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Expression of the collagen receptor glycoprotein VI during megakaryocyte differentiation

BLOOD 2000年第8期96卷 2740-2745页

作者： Berlanga, O Bobe, R Becker, M Murphy, G Leduc, M Bon, C Barry, FA Gibbins, JM Garcia, P Frampton, J Watson, SP Univ Oxford Dept Pharmacol Oxford OX1 3QT England John Radcliffe Hosp Inst Mol Med Oxford OX3 9DU England Inst Pasteur Unite Venins Paris France Univ Reading Sch Anim & Microbial Sci Reading RG6 2AJ Berks England

This study examined the expression of the platelet collagen receptor glycoprotein VI (GPVI) in megakaryocyte cell lines and primary megakaryocytes by reverse transcriptase-polymerase chain reaction and by flow cytometry and ligand blotting using the snake venom toxin convulxin. Expression of GPVI is increased in the megakaryoblastic cell lines HEL and CMK on differentiation with the phorbol ester phorbol 12-myristate Il-acetate (PMA), along with the Fe receptor gamma -chain (FcR gamma -chain). The increase in GPVI expression is associated with marked potentiation of tyrosine phosphorylation and Ca++ elevation in response to convulxin. Syk, linker for activated T cells, and phospholipase C gamma2 (PLC gamma2) are among the proteins tyrosine phosphorylated on convulxin stimulation in PMA-differentiated HEL cells. Studies on primary murine megakaryocytes grown in vitro confirmed that GPVI is up-regulated in parallel with functional activation, assessed by measurement of [Ca++](i), during differentiation. The results demonstrate that expression of GPVI is upregulated along with the FcR gamma -chain during differentiation of megakaryocytes, (C) 2000 by The American Society of Hematology.

关键词：血小板/代谢钙信号/药物作用细胞分化/药物作用细胞系/药物作用细胞系/代谢响尾蛇毒液类/药理学流式细胞术基因表达调控发育期/药物作用外源凝集素类 C型巨核细胞/细胞学巨核细胞/药物作用巨核细胞/代谢血小板膜糖蛋白类/生物合成血小板膜糖蛋白类/遗传学 RNA 信使/生物合成 RNA 信使/遗传学受体 IgG/生物合成受体 IgG/遗传学逆转录聚合酶链反应十四酰佛波乙酯/药理学肿瘤细胞培养的/药物作用增量调节/药物作用动物人类小鼠

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Collagen or collagen-related peptide cause (Ca²⁺)_i elevation and increased tyrosine phosphorylation in human megakaryocytes

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THROMBOSIS AND HAEMOSTASIS 1999年第3期82卷 1153-1159页

作者： Mountford, JC Melford, SK Bunce, CM Gibbins, J Watson, SP Univ Birmingham Queen Elizabeth Med Ctr Dept Med Birmingham B15 2TH W Midlands England Univ Oxford Dept Pharmacol Oxford England Univ Reading Sch Anim & Microbial Sci Reading RG6 2AJ Berks England

Since megakaryocytes are the cellular precursors of platelets we have investigated whether they share responses to platelet agonists, in particular collagen. Although previous studies have reported responses to thrombin in non-human megakaryocytes, through studies of single cell calcium responses and protein tyrosine-phosphorylation we demonstrate for the first time that both isolated human megakaryocytes and CD41/61-positive megakaryocytes derived in culture from CD34+ cells share responses to the platelet agonists collagen, collagen-related peptide and thrombin. The responses to either collagen or CRP were seen only in the most mature megakaryocytes and not in megakaryocyte-like cell lines, suggesting that the response to collagen is a characteristic developed late during megakaryocyte differentiation. Those primary cells offer the opportunity to use many molecular and cellular techniques to study and manipulate signalling events in response to platelet receptor agonists, which cannot be performed in the small, anucleate platelet itself.

