RESUMO
Complex interactions between platelets and activated endothelium occur during the thrombo-inflammatory reaction at sites of vascular injuries and during vascular hemostasis. The endothelial receptor endoglin is involved in inflammation through integrin-mediated leukocyte adhesion and transmigration; and heterozygous mutations in the endoglin gene cause hereditary hemorrhagic telangiectasia type 1. This vascular disease is characterized by a bleeding tendency that is postulated to be a consequence of telangiectasia fragility rather than a platelet defect, since platelets display normal functions in vitro in this condition. Here, we hypothesize that endoglin may act as an adhesion molecule involved in the interaction between endothelial cells and platelets through integrin recognition. We find that the extracellular domain of human endoglin promotes specific platelet adhesion under static conditions and confers resistance of adherent platelets to detachment upon exposure to flow. Also, platelets adhere to confluent endothelial cells in an endoglin-mediated process. Remarkably, Chinese hamster ovary cells ectopically expressing the human αIIbß3 integrin acquire the capacity to adhere to myoblast transfectants expressing human endoglin, whereas platelets from Glanzmann's thrombasthenia patients lacking the αIIbß3 integrin are defective for endoglin-dependent adhesion to endothelial cells. Furthermore, the bleeding time, but not the prothrombin time, is significantly prolonged in endoglin-haplodeficient (Eng +/-) mice compared to Eng +/+ animals. These results suggest a new role for endoglin in αIIbß3 integrin-mediated adhesion of platelets to the endothelium, and may provide a better understanding on the basic cellular mechanisms involved in hemostasis and thrombo-inflammatory events.
Assuntos
Plaquetas/metabolismo , Comunicação Celular , Endoglina/metabolismo , Células Endoteliais/metabolismo , Animais , Plaquetas/citologia , Células CHO , Adesão Celular , Linhagem Celular , Células Cultivadas , Cricetinae , Cricetulus , Endoglina/genética , Células Endoteliais/citologia , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismoRESUMO
Previous studies have shown CD34 family member Podocalyxin is required for epithelial lumen formation in vitro. We demonstrate that Endoglycan, a CD34 family member with homology to Podocalyxin, is produced prior to lumen formation in developing nephrons. Endoglycan localizes to Rab11-containing vesicles in nephron progenitors, and then relocalizes to the apical surface as progenitors epithelialize. Once an apical/luminal surface is formed, Endoglycan (and the actin-binding protein Ezrin) localize to large, intraluminal structures that may be vesicles/exosomes. We generated mice lacking Endoglycan and found mutants had timely initiation of lumen formation and continuous lumens, similar to controls. Mice with conditional deletion of both Endoglycan and Podocalyxin in developing nephrons also had normal tubular lumens. Despite this, Endoglycan/Podocalyxin is required for apical recruitment of the adaptor protein NHERF1, but not Ezrin, in podocyte precursors, a subset of the epithelia. In summary, while CD34 family members appear dispensable for lumen formation, our data identify Endoglycan as a novel pre-luminal marker and suggest lumen formation occurs via vesicular trafficking of apical cargo that includes Endoglycan.
Assuntos
Antígenos CD34/metabolismo , Mucinas/metabolismo , Néfrons/embriologia , Sialoglicoproteínas/metabolismo , Animais , Proteínas do Citoesqueleto/metabolismo , Células Epiteliais/citologia , Camundongos , Camundongos Transgênicos , Mucinas/genética , Néfrons/metabolismo , Fosfoproteínas/metabolismo , Podócitos/citologia , Sialoglicoproteínas/genética , Trocadores de Sódio-Hidrogênio/metabolismoRESUMO
CD40 ligand (CD40L) acts as an immune modulator in activated T cells, and mutations in the extracellular domain are associated to X-linked hyper IgM syndrome. A role for platelet CD40L in mediating thrombotic and inflammatory processes in atherosclerosis has also been reported. Using the Cre/loxP recombination technology we generated four knockout lines of mice with deletion of the Cd40lg gene restricted to the hematopoietic system. Mouse lines with expression of Cre recombinase driven by the Tie2, Vav1, or CD4 promoters showed in vivo ablation of CD40L in leukocytes and platelets. In contrast, in mice with Cre expression driven by the megakaryocyte lineage-restricted Pf4 promoter, abolition of CD40L expression was observed in megakaryocytes cultured in vitro, but not in circulating platelets. Characterization of these animals revealed reduced in vivo thrombogenesis and defective activation of washed CD40L-deficient platelets, suggesting that membrane-bound CD40L is involved in the control of haemostasis acting as a platelet co-activator. In addition, we report the practically absence of CD40L in mouse and human endothelial cells, as well as the detection of an exon 3-deleted CD40L transcript in both platelets and leukocytes of mouse and human origin. Finally, compared with their corresponding littermate floxed controls, Cre+ mice carrying CD40-deficient leukocytes did not exhibit increased IgM levels, and reduction of IgA and IgG levels was statistically significant only in Tie2-Cre+ mice, suggesting that expression of CD40L in an earlier developmental step may be determinant in the regulation of the class switch recombination process.
