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1.
J Thromb Haemost ; 16(1): 44-53, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29117459

RESUMO

Essentials Three dominant variants for the autosomal recessive bleeding disorder type-8 have been described. To date, there has been no phenotype/genotype correlation explaining their dominant transmission. Proline plays an important role in P2Y12R ligand binding and signaling defects. P2Y12R homodimer formation is critical for the receptor function and signaling. SUMMARY: Background Although inherited platelet disorders are still underdiagnosed worldwide, advances in molecular techniques are improving disease diagnosis and patient management. Objective To identify and characterize the mechanism underlying the bleeding phenotype in a Caucasian family with an autosomal dominant P2RY12 variant. Methods Full blood counts, platelet aggregometry, flow cytometry and western blotting were performed before next-generation sequencing (NGS). Detailed molecular analysis of the identified variant of the P2Y12 receptor (P2Y12R) was subsequently performed in mammalian cells overexpressing receptor constructs. Results All three referred individuals had markedly impaired ADP-induced platelet aggregation with primary wave only, despite normal total and surface P2Y12R expression. By NGS, a single P2RY12:c.G794C substitution (p.R265P) was identified in all affected individuals, and this was confirmed by Sanger sequencing. Mammalian cell experiments with the R265P-P2Y12R variant showed normal receptor surface expression versus wild-type (WT) P2Y12R. Agonist-stimulated R265P-P2Y12R function (both signaling and surface receptor loss) was reduced versus WT P2Y12R. Critically, R265P-P2Y12R acted in a dominant negative manner, with agonist-stimulated WT P2Y12R activity being reduced by variant coexpression, suggesting dramatic loss of WT homodimers. Importantly, platelet P2RY12 cDNA cloning and sequencing in two affected individuals also revealed three-fold mutant mRNA overexpression, decreasing even further the likelihood of WT homodimer formation. R265 located within extracellular loop 3 (EL3) is one of four residues that are important for receptor functional integrity, maintaining the binding pocket conformation and allowing rotation following ligand binding. Conclusion This novel dominant negative variant confirms the important role of R265 in EL3 in the functional integrity of P2Y12R, and suggests that pathologic heterodimer formation may underlie this family bleeding phenotype.


Assuntos
Transtornos Plaquetários/genética , Hemorragia/genética , Mutação , Receptores Purinérgicos P2Y12/genética , Adolescente , Transtornos Plaquetários/sangue , Transtornos Plaquetários/diagnóstico , Análise Mutacional de DNA/métodos , Feminino , Predisposição Genética para Doença , Células HEK293 , Hemorragia/sangue , Hemorragia/diagnóstico , Hereditariedade , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Linhagem , Fenótipo , Agregação Plaquetária/genética , Testes de Função Plaquetária , Prolina , Multimerização Proteica , Estrutura Quaternária de Proteína , Receptores Purinérgicos P2Y12/sangue , Receptores Purinérgicos P2Y12/química , Índice de Gravidade de Doença , Relação Estrutura-Atividade , População Branca/genética , Adulto Jovem
2.
Int J Lab Hematol ; 40(2): 187-195, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29143464

RESUMO

INTRODUCTION: Mean platelet volume (MPV) assists the differential diagnosis of inherited thrombocytopenia (IT) but lacks standardisation and varies between automated analysers. Classification of IT based on mean platelet diameter (MPD) has been proposed by an international collaborative study but has not been validated. METHODS: To assess the applicability of MPD to classify forms of IT, digital images of blood films from patients with established genetic causes for IT were generated, and the MPD measured (ZEISS Axio-scanner and Image J software) by a blinded reviewer. Comparison was made to the proposed classification system. RESULTS: Mean platelet volume was measured in thrombocytopenia with different genetic aetiologies, bilallelic BSS (bBSS) (n = 1), monoallelic BSS (mBSS) (n = 2), MYH9-related disorders (MYH9-RD) (n = 11), GFI1B-related thrombocytopenia (RT) (n = 15), FLI1-RT (n = 2), TUBB1-RT (n = 3), ITGA2B/ITGB3-RT (n = 1), RUNX1-RT (n = 2) and controls (n = 54). bBSS and 82% of MYH9-RD samples had MPD >4 µm which correlated with "IT with giant platelets." Only 55% of samples expected in the "large platelet group" had MPD meeting the classification cut-off (MPD >3.2 µm). FLI1-RT MPD were significantly larger than expected whilst ITGA2B/ITGB3-RT MPD were smaller than proposed. MPD in FPD/AML were "normal." CONCLUSION: Platelet MPD measurements are a useful guide to classify IT, but the time taken to record measurements may limit clinical applicability.