关键词：抗原 CD34/血液钙/代谢细胞分化细胞系胶原/药理学胎血/细胞学细胞内液/药物作用细胞内液/代谢巨核细胞/细胞学巨核细胞/药物作用巨核细胞/代谢磷酰化酪氨酸/代谢人类婴儿新生

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PLATELET MORPHOLOGY AFTER ASPIRIN

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AMERICAN JOURNAL OF CLINICAL PATHOLOGY 1975年第4期63卷 554-558页

作者： FAJARDO, LF VET ADM HOSP PALO ALTOCA STANFORD UNIV MED SCHDEPT PATHOL300 PASTEUR DRSTANFORDCA 94305

Previous studies have revealed morphologic platelet abnormalities, particularly inclusions, in patients who have acute malarial infections. To determine the possible role of aspirin (ASA) in the etiology of these abnormalities, the thrombocytes of 14 normal men were examined before and after ingestion of 2 Gm. of ASA. A significant decrease in platelet size was noticed after ASA: however, no ultrastructural alteration of these circulating platelets was found, although functional impairment was manifested in vivo (bleeding time) and in vitro (aggregation). It is concluded that inclusions and other morphologic abnormalities in thrombocytes of malaria patients are not caused by aspirin ingestion.

关键词：阿司匹林/药理学血液凝固试验血小板/药物作用血小板/生理学血小板/超微结构巨核细胞/药物作用巨核细胞/超微结构显微镜检查电子血小板聚集/药物作用时间因素成年人人类男(雄)性

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Hypersensitivity of circulating progenitor cells to megakaryocyte growth and development factor (PEG-rHu MGDF) in essential thrombocythemia

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BLOOD 2000年第10期96卷 3310-3321页

作者： Axelrad, AA Eskinazi, D Correa, PN Amato, D Univ Toronto Dept Anat & Cell Biol Toronto ON M5S 1A8 Canada Univ Toronto Dept Med Biophys Toronto ON M5S 1A8 Canada Mt Sinai Hosp Dept Med Hematol Oncol Toronto ON Canada

Hematopoietic progenitor cells in 2 myeloproliferative disorders, juvenile chronic myelomonocytic leukemia and polycythemia vera, are known to be hypersensitive to cytokines that control normal progenitor cell proliferation, differentiation, and survival in their respective granulocyte/macrophage and erythroid lineages. Because thrombopoietin controls these functions in the normal megakaryocytic lineage, we asked the question: Are megakaryocytic progenitor cells in the myeloproliferative disorder essential thrombocythemia (ET) hypersensitive to thrombopoietin? Peripheral blood mono-nuclear cells from patients with ET, or secondary (reactive) thrombocytosis (2 degreesT), or healthy volunteers were grown in strictly serum-free agarose culture containing interleukin 3 (IL-3) and all-transretinoic acid, with various concentrations of PEG-rHu megakaryocyte growth and development factor (MGDF), The concentration of cytokine at half-maximum colony number served as a measure of progenitor cell sensitivity. Hypersensitivity to PEG-rHu MGDF was found in circulating progenitors from 18 of 20 (90%) informative patients with presumptive diagnosis ET, 1 of 8 (12.5%)2 degreesT patients, and none of the 22 healthy volunteers. Median MGDF sensitivity ratio in ET patients was approximately 53 times greater than in the controls. This hypersensitivity, which was also directed to rHu thrombopoietin, was highly specific with respect to cytokine, disease, and cell lineage. We propose that, despite their single pluripotential cell origin, the different clinicopathologic phenotypes in different chronic myeloproliferative disorders are determined by lineage-restricted hypersensitivities of hematopoietic progenitor cells to endogenous cytokines, This work emphasizes the importance of stringent serum-free conditions for revealing true sensitivities to cytokines, The findings also offer a basis for evolving a positive test for ET, a diagnosis now made essentially by exclusion. (C) 2000 by The A