Assuntos
Aterosclerose/genética , Ligante de CD40/genética , Camundongos Knockout/genética , Proteínas Recombinantes de Fusão/genética , Animais , Aterosclerose/patologia , Aterosclerose/terapia , Plaquetas/citologia , Plaquetas/metabolismo , Ligante de CD40/imunologia , Ligante de CD40/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Imunoglobulina A/genética , Imunoglobulina A/imunologia , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Integrases/genética , Leucócitos/metabolismo , Megacariócitos/imunologia , Megacariócitos/metabolismo , Camundongos , Camundongos Knockout/crescimento & desenvolvimento , Proteínas Recombinantes de Fusão/metabolismoRESUMO
We have generated mouse transgenic lineages for C3G (tgC3G) and C3GΔCat (tgC3GΔCat, C3G mutant lacking the GEF domain), where the transgenes are expressed under the control of the megakaryocyte and platelet specific PF4 (platelet factor 4) gene promoter. Transgenic platelet activity has been analyzed through in vivo and in vitro approaches, including bleeding time, aggregation assays and flow cytometry. Both transgenes are expressed (RNA and protein) in purified platelets and megakaryocytes and do not modify the number of platelets in peripheral blood. Transgenic C3G animals showed bleeding times significantly shorter than control animals, while tgC3GΔCat mice presented a remarkable bleeding diathesis as compared to their control siblings. Accordingly, platelets from tgC3G mice showed stronger activation in response to platelet agonists such as thrombin, PMA, ADP or collagen than control platelets, while those from tgC3GΔCat animals had a lower response. In addition, we present data indicating that C3G is a mediator in the PKC pathway leading to Rap1 activation. Remarkably, a significant percentage of tgC3G mice presented a higher level of neutrophils than their control siblings. These results indicate that C3G plays an important role in platelet clotting through a mechanism involving its GEF activity and suggest that it might be also involved in neutrophil development.
Assuntos
Plaquetas/metabolismo , Fator 2 de Liberação do Nucleotídeo Guanina/genética , Ativação Plaquetária , Animais , Plaquetas/efeitos dos fármacos , Plaquetas/fisiologia , Células Cultivadas , Ativação Enzimática , Feminino , Engenharia Genética , Fator 2 de Liberação do Nucleotídeo Guanina/biossíntese , Humanos , Contagem de Leucócitos , Masculino , Megacariócitos/metabolismo , Megacariócitos/fisiologia , Camundongos , Camundongos Transgênicos , Neutrófilos/fisiologia , Contagem de Plaquetas , Fator Plaquetário 4/genética , Regiões Promotoras Genéticas , Proteína Quinase C/metabolismo , Transdução de Sinais , Acetato de Tetradecanoilforbol/farmacologia , Proteínas rap1 de Ligação ao GTP/metabolismoRESUMO
Podocalyxin (PODXL) is a type I membrane sialomucin, originally described in the epithelial cells (podocytes) of kidney glomeruli. PODXL is also found in extra-renal tissues and in certain aggressive tumors, but its precise pathophysiological role is unknown. Expression of PODXL in CHO cells enhances their adhesive, migratory and cell-cell interactive properties in a selectin and integrin-dependent manner. We aimed at defining the PODXL domains responsible for those cell responses. For this purpose we have analyzed the cell adhesion/migration responses to deletion mutants of human PODXL, and the correlation with the activities of Rac1 and Cdc42 GTPases. The results obtained indicate that integrity of the PODXL ectodomain is essential for enhancing cell adhesion but not migration, while the integrity of the cytoplasmic domain is required for both adhesion and migration. Deletion of the carboxy-terminal DTHL domain (PODXL-ΔDTHL) limited only cell adhesion. The activities of Rac1 and Cdc42 GTPases parallel the PODXL-induced variations in cell adhesion and migration. Moreover, silencing the rac1 gene virtually abolished the effect of PODXL in enhancing cell adhesion.