Assuntos
Plaquetas/patologia , Trombocitopenia/classificação , Transtornos Herdados da Coagulação Sanguínea/diagnóstico , Citodiagnóstico/métodos , Diagnóstico Diferencial , Humanos , Volume Plaquetário Médio , Trombocitopenia/congênito , Trombocitopenia/genética
3.
J Thromb Haemost ; 15(11): 2245-2258, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28880435

RESUMO

Essentials The phenotypes of different growth factor-independent 1B (GFI1B) variants are not established. GFI1B variants produce heterogeneous clinical phenotypes dependent on the site of mutation. Mutation of the first non-DNA-binding zinc-finger causes a mild platelet and clinical phenotype. GFI1B regulates the CD34 promoter; platelet CD34 expression is an indicator of GFI1B mutation. SUMMARY: Background Mutation of the growth factor-independent 1B (GFI1B) fifth DNA-binding zinc-finger domain causes macrothrombocytopenia and α-granule deficiency leading to clinical bleeding. The phenotypes associated with GFI1B variants disrupting non-DNA-binding zinc-fingers remain uncharacterized. Objectives To determine the functional and phenotypic consequences of GFI1B variants disrupting non-DNA-binding zinc-finger domains. Methods The GFI1B C168F variant and a novel GFI1B c.2520 + 1_2520 + 8delGTGGGCAC splice variant were identified in four unrelated families. Phenotypic features, DNA-binding properties and transcriptional effects were determined and compared with those in individuals with a GFI1B H294 fs mutation of the fifth DNA-binding zinc-finger. Patient-specific induced pluripotent stem cell (iPSC)-derived megakaryocytes were generated to facilitate disease modeling. Results The DNA-binding GFI1B variant C168F, which is predicted to disrupt the first non-DNA-binding zinc-finger domain, is associated with macrothrombocytopenia without α-granule deficiency or bleeding symptoms. A GFI1B splice variant, c.2520 + 1_2520 + 8delGTGGGCAC, which generates a short GFI1B isoform that lacks non-DNA-binding zinc-fingers 1 and 2, is associated with increased platelet CD34 expression only, without quantitative or morphologic platelet abnormalities. GFI1B represses the CD34 promoter, and this repression is attenuated by different GFI1B zinc-finger mutations, suggesting that deregulation of CD34 expression occurs at a direct transcriptional level. Patient-specific iPSC-derived megakaryocytes phenocopy these observations. Conclusions Disruption of GFI1B non-DNA-binding zinc-finger 1 is associated with mild to moderate thrombocytopenia without α-granule deficiency or bleeding symptomatology, indicating that the site of GFI1B mutation has important phenotypic implications. Platelet CD34 expression appears to be a common feature of perturbed GFI1B function, and may have diagnostic utility.


Assuntos
Antígenos CD34/genética , Grânulos Citoplasmáticos/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Megacariócitos/metabolismo , Mutação , Proteínas Proto-Oncogênicas/genética , Proteínas Repressoras/genética , Trombocitopenia/sangue , Trombocitopenia/genética , Dedos de Zinco/genética , Antígenos CD34/sangue , Células Cultivadas , Regulação da Expressão Gênica , Predisposição Genética para Doença , Hereditariedade , Heterozigoto , Humanos , Linhagem , Fenótipo , Regiões Promotoras Genéticas , Trombocitopenia/diagnóstico , Transcrição Gênica
6.
J Thromb Haemost ; 11(11): 2039-47, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23927492