关键词：血液循环细胞培养技术/方法队列研究集落形成单位测定细胞因子类/药理学剂量效应关系药物药物过敏/病因学造血干细胞/药物作用巨核细胞/药物作用巨核细胞/病理学巨核细胞/超微结构聚乙烯二醇类/药理学重组蛋白质类/药理学血小板增多出血性/血液血小板增多出血性/病因学血小板生成素/药理学成年人老年人老年人 80以上女(雌)性人类男(雄)性中年人

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Biologic and structural differences of thrombopoietic growth factors

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SEMINARS IN HEMATOLOGY 2000年第2期37卷 19-27页

作者： Begley, CG Basser, RL Western Australian Inst Med Res Perth WA 6000 Australia Ctr Dev Canc Therapeut Parkville Vic Australia Royal Melbourne Hosp Dept Clin Haematol & Med Oncol Melbourne Vic Australia Royal Melbourne Hosp Rotary Bone Marrow Res Labs Melbourne Vic Australia Walter & Eliza Hall Inst Med Res Parkville Vic Australia Ludwig Inst Canc Res CH-1066 Epalinges Switzerland

The search for a thrombopoietic agent has resulted in the identification of numerous cytokines and growth factors with thrombopoietic activity. However, with the exception of interleukin (IL)-11 and thrombopoietin (TPO), the megakaryopoietic activity of most of these molecules has not produced clearly identifiable clinical benefits. Despite the relatively modest effect of IL-11 on megakaryocyte and platelet production in vitro and in vivo, it does reduce the need for platelet transfusions in specialized clinical settings. In contrast, the c-Mpl ligand TPO has been shown to be a potent stimulator of megakaryocyte and platelet production both in vitro and in vivo. Clinical studies are being conducted with two different preparations of the c-Mpl ligand: recombinant human thrombopoietin (rhTPO) and pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF). A recombinant form of the complete human molecule, rhTPO is glycosylated and produced in mammalian cells. PEG-rHuMGDF consists of only the receptor-binding domain linked to a polyethylene glycol (PEG) moiety and is generated in Escherichia coli. Although c-Mpl ligands are still being evaluated, preliminary evidence indicates that these molecules can elevate platelet counts and may be useful in a range of clinical contexts. This report discusses aspects of the biology behind the clinical actions of IL-11 and the c-Mpl ligands.

关键词：血小板/细胞学血小板/药物作用细胞因子类/血液细胞因子类/药理学细胞因子类/生理学生长物质/血液生长物质/药理学生长物质/生理学血细胞生成/药物作用白细胞介素11/血液白细胞介素11/药理学白细胞介素11/生理学巨核细胞/细胞学巨核细胞/药物作用血小板生成素/血液血小板生成素/药理学血小板生成素/生理学动物人类

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Role of the distal half of the c-Mpl intracellular domain in control of platelet production by thrombopoietin in vivo

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MOLECULAR AND CELLULAR BIOLOGY 2000年第2期20卷 507-515页

作者： Luoh, SM Stefanich, E Solar, G Steinmetz, H Lipari, T Pestina, TI Jackson, CW de Sauvage, FJ Genentech Inc Dept Mol Oncol San Francisco CA 94080 USA Genentech Inc Dept Cardiovasc San Francisco CA 94080 USA Genentech Inc Dept Metab & Pharmacokinet San Francisco CA 94080 USA St Jude Childrens Res Hosp Div Expt Hematol Memphis TN 38105 USA