Assuntos
Adesão Celular , Movimento Celular , Sialoglicoproteínas/fisiologia , Proteína cdc42 de Ligação ao GTP/fisiologia , Proteínas rac1 de Ligação ao GTP/fisiologia , Animais , Bioensaio , Células CHO , Cricetinae , Inativação Gênica , Humanos , Deleção de Sequência , Sialoglicoproteínas/genética , Cicatrização , Proteínas rac1 de Ligação ao GTP/genéticaRESUMO
Podocalyxin (Podxl) is a type I membrane mucin-protein of the CD34 family abundantly expressed in kidney epithelial cells (podocytes) where it plays a crucial functional role. Podxl is also expressed in tissues other than kidney, like in brain, but its function is ignored. To investigate the functional role of podocalyxin (Podxl) in brain we produced the specific brain-ablation of the Podxl gen in mice by crossing Podxl(floxed/floxed) mice, generated in our laboratory, to mice with pan-neural expression of recombinase Cre (Cre3). Podxl(-/-) mice show no apparent behavioral phenotype but their brains showed enlargement of ventricular volumes detected in vivo by MR imaging. The pattern of brain vasculature was of normal appearance but the thickness of the main carotid artery was significantly increased. Moreover, the histological analysis showed increased number of choroidal capillaries lining the ventricular spaces. These findings are analyzed in the light of the role likely played by podocalyxin in cell migration and cell-cell recognition during brain development and also on the consistent findings of increased ventricular spaces in human pathological disorders like schizophrenia.
Assuntos
Ventrículos Cerebrais , Sialoglicoproteínas/genética , Animais , Encéfalo/anatomia & histologia , Encéfalo/metabolismo , Encéfalo/patologia , Ventrículos Cerebrais/anatomia & histologia , Ventrículos Cerebrais/patologia , Circulação Cerebrovascular , Feminino , Técnicas de Silenciamento de Genes , Humanos , Rim/citologia , Rim/metabolismo , Imageamento por Ressonância Magnética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Fluxo Sanguíneo Regional , Sialoglicoproteínas/metabolismoRESUMO
Current microtechnologies have shown plenty of room inside a living cell for silicon chips. Microchips as barcodes, biochemical sensors, mechanical sensors and even electrical devices have been internalized into living cells without interfering their cell viability. However, these technologies lack from the ability to trap and preconcentrate cells in a specific region, which are prerequisites for cell separation, purification and posterior studies with enhanced sensitivity. Magnetic manipulation of microobjects, which allows a non-contacting method, has become an attractive and promising technique at small scales. Here, we show intracellular Ni-based chips with magnetic capabilities to allow cell enrichment. As a proof of concept of the potential to integrate multiple functionalities on a single device of this technique, we combine coding and magnetic manipulation capabilities in a single device. Devices were found to be internalized by HeLa cells without interfering in their viability. We demonstrated the tagging of a subpopulation of cells and their subsequent magnetic trapping with internalized barcodes subjected to a force up to 2.57 pN (for magnet-cells distance of 4.9 mm). The work opens the venue for future intracellular chips that integrate multiple functionalities with the magnetic manipulation of cells.