RESUMO

BACKGROUND: GFI1B is a transcription factor important for erythropoiesis and megakaryocyte development but previously unknown to be associated with human disease. METHODS: A family with a novel bleeding disorder was identified and characterized. Genetic linkage analysis and massively parallel sequencing were used to localize the mutation causing the disease phenotype on chromosome 9. Functional studies were then performed in megakaryocytic cell lines to determine the biological effects of the mutant transcript. RESULTS: We have identified a family with an autosomal dominant bleeding disorder associated with macrothrombocytopenia, red cell anisopoikilocytosis, and platelet dysfunction. The severity of bleeding is variable with some affected individuals experiencing spontaneous bleeding while other family members exhibit only abnormal bleeding with surgery. A single nucleotide insertion was identified in GFI1B that predicts a frameshift mutation in the fifth zinc finger DNA-binding domain. This mutation alters the transcriptional activity of the protein, resulting in a reduction in platelet α-granule content and aberrant expression of key platelet proteins. CONCLUSIONS: GFI1B mutation represents a novel human bleeding disorder, and the described phenotype identifies GFI1B as a critical regulator of platelet shape, number, and function.


Assuntos
Transtornos Plaquetários/genética , Plaquetas/patologia , Mutação , Proteínas Proto-Oncogênicas/genética , Proteínas Repressoras/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Plaquetas/metabolismo , Eritrócitos/citologia , Feminino , Mutação da Fase de Leitura , Ligação Genética , Humanos , Masculino , Megacariócitos/citologia , Pessoa de Meia-Idade , Fenótipo , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA , Transfecção , Adulto Jovem
8.
Int J Lab Hematol ; 33(3): 245-50, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21118381

RESUMO

BACKGROUND: Heparin-induced thrombocytopenia (HIT) is a rare complication of heparin therapy resulting from antibody production to platelet factor 4 and heparin complexes (H-PF4). METHODS: We have evaluated four enzyme-linked immunosorbent assay (ELISA)-based screening tests to identify the best assay(s) with the highest specificity but without underdiagnosis of HIT. As functional assays are difficult to perform, ELISAs are useful to provide clinicians with a timely answer. Over a 10-month period, all samples (N=107) referred to our laboratory were tested for HIT antibodies using four commercially available ELISA kits, two detecting IgG/A/M anti-H-PF4 antibodies and the other two IgG specific. RESULTS: Twenty-eight samples were positive by at least one assay; IgGAM ELISAs were found to be more sensitive with 24 samples positive by Asserachrom IgGAM and 23 by Zymutest IgGAM. Only 18 samples were positive by GTI-PF4-IgG and Zymutest IgG. The gold standard serotonin release assay (SRA) was used as a confirmation assay, and 11/28 samples tested positive. All these SRA-positive samples were positive by all four assays. None of the IgGAM-only-positive samples was found to be positive by SRA suggesting a better specificity for the IgG-only assays. CONCLUSION: Our data strongly support the use of IgG-only assays for the detection of HIT antibodies.


Assuntos
Ensaio de Imunoadsorção Enzimática , Heparina/efeitos adversos , Isotipos de Imunoglobulinas/sangue , Trombocitopenia/induzido quimicamente , Trombocitopenia/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Serotonina/metabolismo
10.
Intern Med J ; 39(6): 370-8, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19460057

RESUMO

BACKGROUND: Increasing evidence supports the role of emotional stress in the onset of cardiovascular disease. Although bereavement is a major emotional stress with both acute and more long-term features, the mechanism of its association with cardiovascular risk is not well understood, in particular because of limited studies of acute bereavement. The aim of the study was to identify psychological and behavioural changes in acute bereavement and potential modifiers of these changes. METHODS: Bereaved (n= 62) and non-bereaved individuals (n= 50) were evaluated within 2 weeks and at 6 months following loss using the Centre for Epidemiologic Studies -- Depression, Spielberger State Anxiety and Anger, Social Support Questionnaire and changes in appetite, cigarette and alcohol consumption, cortisol and lipids. RESULTS: Compared with non-bereaved, acutely bereaved had increased symptoms of depression (26.7 +/- 1.7 vs 5.9 +/- 0.7, P < 0.001), anxiety (47.4 +/- 2.0 vs 28.2 +/- 1.4, P < 0.001) and anger (median 16.0 vs 15.0, P < 0.001). Greater depressive symptoms were associated with being unprepared for the death, decreased sleep duration and younger age. Acutely, bereaved slept less than non-bereaved (5.8 +/- 0.2 vs 7.2 +/- 0.2 h, P < 0.001). Reduced sleep time was associated with increased anger and depression and decreased satisfaction with social support. Compared with the non-bereaved, the acutely bereaved had higher cortisol (median 306 vs 266, P= 0.003), reduced appetite (P < 0.001) and lower total cholesterol (median 4.9 vs 5.4, P= 0.006) and low-density lipoprotein (median 2.4 vs 2.9, P < 0.001). CONCLUSION: These results offer insight into the psychological, behavioural and physical changes that may contribute to cardiovascular risk in bereavement.


