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Biomedical subjects

S Bellucci

Publications and source records attributed to S Bellucci.

At least 19 recordsLinked to original sources
PubMed1992

Quantitation of platelet glycoprotein IV (CD36) in healthy subjects and in patients with essential thrombocythemia using an immunocapture assay.

Glycoprotein IV (GPIIb, CD36) is a major platelet membrane glycoprotein which is thought to participate in a number of adhesive reactions and to mediate signal transduction. In order to measure the total content of GPIV in human platelets, we have developed a simple and sensitive solid-phase radioimmunoassay based on the immunocapture of GPIV from Triton X-100-solubilized platelets. FA6-152, a monoclonal antibody to GPIV was coated on microtiter plates and bound antigen was quantified with a radiolabeled polyclonal antibody to GPIV. Using purified GPIV as a standard, the coefficients of variation of the assay were found to be less than 10% at concentrations of GPIV ranging from 0.15 to 0.75 micrograms/ml. The assay was validated by the parallelism obtained between purified GPIV dose-response curves and those obtained with platelet lysates, indicating a similar antigenic activity for GPIV in both samples. The level of GPIV in platelets from healthy donors was 0.23 +/- 0.05 (mean +/- SD, n = 15) micrograms per 100 micrograms of platelet proteins and a mean value of 27,440 +/- 6,200 (SD) molecules per platelet was calculated. The radioimmunoassay could be used to discriminate between the high level of platelet GPIV in patients with essential thrombocythemia (mean +/- SD = 81,850 +/- 27,780 molecules/platelet; n = 8) and the normal GPIV level in patients with secondary thrombocytosis (mean +/- SD = 26,810 +/- 4,030 molecules/platelet; n = 5), thereby demonstrating the clinical usefulness of the assay. The specific increase in platelet GPIV in patients with essential thrombocythemia was confirmed by immunoblot analysis whereas no increase in platelet GPIb or GPIIb-IIIa was observed by this technique.

Adult↗
PubMed1992

A defect of platelet aggregation associated with an abnormal distribution of glycoprotein IIb-IIIa complexes within the platelet: the cause of a lifelong bleeding disorder.

A young Italian man (A.P.) has a lifelong history of bleeding from gums and mucocutaneous tissue. Electron microscopy showed a wide diversity of platelet size including giant forms. In citrated platelet-rich plasma (PRP), platelet aggregation induced by adenosine diphosphate (ADP) and other agonists was much reduced. Both secretion and clot retraction were normal. The aggregation of washed platelets with ADP was improved but remained subnormal, as was aggregation with collagen and thrombin. Fibrinogen-binding was analyzed by flow cytometry using platelets in whole blood or PRP and was markedly decreased. Crossed immunoelectrophoresis of Triton X-100 extracts of (A.P.) platelets showed that GP IIb-IIIa levels were 40% to 50% of normal. Glycoprotein (GP) IIb and GP IIIa were of usual migration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but their labeling was much reduced during lactoperoxidase-catalyzed iodination. Binding to (A.P.) platelets of four different 125I-labeled monoclonal antibodies to GP IIb-IIIa complexes was reduced to 12% to 20% of normal levels. However, when the patient's platelets were stimulated with alpha-thrombin, monoclonal antibody binding showed the same increase (approximately 20,000 sites) as normal platelets. Both flow cytometry and immunocytochemical studies showed that the distribution of residual surface GP IIb-IIIa within the total (A.P.) platelet population was heterogeneous and not related to platelet size. Staining of ultrathin sections confirmed the presence of an internal pool of GP IIb-IIIa. Monoclonal antibodies to other membrane glycoproteins bound normally to (A.P.) platelets. The patient has a selective deficiency of the surface pool of GP IIb-IIIa complexes that is manifested clinically by a mild Glanzmann's thrombasthenia-like syndrome.

Adenosine Diphosphate↗
PubMed1992

New insights into the regulation of megakaryocytopoiesis by haematopoietic and fibroblastic growth factors and transforming growth factor beta 1.

