvascular endothelial cells.
The binding, internalization, and degradation of basic fibroblast growth factor (bFGF) in human omental microvascular endothelial cells (HOME cells) were investigated. Binding studies of bFGF in human endothelial cells have not yet been reported. Basic FGF bound to HOME cells (KD of 42.0 +/- 3.8 pM and 70,526 +/- 6121 binding sites/cell for the high-affinity sites, KD of 0.933 +/- 0.27 nM and 630,252 +/- 172,459 sites/cell for low-affinity binding sites). The number of low-affinity binding sites was found to be variable. Washing the cells with 2 M phosphate-buffered saline removed completely 125I-bFGF bound to low-affinity binding sites but decreased also the high-affinity binding. The majority of the surface-bound 125I-bFGF was removed by washing the cells with acetic acid buffer at pH 3. At 37 degrees C, 30% of the cell-associated 125I-bFGF became resistant to the acidic wash after 90 min, suggesting that this fraction of bound 125I-bFGF was internalized. At this temperature, degradation of the internalized ligand was followed after 1 h by the appearance of three major bands of 15,000, 10,000, and 8,000 Da and was inhibited by chloroquine. These results demonstrated two classes of binding sites for bFGF in HOME cells; the number of high-affinity binding sites being larger than the number reported for bovine capillary endothelial cells. The intracellular processing of bFGF in HOME cells seems to be different from that of heparin binding growth factor-1 in murine lung capillary endothelial cells and of eye-derived growth factor-1 in Chinese hamster fibroblasts.
Binding Sites↗
Thrombin mitogenic responses and protein phosphorylation are different in cultured human endothelial cells derived from large and microvessels.
It is well established that thrombin induces various biological responses in endothelial cells derived from large vessels. However, little is known about the effects of thrombin on the microvasculature. Protein phosphorylation may be one of the mechanisms by which an extracellular stimulus initiates cellular events like proliferation. Therefore, we have compared the effects of either human alpha-thrombin or phorbol esters (TPA) on the proliferation or protein phosphorylation in endothelial cells derived from large vessels (umbilical vein, HUVEC) or microvessels (omental tissue, HOMEC). In HOMEC, thrombin did not stimulate cell proliferation and protein phosphorylation while TPA slightly reduced the cell proliferation and induced the phosphorylation of a 27-kDa protein. In contrast, in HUVEC, thrombin or TPA markedly enhanced the cell proliferation and stimulated the phosphorylation of a 59-kDa protein. These data indicate that (i) endothelial cells from large and small vessels respond differently to thrombin and (ii) there is a complex and as yet unclear relationship between the proliferation and the protein phosphorylation induced by thrombin.
Biological Transport↗
Evidence of endoplasmic reticulum-related Ca2+ ATPase in human microvascular endothelial cells.
We have demonstrated by immunological and molecular methods the presence of a reticulum endoplasmic-related Ca2+-ATPase in human omental microvascular endothelial cells (HOME cells). HOME cells reacted positively with a previously characterized sarcoplasmic reticulum Ca2+-ATPase antibody as demonstrated by indirect immunofluorescence. Western blotting revealed that the antibody recognized a 95-100 kDa protein. 35S-Metabolic labeling led to the detection of a similar protein with which the purified sarcoplasmic reticulum Ca2+-ATPase competed. Dot-blotting experiments indicated that a substantial amount of Ca2+-ATPase was present in HOME cell membranes. In addition, Northern blot analysis using a cDNA probe from cardiac sarcoplasmic reticulum showed the presence of mRNA species of 4-kb. As these experiments were conducted in comparison with cell types with well-defined Ca2+-ATPase in HOME cells.
Animals↗
Platelet-derived growth factor (PDGF) in type 1 diabetes mellitus.