RESUMO
BACKGROUND: Support for the role of transmembrane and membrane-proximal domains of alpha IIb beta 3 integrin in the maintenance of receptor low affinity comes from mutational studies showing that activating mutations can induce constitutive bi-directional transmembrane signaling. DESIGN AND METHODS: We report the functional characterization of a mutant alpha IIb beta 3 integrin carrying the Leu718Pro mutation in the membrane-proximal region of the beta 3 cytoplasmic domain, identified in heterozygosis in a patient with a severe bleeding phenotype and defective platelet aggregation and adhesion. RESULTS: Transiently transfected cells expressed similar levels of normal and mutant alpha IIb beta 3, but surface expression of mutant alpha v beta 3 was reduced due to its retention in intracellular compartments. Cells stably expressing mutant alpha IIb beta 3 showed constitutive binding to soluble multivalent ligands as well as spontaneous fibrinogen-dependent aggregation, but their response to DTT was markedly reduced. Fibrinogen-adherent cells exhibited a peculiar spreading phenotype with long protrusions. Immunofluorescence analysis revealed the formation of alpha IIb beta 3 clusters underneath the entire cell body and the presence of atypical high-density patches of clustered alpha IIb beta 3 containing encircled areas devoid of integrin that showed decreased affinity for the fluorescent lipid analog DiIC(16) and were disrupted in cholesterol-depleted cells. CONCLUSIONS: These findings are consistent with an important role of the membrane-proximal region of beta 3 in modulating alpha IIb beta 3 clustering and lateral redistribution of membrane lipids. Since the beta 3 mutant was associated with a thrombasthenic phenotype in a patient carrying one normal beta 3 allele, these results support a dominant role of clustering in regulating integrin alpha IIb beta 3 functions in vivo.
Assuntos
Integrina beta3/genética , Microdomínios da Membrana/metabolismo , Mutação de Sentido Incorreto , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Trombastenia/patologia , Adulto , Membrana Celular/química , Células Cultivadas , Feminino , Humanos , Lipídeos de Membrana , Trombastenia/genéticaRESUMO
Megakaryocytic differentiation of myelogenous leukemia cell lines induced by a number of chemical compounds mimics, in part, the physiological process that takes place in the bone marrow in response to a variety of stimuli. We have investigated the involvement of mitogen-activated protein kinases (MAPKs) [extracellular signal-regulated protein kinase (ERK1/2) and p38] and phosphoinositide 3-kinase (PI3K) signaling pathways in the differentiated phenotypes of K562 cells promoted by phorbol 12-myristate 13-acetate, staurosporine (STA), and the p38 MAPK inhibitor SB202190. In our experimental conditions, only STA-treated cells showed the phenotype of mature megakaryocytes (MKs) including GPIbalpha expression, DNA endoreduplication, and formation of platelet-like structures. We provide evidence supporting that basal activity, but not sustained activation, of ERK1/2 is required for expression of MK surface markers. Moreover, ERK1/2 signaling is not involved in cell endomitosis. The PI3K pathway exerts dual regulatory effects on K562 cell differentiation: it is intimately connected with ERK1/2 cascade to stimulate expression of surface markers and it is also necessary, but not sufficient, for polyploidization. Finally, apoptosis and megakaryocytic differentiation exhibit different sensitivity to p38 down-regulation: it is required for expression of early specific markers but is not involved in cell apoptosis. The present work with K562 cells provides new insights into the molecular mechanisms regulating MK differentiation. The results indicate that a precise orchestration of signals, including ERK1/2 and p38 MAPKs as well as PI3K pathway, is necessary for acquisition of features of mature MKs.
Assuntos
Diferenciação Celular/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Megacariócitos/citologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Citometria de Fluxo , Humanos , Imidazóis/farmacologia , Células K562 , Megacariócitos/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/fisiologia , Inibidores de Fosfoinositídeo-3 Quinase , Poliploidia , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Transdução de Sinais , Acetato de Tetradecanoilforbol/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologiaRESUMO
The highly sialoglycosylated extracellular domain of podocalyxin (Podxl) is a constituent of the endothelial glycocalyx of most blood vessels but it is unknown if Podxl plays a prominent role in the function of the glycocalyx as a regulator of leukocyte-endothelial adhesion. We have recently found that mice lacking Podxl in the vascular endothelium develop histological lesions compatible with severe vasculitis resulting in organ failure and premature death. In this work, we show that these mice have an increased quantity of resident leukocytes within the peritoneal cavity in both basal and inflammatory conditions. Adhesion of macrophagic cells to lung endothelial cells from Podxl-deficient mice was increased under inflammatory stimuli. Both, chemokine binding and chemokine-mediated adhesion of immune cells were significantly higher in Podxl-deficient endothelial cells. Moreover, glycocalyx function assessed by measuring the anticoagulant capacity of endothelial cell monolayers to inactivate thrombin was significantly altered in the absence of Podxl. Overall, the results suggest that Podxl is an essential component of the glycocalyx and has an important so far unknown role in preventing leukocyte-endothelial cell adhesion under resting and inflammatory conditions.