Assuntos
Luto , Doenças Cardiovasculares/etiologia , Doenças Cardiovasculares/psicologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Doenças Cardiovasculares/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Fatores de Risco , Estresse Psicológico/sangue , Estresse Psicológico/complicações , Estresse Psicológico/psicologia , Fatores de Tempo
11.
J Thromb Haemost ; 7(4): 573-81, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19192119

RESUMO

BACKGROUND: Depression is associated with an increased risk of cardiovascular disease (CVD). Although the mechanism is uncertain, prothrombotic and inflammatory factors may play a role. OBJECTIVES: As platelets play a key role in CVD, we determined first, whether depressed individuals had more activated platelets than non-depressed individuals and second, whether treatment of depression reduced platelet activation levels. PATIENTS/METHODS: We recruited 108 depressed outpatients and 45 control subjects all without a history of CVD. After psychological assessment, the depressed patients were offered treatment with medication and/or psychotherapy. Flow cytometric markers of platelet activation and level of depression were assessed at baseline and at 4 weeks and 6 months after treatment. RESULTS: Depression was associated with increased platelet activation with a higher number of circulating CD62p (0.76x10(9) L(-1) vs. 0.46, P=0.019) and CD63 (P=0.05) positive platelets compared with controls. Patients with depression also had more circulating platelet-leukocyte aggregates than controls (P<0.001). There was a positive correlation between the severity of depression and the level of platelet activation. Platelets from depressed patients were also hyperreactive to adenosine 5 -diphosphate (ADP) stimulation with increased CD62p and CD63 exposure (P=0.003 and 0.019, respectively). Six months of treatment resulted in a reduced number of circulating CD62p and CD63 positive platelets (29.84% and 53.38% decrease) and a 20.9% reduction in CD63 exposure after ADP activation. CONCLUSIONS: Depression is associated with increased in vivo platelet activation and resolution of depression using psychotherapy and/or medication reduces platelet activation. These findings provide insights into the link between depression and cardiovascular risk.


Assuntos
Depressão/sangue , Depressão/terapia , Ativação Plaquetária , Adulto , Antidepressivos/farmacologia , Antidepressivos/uso terapêutico , Antígenos CD/análise , Doenças Cardiovasculares/etiologia , Estudos de Casos e Controles , Depressão/complicações , Feminino , Citometria de Fluxo , Humanos , Masculino , Pessoa de Meia-Idade , Selectina-P/análise , Ativação Plaquetária/efeitos dos fármacos , Glicoproteínas da Membrana de Plaquetas/análise , Psicoterapia , Tetraspanina 30
12.
Intern Med J ; 37(6): 409-11, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17535386

RESUMO

Inherited deficiency of protein S (PS) is a rare but accepted risk factor for venous thromboembolism. There is accumulating evidence that inherited PS deficiency may be associated with a variety of adverse obstetric events. Acquired PS deficiency may be caused by a variety of clinical states including normal pregnancy. We conducted a retrospective audit of the results of screening for PS deficiency through our reference laboratory. The majority of patients in this audit with significantly reduced (<50%) free functional PS levels had a major confounding factor likely to cause acquired PS deficiency, most frequently pregnancy. Recommendations for PS testing for the diagnosis of hereditary PS deficiency include deferring testing until at least 40 days post-partum. It appears that these recommendations are not being adhered to leading to difficulty in the interpretation of results.