In 10 patients with type 1 diabetes mellitus, platelet-derived growth factor (PDGF) was assessed using an in vitro assay based on the stimulation of DNA synthesis in the 3T3 mouse fibroblast cell line. beta-Thromboglobulin (beta-TG) was used as an index for platelet alpha-granules content. Platelet beta-TG content and PDGF were markedly decreased in diabetic patients while plasma beta-TG was increased as compared with control subjects. In diabetic patients, a significant negative correlation was found between plasma beta-TG level and beta-TG total platelet content, associated with a significant positive correlation between platelet beta-TG content and PDGF. These results suggest that PDGF release might be increased in diabetic subjects. This may account in part for the cell proliferation observed in diabetic angiopathy.
Adult↗
Prelining of polytetrafluoroethylene grafts with cultured human endothelial cells isolated from varicose veins.
Prelining graft material with autologous functioning endothelial cells might be one of the ultimate requirements to obtain a biocompatible surface. Accordingly, endothelial cells from stripped varicose veins were enzymatically harvested and grown on a fibronectin matrix. Proliferation was investigated in defined medium supplemented with various concentrations of endothelial cell growth supplement (ECGS) (25, up to 150 micrograms/ml) and heparin (10(-8), up to 10(-5)mol/L): optimal growth required both 150 micrograms/ml of ECGS and 10(-5)mol/L heparin. Under these conditions, cell culture achieved cell densities at a confluence of 1.2 +/- 1.1 10(5) cells/cm2 with a doubling time of 1 day. During subcultivation cultured cells consistently exhibited characteristic cobblestone morphology and immunofluorescent staining for factor VIII-related antigen, whereas prostacyclin production determined by enzyme-linked immunosorbent assay for 6-keto-prostaglandin F1 alpha reached 21.1 +/- 1.2 ng/10(6) cells after 15-minute stimulation with 1 U/ml of thrombin. Heparin-containing culture medium-endothelial cell interactions were particularly studied, and with iodine 125-heparin, binding was demonstrated with an apparent dissociation constant (Kd) of 0.36 +/- 0.04 mumol/L. A cold storage technique at -80 degrees C was sought, and freezed cells were used to coat in vitro polytetrafluoroethylene grafts. Protein-treated material allowed cell attachment and growth to a confluent monolayer as assayed by light and scanning electron microscopy. These data validate the feasibility of prelining grafts in vitro with autologous functioning endothelial cells. This approach may be useful in improving the performance of small-caliber vascular grafts according to prostacyclin production and surface-bound heparin of these cells.
Adult↗
Heparin stimulates fibroblasts growth induced by platelet derived growth factor.
We have demonstrated that 125I unfractionated heparin binds to cultured human skin fibroblasts with a Kd of 1.16 10(-8) M and is internalized partly. A low molecular weight heparin fraction (PK 10169) competed (50%) with 125I unfractionated heparin, but to a lesser extent than cold unfractionated heparin (90%). When the cell proliferation was induced by pure PDGF, heparin markedly potentiated the fibroblast growth. Similar stimulation was observed when the growth was induced by FGF or EGF. Low molecular weight heparin enhanced the fibroblast proliferation induced by PDGF but to a lesser extent than unfractionated heparin. Chondroitin sulfate has no effect. PDGF did not modify the heparin binding on fibroblast cultures either at 4 degrees C or 37 degrees C and did not alter the process of heparin internalization. PDGF binding to the cultured fibroblast (Kd 10.1 +/- 3.4 10(-10) M) was not modified by the presence of heparin when studied at 4 degrees C or 37 degrees C.
Cell Division↗
Binding of heparin to human microvascular endothelial cells and the effect on proliferation.