Assuntos
Comunicação Celular , Endotélio Vascular/fisiologia , Leucócitos/fisiologia , Pulmão/irrigação sanguínea , Sialoglicoproteínas/fisiologia , Animais , Adesão Celular , Células Cultivadas , Quimiocinas/metabolismo , Endotélio Vascular/citologia , Humanos , Leucócitos/citologia , Pulmão/metabolismo , Camundongos , Camundongos Knockout , Transdução de SinaisRESUMO
Glanzmann thrombasthenia is a rare bleeding disorder that can present life-threatening bleeding. Our patients develop antiplatelet antibodies that become refractory to any pharmacological treatment. Allogeneic hematopoietic stem-cell transplantation is the only currently curative procedure, but has major risks mainly in adult; indeed, our patient died.
RESUMO
BACKGROUND: Podocalyxin (podxl) is a heavily glycosylated transmembrane protein mainly found on the apical membrane of rat podocytes and also in endothelial, hematopoietic, and tumor cells. Despite of its interest no much is known about the transcriptional regulation of podxl in different cells. Thus, we aimed at studying the functional features of the 5'-regulatory region of the human Podxl gene. RESULTS: The promoter region of the human Podxl gene has been cloned and its structure and function were analyzed. The primary DNA sequence is rich in G+C and is devoid of TATA or CAAT boxes. The sequence contains recognition sites for several putative transcription factors; however, the basic promoter activity seems to rely entirely on Sp1 transcription factor since supershift analysis was positive only for this factor. The region encompassed by 66 to -111 nts conferred the minimal transcriptional activity that increases as the number of Sp1 sites augmented with the length of the promoter fragment. In Sp1-lacking insect cells the Podxl promoter constructs showed activity only if cotransfected with an Sp1 expression plasmid. Finally, mutation of the Sp1 sites reduced the promoter activity. We analyzed whether methylation of the CpG dinucleotides present in the first approximately 600 nts of the promoter region of Podxl could explain the variable rates of expression in different types of cells. Inactivation of methyltransferases by 5'-aza-2'deoxicitidine showed a dose-dependent increase in the podxl content. Moreover, in vitro methylation of the promoter constructs -111,-181 and -210 led to an almost complete reduction of the promoter activity. A correlation was found between the degree of methylation of the CpG promoter dinucleotides and the rate of podxl expression in different cell lines. CONCLUSION: Our results indicate that transcriptional regulation of Podxl is supported primarily by Sp1 site(s) and that DNA-methylation of the CpG promoter islands contributes to control the tissue specific expression of podxl.
Assuntos
Ilhas de CpG , Metilação de DNA , Regulação da Expressão Gênica , Regiões Promotoras Genéticas , Sialoglicoproteínas/genética , Fator de Transcrição Sp1/fisiologia , Região 5'-Flanqueadora , Animais , Sequência de Bases , Linhagem Celular , Drosophila/citologia , Humanos , Dados de Sequência Molecular , Sialoglicoproteínas/metabolismoRESUMO
BACKGROUND AND OBJECTIVES: Glanzmann thrombasthenia is an autosomal recessive bleeding disorder characterized by a life-long hemorrhagic tendency and absent or severely reduced platelet aggregation in response to agonists, caused by quantitative or qualitative abnormalities in the platelet fibrinogen receptor, integrin alphaIIb beta3. The aim of this study was to identify the molecular genetic defect and determine its functional consequences in a patient with type II Glanzmann thrombasthenia. DESIGN AND METHODS: The expression of platelet alphaIIb beta3 was determined by flow cytometry and western blotting. Mutations were identified by sequencing both cDNA and genomic DNA. Functional characterization was assessed by exontrap and transient transfection analysis. RESULTS: Flow cytometry and western blot analysis revealed markedly reduced levels of platelet alphaIIb beta3, which may account for the residual fibrinogen binding detected upon platelet activation. Sequencing of genomic DNA revealed the presence of two mutations in the alphaIIb gene: a C1750T transition in the last codon of exon 17 changing Arg553 to STOP, and a C2829T transition in exon 27 that changes Pro912 to Leu. Sequence analysis of reversely transcribed alphaIIb mRNA did not detect cDNA from the C1750T mutant allele, and revealed a significant increase of the physiological splicing out of exon 28 in the cDNA carrying the C2829T mutation. Transient expression of [912Leu]alphaIIb in CHO-b3 cells showed a marked reduction in the rate of surface expression of alphaIIb beta3. INTERPRETATION AND CONCLUSIONS: The results suggest that the thrombasthenic phenotype is the result of reduced availability of alphaIIb-mRNA, enhanced expression of exon 28-deleted transcripts, and defective processing of [912Leu]alphaIIb.