Assuntos
Técnicas de Laboratório Clínico , Deficiência de Proteína S/diagnóstico , Feminino , Humanos , Gravidez , Estudos Retrospectivos
13.
J Thromb Haemost ; 5(3): 528-34, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17166248

RESUMO

BACKGROUND: Routinely available coagulation assays are not capable of detecting clinically defined hypercoagulable states. A number of global coagulation assays have been developed with the potential to evaluate hypercoagulability, which predisposes to the common clinical events of arterial and venous thromboembolism (VTE). OBJECTIVES: We hypothesized that the overall hemostatic potential (OHP) assay would show abnormal fibrin generation and lysis in patients with clinically defined hypercoagulable states. METHODS: We used the OHP assay as described by Blombäck and colleagues [1,2] in 161 clinically hypercoagulable patients with arterial or VTE, pregnancy complications or autoimmune disease. Eighty patients had associated antiphospholipid antibodies (APLA). Ninety-eight normal plasma donors were tested for comparison. RESULTS: We derived three new assay parameters for correlation with hypercoagulable states: the maximum optical density, maximum slope, and delay in onset of fibrin generation. We found significantly different assay results for all patients' parameters examined when compared with controls, indicating both increased fibrin generation and reduced fibrinolysis in hypercoagulable patients. The findings were similar whether samples were collected in association with an acute thrombotic event or not. Estimated assay sensitivity for detection of a clinically defined hypercoagulable state was 96%. CONCLUSIONS: The OHP assay is a simple, inexpensive global test that is useful for assessing patients with hypercoagulable states including APLA. OHP results are significantly abnormal in hypercoagulable groups compared with controls, indicating that both increased fibrin generation and reduced fibrinolysis contribute to hypercoagulable states. The assay may ultimately assist in tailoring clinical management to patients' individual requirements.


Assuntos
Testes de Coagulação Sanguínea/métodos , Fibrina/metabolismo , Fibrinólise , Trombofilia/sangue , Trombofilia/diagnóstico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Síndrome Antifosfolipídica/sangue , Síndrome Antifosfolipídica/complicações , Estudos de Casos e Controles , Feminino , Hemostasia , Humanos , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Valor Preditivo dos Testes , Gravidez , Complicações Hematológicas na Gravidez/sangue , Complicações Hematológicas na Gravidez/diagnóstico , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Tromboembolia/sangue , Tromboembolia/complicações , Trombofilia/etiologia , Fatores de Tempo , Trombose Venosa/sangue , Trombose Venosa/complicações
14.
J Dent Res ; 85(1): 74-8, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16373685

RESUMO

Prior studies of a link between periodontal and cardiovascular disease have been limited by being predominantly observational. We used a treatment intervention model to study the relationship between periodontitis and systemic inflammatory and thrombotic cardiovascular indicators of risk. We studied 67 adults with advanced periodontitis requiring full-mouth tooth extraction. Blood samples were obtained: (1) at initial presentation, immediately prior to treatment of presenting symptoms; (2) one to two weeks later, before all teeth were removed; and (3) 12 weeks after full-mouth tooth extraction. After full-mouth tooth extraction, there was a significant decrease in C-reactive protein, plasminogen activator inhibitor-1 and fibrinogen, and white cell and platelet counts. This study shows that elimination of advanced periodontitis by full-mouth tooth extraction reduces systemic inflammatory and thrombotic markers of cardiovascular risk. Analysis of the data supports the hypothesis that treatment of periodontal disease may lower cardiovascular risk, and provides a rationale for further randomized studies.