We have studied the binding of 125I-heparin to human omental microvascular endothelial cells (HOME cells) and investigated its effect on cell proliferation. At 20 degrees C, the binding reached a steady state from 4 hours onwards. Saturation of 125I-heparin binding occurred at 200 nM. Scatchard analysis indicated one class of binding sites (KD = 0.023 microM, 2 X 10(-6) sites/cell). Using fractionated heparin and other sulfated polysaccharides it was demonstrated that the binding was dependent on the charge and the molecular weight of the compounds. The binding was followed by the partial internalization of the bound ligand (23.8%). Heparin and Stipocus Japonicus mucopolysaccharide (SJAMP) inhibited the proliferation of exponentially growing HOME cells and human umbilical vein endothelial cells (HUVE cells). However, serum-deprived HOME cells were not inhibited by heparin.
Binding, Competitive↗
Thrombin-induced platelet factor Va formation in patients with a gray platelet syndrome.
The present study was initiated to establish the functional factor V concentration in platelets of patients with a mild bleeding disorder ascribed to a gray platelet syndrome. This inherited platelet disorder has been characterized by a specific deficiency of alpha-granules and subsequent deficiencies in the alpha-granule proteins. We found that the concentration of plasma factor V was slightly decreased (70% of normal values). In contrast, platelet factor Va formation was severely impaired. Besides a much lower factor V content than in control platelets (10-20% of normal), the dependency of platelet factor Va formation on thrombin concentration was altered. Increasing the thrombin concentration 4-fold compared to the concentration that results in maximal factor Va generation from normal platelets did not result in a maximal factor Va formation from gray platelets. When a suspension of washed gray platelets was incubated with a prostacyclin analogue prior to the stimulation with thrombin, a 10-fold lower factor Va activity was measured. Thus, thrombin-induced factor Va formation in a suspension of gray platelets is the result of a release reaction, followed by the thrombin-catalyzed activation of released factor V. Whereas the kinetics of the former reaction are apparently impaired, the kinetics of the latter one were found to be identical to those observed for normal platelet and plasma factor V activation.
Blood Platelet Disorders↗
Alpha-interferon in hairy cell leukaemia: direct effects on hairy cells or indirect cytotoxicity?
This report presents the results of a clinical trial on 53 patients with hairy cell leukaemia using low-dose alpha-interferon as therapy. Improvement of cytopenia and/or bone marrow hairy cell infiltration occurred in all but one patient. Often, blood and bone marrow improvements were dissociated and, after 7 or 13 months of therapy, complete remission was only observed in about 40% of patients. Recurrence of the disease was observed in some cases after cessation of therapy. A summary of the following results is presented: alpha-interferon receptor analysis, oncogene expression, study of the sensitivity of hairy cells to natural killer cells, and the effects of interferon on T-cell receptor gene rearrangement and on the function of T-cell clones. Results are also presented which show that myelofibrosis may be due to a release of platelet derived growth factor. The immunological findings and oncogene expression in 2 patients with a variant form of hairy cell leukaemia for which resistance to therapy was observed are also described. All the results show that interferon acts on hairy cells and are consistent with a direct effect by interferon in the treatment of hairy cell leukaemia.
Blood Platelets↗
Platelet acquired defect in PDGF and beta thromboglobulin content in hairy cell leukaemia: improvement after interferon therapy.
To investigate the platelet contribution to the development of myelofibrosis in hairy cell leukaemia (HCL), we have studied two platelet alpha granule components in 15 patients with HCL before chemotherapy: mitogenic activity was measured by 3H thymidine incorporation in BALB/C 3T3 cells and beta thromboglobulin (beta TG) assayed by radioimmunoassay (RIA). Platelet mitogenic activity and beta TG content were significantly decreased in the patients as compared to the control subjects. The nine patients who were treated with recombinant human interferon (IFN alpha A) were restudied after 4 months of therapy. The levels of both mitogenic activity and beta TG platelet content were significantly increased after IFN alpha-treatment with a complete response in five of the nine treated patients, a partial response in two and no response in the two others. HCL seemed therefore to be responsible for an acquired platelet alpha granule defect. As in the grey platelet syndrome a relationship between this abnormal platelet granule storage and the development of myelofibrosis is suggested in HCL.