The cytokine thrombopoietin (TPO) controls the formation of megakaryocytes and platelets from hematopoietic stem cells. TPO exerts its effect through activation of the c-Mp1 receptor and of multiple downstream signal transduction pathways. While the membrane-proximal half of the cytoplasmic domain appears to be required for the activation of signaling molecules that drive proliferation, the distal half and activation of the mitogen-activated protein kinase pathway have been implicated in mediating megakaryocyte maturation in vitro. To investigate the contribution of these two regions of c-Mp1 and the signaling pathways they direct in mediating the function of TPO in vivo, we used a knock-in (KI) approach to delete the carboxy-terminal 60 amino acids of the c-Mp1 receptor intracellular domain. Mice lacking the C-terminal 60 amino acids of c-Mp1 (Delta 60 mice) have normal platelet and megakaryocyte counts compared to wild-type mice. Furthermore, platelets in the KI mice are functionally normal, indicating that activation of signaling pathways connected to the C-terminal half of the receptor is not required for megakaryocyte differentiation or platelet production. However, Delta 60 mice have an impaired response to exogenous TPO stimulation and display slower recovery from myelosuppressive treatment, suggesting that combinatorial signaling by both ends of the receptor intracellular domain is necessary for an appropriate acute response to TPO.

关键词：血细胞计数血小板/细胞学血小板/药物作用血小板/生理学细胞分化/药物作用细胞分裂/药物作用细胞培养的外显子/遗传学纤维蛋白原/代谢血细胞生成造血干细胞/细胞学造血干细胞/药物作用巨核细胞/细胞学巨核细胞/药物作用小鼠转基因肿瘤蛋白质类磷酰化/药物作用磷酸酪氨酸/代谢血小板活化倍性原癌基因蛋白质类/化学原癌基因蛋白质类/遗传学原癌基因蛋白质类/代谢受体细胞因子/化学受体细胞因子/遗传学受体细胞因子/代谢受体血小板生成素序列缺失/遗传学信号传导/药物作用血小板生成素/药理学时间因素动物小鼠

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A novel megakaryocyte differentiation factor from mouse placenta

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TRENDS IN CARDIOVASCULAR MEDICINE 1999年第6期9卷 167-171页

作者： Lin, JD Linzer, DIH Northwestern Univ Dept Biochem Mol Biol & Cell Biol Evanston IL 60208 USA

Megakaryocyte differentiation and subsequent platelet production are regulated by a network of growth factors and cytokines. We hypothesized that pregnancy-specific regulatory factors also may participate in the modulation of megakaryocytopoiesis and thrombopoiesis. We identified a mouse placental prolactin-like protein hormone with an activity similar to interleukin 6 in targeting megakaryocytes and inducing cell differentiation. The receptor for this placental hormone is present on megakaryocytes from pregnant and nonpregnant female mice and from male mice, and from humans, suggesting that this signaling pathway (if not necessarily this particular ligand) is broadly functional both in terms of physiologic state and evolution. Thus, studying the biologic activities of the large family of placental prolactin-like proteins represents a potentially valuable approach to the discovery of novel hematopoietic signaling pathways. (Trends Cardiovasc Med 1999;9:167- 171).(C) 1999, Elsevier Science Inc.

关键词：细胞分化/药物作用细胞培养的巨核细胞/细胞学巨核细胞/药物作用巨核细胞/代谢膜糖蛋白类/代谢胎盘激素类/药理学催乳素/药理学蛋白质结合信号传导/药物作用动物女(雌)性男(雄)性小鼠妊娠

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Characterization of hematopoietic lineage-specific gene expression by ES cell in vitro differentiation induction system

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BLOOD 2000年第3期95卷 870-878页

作者： Era, T Takagi, T Takahashi, T Bories, JC Nakano, T Osaka Univ Dept Mol Cell Biol Microbial Dis Res Inst Suita Osaka 5650871 Japan Hop St Louis U462 INSERM Paris France