Assuntos
Éxons/genética , Triagem de Portadores Genéticos , Mutação de Sentido Incorreto , Glicoproteína IIb da Membrana de Plaquetas/genética , Trombastenia/genética , Animais , Células CHO , Pré-Escolar , Cricetinae , Feminino , Humanos , Trombastenia/classificaçãoRESUMO
Podocalyxin (Podxl) has an essential role in the development and function of the kidney glomerular filtration barrier. It is also expressed by vascular endothelia but perinatal lethality of podxl(-/-) mice has precluded understanding of its function in adult vascular endothelial cells (ECs). In this work, we show that conditional knockout mice with deletion of Podxl restricted to the vascular endothelium grow normally but most die spontaneously around three months of age. Histological analysis showed a nonspecific inflammatory infiltrate within the vessel wall frequently associated with degenerative changes, and involving vessels of different caliber in one or more organs. Podxl-deficient lung EC cultures exhibit increased permeability to dextran and macrophage transmigration. After thrombin stimulation, ECs lacking Podxl showed delayed recovery of VE-cadherin cell contacts, persistence of F-actin stress fibers, and sustained phosphorylation of the ERM complex and activation of RhoA, suggesting a failure in endothelial barrier stabilization. The results suggest that Podxl has an essential role in the regulation of endothelial permeability by influencing the mechanisms involved in the restoration of endothelial barrier integrity after injury.
Assuntos
Células Endoteliais/metabolismo , Sialoglicoproteínas/metabolismo , Animais , Permeabilidade Capilar , Genótipo , Inflamação , Camundongos , Camundongos KnockoutRESUMO
We report a novel genetic defect in a patient with type I Glanzmann thrombasthenia. Flow cytometry analysis revealed undetectable levels of platelet glycoproteins alphaIIb and beta3, although residual amounts of both proteins were detectable in immunoblotting analysis. Sequence analysis of reversely transcribed platelet beta3 mRNA showed a 100-base pair deletion in the 3'-boundary of exon 11, that results in a frame shift and appearance of a premature STOP codon. Analysis of the corresponding genomic DNA fragment revealed the presence of a homozygous C1815T transition in exon 11. The mutation does not change the amino acid residue but it creates an ectopic consensus splice donor site that is used preferentially, causing splicing out of part of exon 11. The parents of the proband, heterozygous for this mutation, were asymptomatic and had reduced platelet content of alphaIIbbeta3. PCR-based relative quantification of beta3 mRNA failed to detect the mutant transcript in the parents and showed a marked reduction in the patient. The results suggest that the thrombasthenic phenotype is, mainly, the result of the reduced availability of beta3-mRNA, most probably due to activation of the nonsense-mediated mRNA decay mechanism. They also show the convenience of analyzing both genomic DNA and mRNA, in order to ascertain the functional consequences of single nucleotide substitutions.