Blood Platelets↗
Megakaryocytes and myelofibrosis in gray platelet syndrome.
Studies have been carried out with cultured human megakaryocytes derived from Gray Platelet Syndrome blood and have demonstrated that MK-DGF and MK-F4 are present in the early stages of megakaryocyte maturation (day 5-6) but disappear in the late phase (day 9-11). In Gray platelets total mitogenic activity, PDGF and PF4 amounts were reduced while beta TG and PF4 plasma levels were slightly increased. These findings indicate a possible relationship between the loss of alpha granule contents from the megakaryocytes in the marrow and the myelofibrosis found in Gray platelet disorder and possibly associated in other diseases with platelet or megakaryocyte abnormalities.
Blood Platelet Disorders↗
[Course of the manifestations associated with the antiprothrombinase circulating anticoagulant].
Fifteen patients (9 females and 6 males) with the "lupus type" circulating anticoagulant have been studied. The underlying disease was an auto-immune disorder in 11 cases and a malignant hemopathy in 4 cases. The manifestations frequently associated with the lupus inhibitor, such as thrombosis, thrombocytopenia and false-positive VDRL test were analysed. Hemorrhagic syndrome occurred only when thrombocytopenia or acquired abnormality of Willebrand's factor was present. Thrombotic events (8 cases) were frequent. Deep venous thrombosis was complicated with pulmonary embolism in 4 patients. Platelet abnormality, decreased fibrinolytic response or acquired Willebrand's syndrome were found in all patients with a thrombotic event. These different manifestations followed diverging courses in some patients with persistent thrombocytopenia although the anticoagulant had disappeared in 3 cases, negativation of the false-positive VDRL test while the anticoagulant remained unchanged in 1 case, occurrence of a thromboembolic episode although the anticoagulant had disappeared in 1 case.
Adult↗
Human platelet-tumor cell interactions vary with the tumor cell lines.
Platelets may promote the development of metastasis, and tumor cells that aggregate platelets are believed to be more malignant. We studied three different human mammary carcinoma cell lines, which had different interactions with human platelet-rich plasma (PRP). The MCF-7 and the T47-D cell lines induced an adenosine diphosphate (ADP)-mediated platelet aggregation. The third cell line, MDA-MB 231 did not induce any platelet aggregation. On the contrary, this cell line inhibited ADP- and arachidonic acid-induced platelet aggregation. This inhibiting activity is mainly adenosine-mediated. The mechanism by which platelets may contribute to the dissemination of cancer could be related to platelet growth factors. MCF-7 and T47-D cell lines induced a release of platelet-derived growth factor (PDGF). On the contrary, the MDA-MB 231 cell line did not induce any platelet release. The role of these platelet growth factors in tumor cell growth is discussed.
6-Ketoprostaglandin F1 alpha↗
Increased synthesis of fibrinolytic inhibitor induced by muramyl dipeptide derivatives in human cultured endothelial cells.
A decreased fibrinolytic activity induced by bacterial products and some muramyl peptides has been previously demonstrated in macrophages preparations. Since vascular endothelial cells are important for the fibrinolytic balance, we have studied the effects of MDP derivatives on cultured endothelial cells. The supernatant of MDP and murabutide treated cell cultures exhibited an increased fibrinolytic inhibitory activity when tested with urokinase. The MDP(D-D)-treatment had no effect. This increased inhibitory activity was detectable in the supernatant after a 6 h treatment and was suppressed by the addition of puromycin to the cell cultures. Furthermore, the endothelial cell culture supernatant also reduced the lytic activity of the human plasma plasminogen activator induced by venostasis. This was enhanced by MDP treatment of the cultures. These in vitro results suggested that adjuvant-active muramyl peptides may regulate the fibrinolytic balance at the vessel wall level. This could be of possible significance in the transendothelial cell migration where the role of plasminogen activator(s) has been involved.
Acetylmuramyl-Alanyl-Isoglutamine↗