Effects of cytokines on murine megakaryocyte (MK) colony formation from either unfractionated marrow cells or purified early haematopoietic cells were studied. Recombinant interleukin-3 (IL3), interleukin-6 (IL6), granulocyte-macrophage colony-stimulating factor (GM-CSF), erythropoietin (Epo) and acidic and basic fibroblast growth factor (aFGF and bFGF) each was able to stimulate MK colony growth although they varied somewhat in their potential. IL6 and FGFs, in addition to their effect on MK colony growth, increased the size of individual MK. The combination of IL3 with IL6 or FGF resulted in an additive action. Monoclonal anti-IL6 antibody completely neutralized the activity of mouse IL6 and FGFs but had no effect on human IL6, mouse IL3 and GM-CSF. When using purified lineage negative marrow cells, only IL3 and IL6 promoted MK colony formation. Transforming growth factor beta 1 (TGF-beta 1) at 10-200 pg/ml selectively inhibited IL3-induced MK colony formation, and at 0.2-0.5 ng/ml it still had no obvious effect on the activity of IL6 or GM-CSF but caused an inhibition of FGF-induced MK colony formation. These data suggest that differential mechanisms are involved in the regulation of megakaryocytopoiesis by IL3, IL6, FGFs and GM-CSF, and that TGF-beta 1 negatively regulates MK development mainly by interfering with the action of IL3.

Animals↗
PubMed1992

Identification of a normal human bone marrow cell population co-expressing megakaryocytic and erythroid markers in culture.

A population of haematopoietic cells co-expressing glycoprotein IIIa (GPIIIa), which has been shown to be present in the megakaryocyte-platelet lineage, and glycophorin A, which has been shown to be specific for the erythroid lineage, has been identified in normal bone marrow cultures using double immunofluorescence staining. The cells showing this phenotype have the size of lymphocytes and appear at an early time (day 1 to d 6) of culture. We have also detected a type of mixed megakaryocyte (MK) cluster containing this phenotype on d 3 or 4 in most normal marrow samples. Such a cell phenotype was not detected after 6 d of culture. It is thus the first time that this phenotype, although already described for several human neoplastic cell lines, has been observed in several normal human bone marrow cultures. Cells expressing this phenotype may represent haematopoietic cells, common to megakaryocytic and erythroid lineages, at a very early stage of differentiation.

Biomarkers↗
PubMed1992

Inhibitory effect of platelet factor 4 (PF4) on the growth of human erythroleukemia cells: proposed mechanism of action of PF4.

The effect of platelet factor 4 (PF4) on the growth of human erythroleukemia cell line (HEL) and the binding characteristics of iodine 125-labeled PF4 to cells were studied to determine the mechanism of action of PF4. HEL cells were cocultured with various doses of PF4 in either a plasma clot system for colony assay or a liquid system for tritiated thymidine incorporation. A significant inhibition of HEL colony growth and tritiated thymidine incorporation was seen at PF4 doses of 1 microgram/ml and 0.5 microgram/ml, respectively. The inhibitory effect of PF4 could be abrogated by the addition of heparin (5 to 10 micrograms/ml). Enzyme-linked immunosorbent assay showed that PF4 had no obvious effect on the expression of platelet glycoprotein IIb/IIIa of HEL cells. Binding of 125I-PF4 to HEL cells reached equilibrium within 20 to 30 minutes with dissociation constant of 1.3 x 10(-10)M and Bmax of 6.3 pmol/10(5) cells and was inhibited by an excess of unlabeled PF4, beta TG, and heparin but was not affected by PMA, IL-3, IL-6, GM-CSF, and interferon-alpha. PF4 did not affect the binding of 125I-IL-3 and 125I-IL-6 to HEL cells. These data demonstrate that PF4 inhibits the growth of HEL cells by specific binding to HEL cells and suggest that the action of PF4 may be associated with the heparin-binding sites of the molecule.

Cell Division↗
PubMed1992

[Platelets and aging].

Platelets seem involved in pathogenesis of atherosclerosis and Alzheimer disease which frequency increases with population ageing. Platelet hyperactivation may contribute to atherosclerosis by release of factors, which increase fibroblast and smooth muscle cell proliferation and perhaps lipid deposition. Many studies evidenced an increased platelet activation with ageing concomitantly to an increase of some coagulation factors, and an impaired response of endothelial cells leading to a prethrombotic state and facilitating the occurrence of atherosclerosis. On the other hand, in Alzheimer disease, a deposit of amyloid beta protein responsible for vascular and neuronal damage was evidenced. Platelet activation is responsible for the release of an amyloid beta protein precursor (the protease nexin 2). An increased platelet activation as demonstrated with aging, may thus explained the increased occurrence of Alzheimer disease.

Aged↗
PubMed1991

Platelet thrombospondin and glycoprotein IV abnormalities in patients with essential thrombocythemia: effect of alpha-interferon treatment.