Assuntos
Integrina beta3/genética , Mutação , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética , Trombastenia/etiologia , Trombastenia/genética , Alelos , Processamento Alternativo , Animais , Anticorpos Monoclonais/química , Plaquetas/metabolismo , Células CHO , Pré-Escolar , Códon sem Sentido , Códon de Terminação , Cricetinae , DNA/genética , Éxons , Saúde da Família , Pai , Feminino , Fibrinogênio/química , Citometria de Fluxo , Mutação da Fase de Leitura , Técnicas Genéticas , Homozigoto , Humanos , Imunoprecipitação , Masculino , Camundongos , Modelos Genéticos , Mães , Fenótipo , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Análise de Sequência de DNARESUMO
This work reports the functional studies of CHO cells coexpressing alpha-adrenergic (alphaAR) and human fibrinogen (Fg) receptors (integrin alphaIIbbeta3). Stimulation of these cells with alpha-agonists produced a transient rise in the free cytosolic calcium (Ca(++)) accompanied by enhanced binding to soluble Fg, and these effects were prevented by specific alphaAR antagonists. The alpha-adrenergic-induced activation of alphaIIbbeta3 in CHO-alphaIIbbeta3-alphaAR increased the rate of adhesion and extension of cells onto Fg coated plates, and also induced a soluble Fg- and alphaIIbbeta3-dependent formation of cell aggregates, whereas no effects were observed by the stimulation of CHO-alphaIIbbeta3 cells. alpha-Adrenergic antagonists, the ligand mimetic peptide RGDS, pertussis toxin (PTX), or EDTA, they all prevented the alpha-adrenergic stimulation of adhesion and aggregation. However, inhibition of PKC prevented the alpha-adrenergic stimulation of cell adherence, whereas blocking the intracellular Ca(++) mobilization impeded the stimulation of cell aggregation. The alpha-adrenergic activation was associated with phosphorylation of a protein of approximately 100 kDa and proteins of the MAPK family. The former was selectively phosphorylated by alpha-adrenergic stimulation whereas the latter were phosphorylated by the binding of cells to Fg and markedly intensified by alpha-adrenergic stimulation.
Assuntos
Complexo Glicoproteico GPIIb-IIIa de Plaquetas/química , Receptores Adrenérgicos alfa/metabolismo , Actinas/química , Animais , Anticorpos Monoclonais/química , Células CHO , Cálcio/metabolismo , Adesão Celular , Cricetinae , Meios de Cultura Livres de Soro/farmacologia , Citosol/metabolismo , Relação Dose-Resposta a Droga , Ácido Edético/farmacologia , Fibrinogênio/química , Citometria de Fluxo , Regulação da Expressão Gênica , Ligantes , Microscopia de Fluorescência , Oligopeptídeos/química , Peptídeos/química , Toxina Pertussis/farmacologia , Fosforilação , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Ligação Proteica , Proteína Quinase C/metabolismoAssuntos
Transtornos Herdados da Coagulação Sanguínea/genética , Fibrinogênio/genética , Fibrinogênios Anormais/genética , Hemorragia/genética , Mutação , Trombose/genética , Arteriopatias Oclusivas/sangue , Arteriopatias Oclusivas/genética , Transtornos Herdados da Coagulação Sanguínea/sangue , Testes de Coagulação Sanguínea , Feminino , Fibrinogênio/metabolismo , Testes Genéticos , Traumatismos da Mão/complicações , Hemorragia/sangue , Hemorragia/terapia , Heterozigoto , Humanos , Menorragia/genética , Pessoa de Meia-Idade , Peso Molecular , Linhagem , Fenótipo , Hemorragia Pós-Parto/genética , Trombose/sangue , Trombose/terapiaRESUMO
This work aimed at elucidating the molecular genetic defect in two related patients with Bernard-Soulier syndrome (BSS) phenotype. Flow cytometric analysis revealed undetectable levels of platelet glycoproteins (GP), Ibalpha and IX, although plasma glycocalicin was detectable in both cases. The complete sequencing of GPIbalpha, GPIbbeta, and GPIX revealed the presence of a single point mutation, a G to A substitution, in codon 30 of GPIbbeta, that changes Cys5 to Tyr. The parents and sibling of the patients, heterozygotes for this mutation, were asymptomatic and they all showed a reduced platelet content of GPIbalpha and GPIX. Transient transfection of the mutant GPIbalpha subunit failed to render surface expression of GPIbalpha and exerted a dominant-negative effect on the surface exposure of the GPIb-IX complex. Metabolic labelling and immunoprecipitation analysis of transfected cells indicated that [5Tyr]GPIbbeta may associate with GPIX and GPIbalpha, but the maturation of the GPIb-IX complex is impaired. Substitution of either Cys5 or Cys7 by Ala failed to show surface expression of GPIb-IX, suggesting that the Cys5- Cys7 disulfide loop in GPIbbeta is essential for the efficient processing and trafficking of GPIb-IX complexes toward the plasma membrane. Our findings indicate that the identified novel GPIbbeta mutation is responsible for the BSS phenotype of the patients and provide an explanation for the molecular mechanism underlying the reduced platelet content of GPIb-IX complex in the heterozygous individuals.