Assuntos
Cardiopatias/sangue , Mediadores da Inflamação/sangue , Periodontite/terapia , Trombose/sangue , Extração Dentária , Adulto , Proteína C-Reativa/análise , Estudos de Coortes , Diabetes Mellitus/sangue , Feminino , Fibrinogênio/análise , Seguimentos , Humanos , Hiperlipidemias/sangue , Hipertensão/sangue , Contagem de Leucócitos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Inibidor 1 de Ativador de Plasminogênio/sangue , Contagem de Plaquetas , Fatores de Risco , Fumar/sangue , Ativador de Plasminogênio Tecidual/sangue
15.
Tissue Antigens ; 63(1): 34-40, 2004 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-14651521

RESUMO

The frequencies of human platelet antigens (HPA) are variable among different ethnic groups. Platelet phenotyping and genotyping in different populations are important to the clinical implications of antiplatelet alloimmunization. No report on HPA prevalence has been published concerning the Vietnamese Kinh and Ma'ohis Polynesian populations. Recent anthropological and genetic marker studies suggest that these two groups have a common origin in East Asia, so we have conducted a combined study concerning the frequency of HPA-1 to HPA-11w systems (excluding HPA-8w) and Gov in these two populations. The results demonstrate a similar pattern of prevalence between Ma'ohis and most of the Asian populations. However, it should be noted that the frequency of HPA-2 is closer to northern Caucasian frequencies than to Asian frequencies. The population of Kinh shows an HPA distribution that is closer to the Chinese population than to the northeastern Thais except for HPA-3, closer to the Indonesian population. Given HPA-3 gene frequency distribution fetomaternal incompatibility could occur more frequently with the risk of alloantibody production.


Assuntos
Antígenos de Plaquetas Humanas/genética , Etnicidade/genética , Frequência do Gene , População/genética , Adulto , Feminino , Genótipo , Humanos , Masculino , Polimorfismo de Fragmento de Restrição , Polinésia , Vietnã
16.
Biochem J ; 357(Pt 2): 529-36, 2001 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-11439104

RESUMO

Using green fluorescent protein (GFP) as an autofluorescent tag, we report the first successful visualization of a beta3 integrin in a living cell. GFP fused in frame to the cytoplasmic tail of either alphaIIb or beta3 allowed normal expression, heterodimerization, processing and surface exposure of alphaIIbGFPbeta3 and alphaIIb(beta3)GFP receptors in Chinese hamster ovary (CHO) cells. Direct microscopic observation of the autofluorescent cells in suspension following antibody-induced alphaIIb(beta3) capping revealed an intense autofluorescent cap corresponding to unlabelled immunoclustered GFP-tagged alphaIIb(beta3). GFP-tagged alphaIIbbeta3 receptors mediated fibrinogen-dependent cell adhesion, were readily detectable in focal adhesions of unstained living cells and triggered p125(FAK) tyrosine phosphorylation similar to wild-type alphaIIb(beta3) (where FAK corresponds to focal adhesion kinase). However, GFP tagged to beta3, but not to alphaIIb, induced spontaneous CHO cell aggregation in the presence of soluble fibrinogen, as well as binding of the fibrinogen mimetic monoclonal antibody PAC1 in the absence of alphaIIb(beta3) receptor activation. Time-lapse imaging of living transfectants revealed a characteristic redistribution of GFP-tagged alphaIIb(beta3) during the early stages of cell attachment and spreading, starting with alphaIIb(beta3) clustering at the rim of the cell contact area, that gradually overlapped with the boundary of the attached cell, and, with the onset of cell spreading, to a reorganization of alphaIIb(beta3) in focal adhesions. Taken together, our results demonstrate that (1) fusion of GFP to the cytoplasmic tail of either alphaIIb or beta3 integrin subunits allows normal cell surface expression of a functional receptor, and (2) structural modification of the beta3 integrin cytoplasmic tail, rather than the alphaIIb subunit, plays a major role in alphaIIb(beta3) affinity modulation. With the successful direct visualization of functional alphaIIb(beta3) receptors in living cells, the generation of autofluorescent integrins in transgenic animals will become possible, allowing new approaches to study the dynamics of integrin functions.