Platelet aggregability and some biochemical parameters were evaluated in seven patients with essential thrombocythemia (ET) compared with seven patients with secondary thrombocytosis (ST). Defective platelet aggregation with one or more agonists was seen in five patients with ET whereas aggregation was increased in two other patients. In addition, three patients with ET demonstrated spontaneous platelet aggregation in citrated plasma. This was associated with increased level of thrombospondin (TSP) in the plasma membrane. Interestingly, the presence of a proteolyzed 160 kDa form of TSP was detected in all patients with ET, whereas it was never found in patients with ST. Furthermore, three patients with ET demonstrated increased levels of platelet surface glycoprotein IV (GP IV), the putative receptor for TSP in the plasma membrane. In two of these patients, this correlated with increased surface expression of TSP and spontaneous platelet aggregation. The results suggest a possible link between the increased number of plasma membrane GP IV molecules, the spontaneous expression of TSP on the platelet surface and platelet hyperaggregability in some ET patients. The levels of plasma membrane GP IV and platelet surface-associated TSP tended to be normalized during alpha-interferon treatment, whereas the presence of an altered form of TSP persisted. This last parameter might be of practical usefulness in the characterization of the disease, permitting a clear distinction from ST.

Aged↗
PubMed1991

Studies of in vitro megakaryocytopoiesis in adult immune thrombocytopenic purpura (ITP).

In vitro megakaryocytopoiesis was studied in 8 patients with chronic immune thrombocytopenia (ITP). A significant increase of megakaryocyte (MK) colony formation was observed in 5/5 patients studied. Furthermore, the serum of these 8 patients was able to enhance MK colony formation by normal marrow cells. This effect was neither due to a decrease of inhibitors of megakaryocytopoiesis such as betathromboglobulin (beta TG) nor to the IgG fraction of patients' serum. In addition, the level of interleukin 6, which is above all a stimulus for MK maturation, was found within the normal range in 8/8 patients tested. These data suggest that in chronic ITP there is an increase of MK progenitor cell number which may be due to an increased level of MK colony-stimulating activity.

Adult↗
PubMed1991

In vivo inhibition of megakaryocyte and platelet production by platelet factor 4 in mice.

The in vivo effect of human platelet factor 4 (PF4) on murine megakaryocytopoiesis and thrombopoiesis was studied. Administration of PF4 induced a dose-dependent decrease in the numbers of megakaryocytes and their progenitor cells (CFU-MK), continuing for 1 week after the injection. However, the size of megakaryocytes and their colonies was not changed following PF4 injection. Platelet levels were significantly decreased at days 3-4. The number of CFU-GM was decreased at days 1-2. White blood cells and hemoglobin were unaffected by PF4. These data indicate that PF4 inhibits megakaryocyte and platelet production in vivo by acting on the early stage of megakaryocyte development.

Animals↗
PubMed1991

Association of autoimmune thrombocytopenic purpura (AITP), Graves' disease and ovarian carcinoma.

We report here the case of a patient suffering sequentially from autoimmune thrombocytopenic purpura (AITP), Grave's disease and an ovarian carcinoma. An autoimmune mechanism was indicated by the presence of platelet associated immunoglobulins and the detection of antithyroid microsomal antibodies. However, the exact mechanism associating these autoimmune manifestations with the ovarian tumour remains unexplained. Specific therapy for hyperthyroid led to a moderate increase of the platelet count. But complete remission of AITP now lasting for more than eight years was only obtained by ablation of the ovarian tumour.

Autoimmune Diseases↗
PubMed1991

Megakaryocytopoiesis: characterization and regulation in normal and pathologic states.

Data concerning megakaryocytopoiesis and its regulation were summarized in this report. Critical analysis of these data indicates that: (i) megakaryocytopoiesis is a complex, multiple-stage cellular and biologic process; (ii) the survival, proliferation and differentiation of progenitor cells into immature megakaryocytes are regulated mainly by interleukin-3, granulocyte-macrophage colony-stimulating factor and an as yet uncharacterized megakaryocyte colony-stimulating factor, and the maturation of immature megakaryocytes to produce platelets is regulated primarily by interleukin-6 and thrombopoietin; (iii) optimal megakaryocyte development needs adequate interactions of several growth factors with target cell population and hematopoietic microenvironment; (iv) megakaryocytopoietic inhibition is controlled essentially by megakaryocyte-platelet products such as transforming growth factor-beta, and platelet factor 4 and its related proteins; interferon-alpha and -gamma also are able to play an inhibitory role; and (v) expansion or decrease of either normal or neoplastic megakaryocyte progenitor cells, change of platelet mass and abnormalities of growth factor levels in hematopoietic tissue might result in an abnormal megakaryocytopoiesis.