Assuntos
Síndrome de Bernard-Soulier/genética , Cistina , Complexo Glicoproteico GPIb-IX de Plaquetas/química , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Adulto , Dissulfetos , Saúde da Família , Feminino , Homozigoto , Humanos , Masculino , Complexo Glicoproteico GPIb-IX de Plaquetas/genética , Mutação Puntual , Dobramento de Proteína , Estrutura Terciária de Proteína/genética , Transporte Proteico , IrmãosRESUMO
We report the molecular genetic analysis of a patient with thrombasthenic phenotype. The lack of surface platelet GPIIb-IIIa complexes and the presence of GPIIIa suggested it was a case of type I Glanzmann's thrombasthenia due to a mutation in GPIIb. Single stranded conformational polymorphism analysis (SSCP) of exon-19 of GPIIb showed polymorphic DNA bands. The DNA sequence of exon-19 revealed the presence of a homozygous C1882T transition that changes residue R597 to STOP codon. Since no other mutations were found in either GPIIb or GPIIIa it is concluded that the C1882T substitution in GPIIb is responsible for the thrombasthenic phenotype of the patient. The lack of platelet GPIIb-mRNA in the proband indicates instability of the [C1882T]GPIIb-mRNA. Coexpression of normal GPIIIa and GPIIb(delta597-1008) in CHO cells failed to show surface expression of GPIIb(delta597-1008)-IIIa complexes. Immunoprecipitation analysis demonstrated that GPIIb(delta597-1008) may indeed complex GPIIIa; however, the association is either unstable or incapable of progressing along the secretory pathway.
Assuntos
Códon sem Sentido , Transtornos Hemorrágicos/genética , Integrina beta3/metabolismo , Glicoproteína IIb da Membrana de Plaquetas/genética , Deleção de Sequência , Trombastenia/genética , Animais , Células CHO , Criança , Códon de Terminação/genética , Cricetinae , Cricetulus , Análise Mutacional de DNA , Éxons/genética , Humanos , Masculino , Linhagem , Fenótipo , Glicoproteína IIb da Membrana de Plaquetas/química , Glicoproteína IIb da Membrana de Plaquetas/metabolismo , Polimorfismo Conformacional de Fita Simples , Testes de Precipitina , Mapeamento de Interação de Proteínas , RNA Mensageiro/genética , Proteínas Recombinantes de Fusão/genética , TransfecçãoRESUMO
We report the analysis of a variant case of thrombasthenic phenotype that is a compound heterozygote for two mutations located within the metal ion dependent adhesion site (MIDAS) of the beta3 subunit. The patient inherited a maternal allele carrying the Met124Val substitution and a paternal allele that changes Asp119 to Tyr. Phenotyping of the human platelet antigen 1 (HPA-1) showed that the platelet alphaIIbbeta3 complex in the patient was mostly accounted for by the Asp 119Tyr allele that does not bind to fibrinogen (Fg). The patient showed agonistinduced binding of platelets to Fg but neither binding to PAC-1 nor cell aggregation could be detected, most likely due to the minute expression (< or = 5%) of alphaIIb(124Val)beta3 receptors. CHO cells expressing (124Val)beta3 showed a diminished surface expression of alphaIIbbeta3, enhanced adhesion to immobilized Fg, and spontaneous aggregation in the presence of soluble Fg, suggesting that (124Val)beta3 may confer constitutive activity to the alphaIIb(124Val)beta3 receptors. A distinct feature of these cells is the failure of DTT to enhance the binding to soluble Fg and the formation of cell aggregates. The substitution of (124Met)beta3 by either a polar or a positively charged amino acid restored the surface exposure and function of the alphaIIbbeta3 receptors whereas a negatively charged residue did not.