The continuous generation of mature blood cells from hematopoietic progenitor cells requires a highly complex series of molecular events. To examine lineage-specific gene expression during the differentiation process, we developed a novel method combining LacZ reporter gene analysis with in vitro hematopoietic differentiation induction from mouse embryonic stem cells. For a model system using this method, we chose the erythroid and megakaryocytic differentiation pathways. Although erythroid and megakaryocytic cells possess distinct functional and morphologic features, these 2 lineages originate from bipotential erythro-megakaryocytic progenitors and share common lineage-restricted transcription factors. A portion of the 5' flanking region of the human glycoprotein IIb (alpha IIb) integrin gene extending from base -598 to base +33 was examined in detail. As reported previously, this region is sufficient for megakaryocyte-specific gene expression. However, previous reports that used human erythro-megakaryocytic cell lines suggested that one or more negative regulatory regions were necessary for megakaryocyte-specific gene expression. Our data clearly showed that an approximately 200-base enhancer region extending from -598 to -400 was sufficient for megakaryocyte-specific gene expression, This experimental system has advantages over those using erythro-megakaryocytic cell lines because it recapitulates normal hematopoietic cell development and differentiation. Furthermore, this system is more efficient than transgenic analysis and can easily examine gene expression with null mutations of specific genes. (C) 2000 by The American Society of Hematology.

关键词：结合部位细胞分化/药物作用细胞谱系/遗传学 DNA结合蛋白质类/代谢增强子元件(遗传学) 红细胞/细胞学红系祖细胞/细胞学红系祖细胞/药物作用红系细胞特异性DNA结合因子类红细胞生成/药物作用红细胞生成素/药理学基因表达调控/药物作用基因合成血细胞生成/药物作用乳糖操纵子巨噬细胞集落刺激因子/药理学巨噬细胞/细胞学巨核细胞/细胞学巨核细胞/药物作用肽延伸因子1/遗传学血小板糖蛋白GPⅡb-Ⅲa复合物/遗传学原癌基因蛋白质类/代谢原癌基因蛋白质c-ets 重组融合蛋白质类/生物合成重组融合蛋白质类/遗传学调控序列核酸干细胞/细胞学干细胞/药物作用血小板生成素/药理学转录因子/代谢动物人类小鼠

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132Ge体外对受照小鼠CFU-GM的影响

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中华放射医学与防护杂志 1993年第2期13卷 85页

作者：吴学东张肇和陈洪文第一军医大学南方医院，广州　510516

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Repression of A TAF_II32 isoform as part of a program of genes regulated during Mpl ligand-induced megakaryocyte differentiation

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 1999年第1期262卷 55-59页

作者： Thompson, A Ravid, K Boston Univ Sch Med Dept Biochem Boston MA 02118 USA Boston Univ Sch Med Whitaker Cardiovasc Inst Boston MA 02118 USA

Circulating platelets, essential for thrombosis and hemostasis, originate from megakaryocytes. Megakaryocyte growth, differentiation and survival processes are regulated by the c-Mpl receptor ligand. In the current study we used differential display to identify part of the program of genes regulated during Mpl ligand-induced murine megakaryocyte differentiation. Several of the genes, including the retinoblastoma binding protein p84, were found to be induced, while others were repressed. One such repressed gene was identified as a TATA-binding protein (TBP)-Associated Factor (TAF) family member, TAF(II)32, previously reported to be upregulated during apoptosis, Our analysis of various cell types suggested that the previously identified species homologs, human TAF(II)32 and murine TAF(II)32, are in fact different isoforms, which We propose to re-name TAF(II)32 alpha and TAF(II)32 beta, respectively. Only the TAF(II)32 beta isoform is regulated during Mpl ligand-induced megakaryocyte differentiation, which suggests individual roles for the two forms. (C) 1999 Academic Press.

关键词：生物学标记细胞分化/药物作用细胞分化/遗传学细胞培养的克隆分子减量调节/药物作用巨核细胞/细胞学巨核细胞/药物作用巨核细胞/代谢分子序列数据蛋白质亚型/遗传学 RNA 信使/遗传学 RNA 信使/代谢序列同源性 TATA结合蛋白质相关因子类血小板生成素/药理学时间因素反式激活(遗传学)/药物作用转录因子TFIID 转录因子 TFⅡ类/遗传学动物人类小鼠

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