Hematologic Diseases↗
PubMed1990

Negative regulation of human megakaryocytopoiesis by human platelet factor 4 (PF4) and connective tissue-activating peptide (CTAP-III).

We have previously reported that human platelet factor 4 (PF4) and beta-thromboglobulin inhibit human megakaryocyte (meg) colony formation in vitro. Here, we report further findings concerning the effect of PF4 as well as another platelet-derived factor: connective tissue-activating peptide (CTAP-III). Addition of these factors (2.5-10 micrograms/ml) into normal marrow cultures resulted in a significant decrease of meg colonies, especially the mixed-meg colonies, BFU-meg and large CFU-meg, suggesting that they inhibit both proliferation and maturation of meg progenitor cells, with a predominant effect on earlier progenitor cells. Comparison of the effects of the two factors showed that their major effects were similar, with some difference in inhibitory degree. These results indicate that both PF4 and CTAP-III are potent inhibitors of meg colony formation and involved in negative autocrine regulation of megakaryocytopoiesis.

Bone Marrow Cells↗
PubMed1990

Negative regulation of human megakaryocytopoiesis by human platelet factor 4 and beta thromboglobulin: comparative analysis in bone marrow cultures from normal individuals and patients with essential thrombocythaemia and immune thrombocytopenic purpura.

The effect of human platelet factor 4 (PF4) and beta-thromboglobulin (BTG) on megakaryocyte colony formation in normal subjects as well as in essential thrombocythaemia (ET) and in immune thrombocytopenic purpura (ITP) was studied. Both PF4 and BTG were found to be capable of inhibiting the development of isolated megakaryocytes and their colonies in normal marrow cultures in a dose-dependent fashion. A significant 50% inhibition was seen at a PF4 or BTG concentration of 1-2.5 micrograms/ml, and complete inhibition in the range of 5-10 micrograms PF4 or BTG/ml. The two platelet proteins had similar effects on megakaryocyte development. A combination of PF4 and BTG resulted in an additive effect. Antibodies against PF4 or BTG could effectively neutralize the inhibitory effect of PF4 or BTG respectively. In ET and ITP, in vitro megakaryocyte development was also inhibited by PF4 and BTG in a similar way to that seen in normal subjects, suggesting that the responsiveness of megakaryocyte progenitors to PF4 and BTG is normal in these two disorders. PF4 and BTG did not affect the growth of colony forming units granulocyte-macrophage (CFU-GM) except at very high concentration (greater than or equal to 10 micrograms/ml) but they did inhibit erythroid colony formation by normal and ET burst forming units erythroid (BFU-E). However, the inhibition of BFU-E by PF4 and BTG was dose-related, and a 50% inhibition required a PF4 or BTG dose ranging from 5 to 10 micrograms/ml. These results indicate that PF4 and BTG are involved in negative regulation of normal and pathologic megakaryocytopoiesis and that their inhibition acts predominantly on the megakaryocytic lineage.

Autoimmune Diseases↗
PubMed1990

Platelet aggregation is not necessary for occurrence of Raynaud's phenomenon: a clinical history and laboratory results.

"Raynaud's phenomenon (R.P.) is characterized by a spastic vasoconstriction of hand and/or feet extremities leading to painful ischemic attacks. In the pathogeny of this syndrome the role of a platelet hyperactivation was evidenced. Nevertheless, the responsibility of platelet aggregation remains controversial. We report here the case of a patient presenting with a Glanzmann thrombasthenia defined by the complete lack of platelet aggregation, and showing a typical RP which was confirmed by the measurement of hand temperature and the decrease of digital and hand blood flow. Thus, the description of this case shows that platelet aggregation by itself is not necessary for the occurrence of RP, and encourages for therapeutic purposes the development of vasodilatators rather than platelet antiaggregants agents."

Female↗
PubMed1990

Regulation of human megakaryocytopoiesis.

Megakaryocytopoiesis is a complex, highly regulated cellular and biologic process which leads to the production of platelets. The proliferation of megakaryocyte (MK) progenitors is mainly regulated by interleukin-3, granulocyte-macrophage colony-stimulating factor and an as yet uncharacterized MK colony-stimulating factor. The maturation of MKs to produce platelets is essentially regulated by interleukin-6 and thrombopoietin. Optimal megakaryocytopoiesis is controlled by appropriate combinations of positive and negative influence. Megakaryocytopoietic inhibition is controlled by transforming growth factor beta, platelet factor 4 and its related proteins, interferon-alpha and -gamma.

Blood Platelets↗