Assuntos
Antígenos CD/genética , Proteínas Luminescentes/genética , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética , Glicoproteínas da Membrana de Plaquetas/genética , Animais , Antígenos CD/fisiologia , Células CHO , Adesão Celular/fisiologia , Agregação Celular/fisiologia , Movimento Celular/fisiologia , Cricetinae , Fibrinogênio/fisiologia , Genes Reporter , Proteínas de Fluorescência Verde , Integrina beta3 , Microscopia de Vídeo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/fisiologia , Glicoproteínas da Membrana de Plaquetas/fisiologia , Reação em Cadeia da Polimerase , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/fisiologia , Transfecção
17.
Thromb Haemost ; 86(6): 1425-34, 2001 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11776310

RESUMO

We have investigated the effect of a new Leu196Pro mutation, identified in the MIDAS-like domain of the beta3 integrin subunit in a patient with type II Glanzmann thrombasthenia, on beta3 integrin receptor function. Expression of the mutant beta3Pro196 subunit in CHO cells, either associated with recombinant human alphaIIb or alphav, resulted in normal biosynthesis of beta3 and heterodimerization with alphav or alphaIIb, but selectively interfered with alphaIIbbeta3 maturation and transport to the cell surface. Functional analysis of the beta3 mutant receptors revealed strong inhibition of alphavbeta3-mediated cell spreading on immobilized fibrinogen, focal contact formation, p125FAK phosphorylation and fibrin clot retraction, as opposed to normal alphaIIbbeta3-mediated cell interaction with immobilized fibrinogen, focal contact translocation and signaling. In contrast, antibody- or DTT-activated mutant aIIbbeta3 was unable to bind soluble fibrinogen or the ligand mimetic PAC-1 monoclonal antibody, but underwent a conformational change following RGD peptide binding as demonstrated by AP5-LIBS epitope expression. These results suggest that (1) the highly conserved TL196T motif in the beta3 integrin subunit is located in a domain structurally important for the exposure of a functional binding site for soluble fibrinogen; and (2) that the MIDAS-like contact site in beta3 is not involved in alphaIIbbeta3-mediated cell adhesion to immobilized fibrinogen, while it is essential for alphavbeta3-mediated interaction with this ligand.


Assuntos
Substituição de Aminoácidos , Antígenos CD/genética , Mutação de Sentido Incorreto , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética , Glicoproteínas da Membrana de Plaquetas/genética , Mutação Puntual , Receptores de Vitronectina/genética , Trombastenia/genética , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Antígenos CD/química , Antígenos CD/imunologia , Antígenos CD/fisiologia , Células CHO , Retração do Coágulo , Códon/genética , Cricetinae , Cricetulus , Dimerização , Feminino , Fibrinogênio/metabolismo , Humanos , Integrina beta3 , Dados de Sequência Molecular , Adesividade Plaquetária/genética , Adesividade Plaquetária/fisiologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/fisiologia , Glicoproteínas da Membrana de Plaquetas/química , Glicoproteínas da Membrana de Plaquetas/imunologia , Glicoproteínas da Membrana de Plaquetas/fisiologia , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Receptores de Vitronectina/fisiologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/fisiologia , Relação Estrutura-Atividade
18.
Blood ; 90(2): 669-77, 1997 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-9226167

RESUMO

Glanzmann's thrombasthenia (GT) is a recessive autosomal bleeding disorder characterized by abnormal platelet aggregation due to a qualitative or quantitative defect of the glycoprotein (GP) IIb-IIIa complex (integrin alphaIIb beta3). We describe a new mutation in the GPIIIa gene responsible for type I GT in a consanguineous Algerian family. A discordance between phenotyping and genotyping of the GPIIIa-related HPA-1 platelet alloantigen system in three family members heterozygous for the disease suggested a genetic defect in the GPIIIa gene and a normal GPIIb gene. Sequence analysis of amplified genomic DNA fragments showed a 6-bp deletion in exon 7 of the GPIIIa gene resulting in the amino acid deletion/substitution (Ile325pro326Gly327 --> Met) and creating a new BspHI restriction site. Expression of the mutated integrin beta3 subunit cDNA in Chinese hamster ovary cells showed that the cDNA gene was transcribed into a full-length beta3 protein with an apparent molecular weight identical to wild-type beta3 and accumulated as a single-chain molecule in the cell cytoplasm. The absence of heterodimeric complex formation of the mutant beta3 protein with endogenous alpha v was shown by immunoprecipitation experiments, intracellular immunofluorescent labeling, and a semiquantitative enzyme-linked immunosorbent assay using the alpha vbeta3 complex-specific monoclonal antibodies LM609 and 23C6. Substitution of the methionine residue by a proline, present at position 326 of wild-type beta3, did not restore the ability of the recombinant mutant beta3 protein to associate with alpha v , suggesting that the Ile-Pro-Gly motif is located in a beta3 domain important for integrin subunit interaction. The association of a BspHI restriction site with this newly identified mutation has allowed allele-specific restriction analysis of Algerian GT individuals and the identification of two new unrelated type I patients exhibiting the same mutation, suggesting that the described mutation might be significant in this population and that BspHI restriction analysis will provide a useful screening assay for antenatal diagnosis and genetic counselling.


Assuntos
Antígenos CD/química , Antígenos CD/genética , Glicina , Isoleucina , Glicoproteínas da Membrana de Plaquetas/química , Glicoproteínas da Membrana de Plaquetas/genética , Polimorfismo Conformacional de Fita Simples , Prolina , Deleção de Sequência , Trombastenia/genética , Argélia/etnologia , Sequência de Aminoácidos , Animais , Antígenos CD/biossíntese , Sequência de Bases , Células CHO , Consanguinidade , Cricetinae , Primers do DNA , Feminino , França , Humanos , Lactente , Integrina beta3 , Masculino , Linhagem , Glicoproteínas da Membrana de Plaquetas/biossíntese , Reação em Cadeia da Polimerase , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Trombastenia/sangue , Transfecção
19.
Blood ; 89(7): 2422-8, 1997 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-9116286

RESUMO

The heterodimeric complex glycoprotein (GP)IIb-IIIa, the fibrinogen receptor of platelets, carries numerous alloantigen systems. These polymorphisms are responsible for the immune response after transfusion or during pregnancy. In the latter case, the mother develops an antibody against an epitope present on fetal platelets, and this results in platelet destruction in the fetus. In this report, we describe the molecular characterization of a new alloantigen (La(a)) on GPIIIa responsible for neonatal alloimmune thrombocytopenia (NAIT). Using polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP) and DNA sequencing, we found a point mutation (G to A) in a heterozygous state on the GPIIIa gene leading to amino acid substitution Arg to Gln at position 62 of the mature protein. Transient expression of GPIIb-IIIa complexes in Cos-7 cells using wild-type or mutated GPIIIa cDNA allowed us to demonstrate that this mutation was responsible for expression of the La(a) epitope.


Assuntos
Antígenos de Plaquetas Humanas/genética , Plaquetas/imunologia , Transfusão Feto-Materna , Imunidade Materno-Adquirida , Isoanticorpos/imunologia , Mutação Puntual , Trombocitopenia/imunologia , Adulto , Animais , Sítios de Ligação , Células COS , Feminino , Humanos , Recém-Nascido , Masculino , Oligopeptídeos/metabolismo , Reação em Cadeia da Polimerase , Polimorfismo Genético , Polimorfismo Conformacional de Fita Simples , Gravidez , Trombocitopenia/congênito , Trombocitopenia/genética
20.
Hematol Cell Ther ; 38(4): 355-7, 1996 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-8891728

RESUMO

An increased bleeding time and a prolonged APTT (activated partial thromboplastin time) observed in a 48-year-old woman led to the discovery of SLE confirmed by immunological tests. Platelet function and analysis of membrane glycoproteins revealed an isolated impaired collagen induced platelet aggregation and allowed the detection of autoantibodies directed against GpIa/IIa and GpIb/IX.


Assuntos
Antígenos CD/imunologia , Autoanticorpos/sangue , Plaquetas/efeitos dos fármacos , Colágeno/farmacologia , Lúpus Eritematoso Sistêmico/diagnóstico , Complexo Glicoproteico GPIb-IX de Plaquetas/imunologia , Feminino , Humanos , Integrina alfa2 , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/imunologia , Pessoa de Meia-Idade , Ativação Plaquetária/efeitos dos fármacos , Ativação Plaquetária/imunologia
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