BACKGROUNDGlanzmann thrombasthenia (GT) is an autosomal recessive bleeding disorder characterized by the tendency to hemorrhage and the inability of platelets to aggregate in response to agonists. GT is caused by a defect of the platelet glycoprotein IIb/IIIa complex. The objective of this study was to describe the clinical features and the genetic cause of GT in a 6-year-old girl from south China.

METHODSA three-generation family was studied. The proband patient aged 6 years and her parents undertook examinations of platelet counts, blood film, bleeding time, platelet aggregation, and flow cytometry. All coding exons of the ITGA2B and ITGB3 genes were amplified by polymerase chain reaction (PCR), and direct sequencing was performed for mutational screening on the patient and normal controls consisted of 52 healthy blood donors. Reverse transcription PCR was conducted to test for exon skipping.

RESULTSThe proposita patient showed dispersing platelets, prolonged bleeding time, and severely reduced platelet aggregation in response to the physiological agonists adenosine diphosphate (ADP), epinephrine, collagen, and ristocetin. Flow cytometric measurements showed that the contents of alphaIIb and beta3 were significantly decreased. Sequencing results demonstrated two different types of heterozygous mutations existed in the alphaIIb gene (c.2930delG and IVS15-1delG). The compound mutations were also confirmed in the patient's mother and father separately.

CONCLUSIONSThe alphaIIbbeta3 deficiency of the proband was caused by two compound ITGA2B mutations, which were first reported in Chinese GT patients. The IVS15-1delG was first confirmed to cause an exon skipping.

Asian Continental Ancestry Group ; Child ; Female ; Flow Cytometry ; Heterozygote ; Humans ; Integrin alpha2 ; genetics ; Integrin beta3 ; genetics ; Mutation ; Pedigree ; Reverse Transcriptase Polymerase Chain Reaction ; Thrombasthenia ; genetics ; metabolism ; pathology

BACKGROUNDTo express and purify human alpha3-integrin to serve as the antigen to prepare its antibody and to separate the Vero cell clones without expression of alpha3-integrin.

METHODSThe human alpha3-integrin gene was amplified by using RT-PCR, then subcloned into a pQE30 expression vector and expressed in E. coli. The gene expression was confirmed by Western blot assay. Rabbit was inoculated with purified antigen to stimulate the antibody generation. The target Vero cells were separated by negative selection using antibody plus complement mediated cytolysis. The separated cell clones were confirmed by immunofluorescence and Western blot assay.

RESULTSThe alpha3- integrin gene was cloned and expressed effetively, Western blot assay revealed that the expressed protein held good immune reactivity. High titer antibody was generated. However the expression of alpha3-integrin was not detected on Vero, VeroE6, Hep-2, 2BS and 293 cells.

CONCLUSIONSThe results of the study suggested that hantavirus has other receptors on Vero cells beside alpha 3-integrin.

Animals ; Cercopithecus aethiops ; Cloning, Molecular ; Gene Expression ; Hantavirus ; Integrin beta3 ; biosynthesis ; genetics ; immunology ; Rabbits ; Receptors, Virus ; Vero Cells ; metabolism

OBJECTIVETo study the association of integrin alpha-2 (ITGA2) gene C807T, integrin beta-3 (ITGB3) gene T176C polymorphisms with ischemic stroke and the effect of the polymorphisms on plasma lipid and lipoprotein levels.

METHODSPolymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and DNA sequencing were used to detect the integrin genotypes in 265 patients with ischemic stroke and 280 healthy controls. The plasma lipid and lipoprotein levels were measured by routine method.

RESULTSPlasma total cholesterol (TC), triacylglycerol (TG) and low density lipoprotein-cholesterol (LDL-C) in the patients with ischemic stroke were significantly higher than those in the controls (P< 0.05). The distributions of the ITGB3 gene T176C polymorphism were not different between the ischemic stroke group and control group, but the ITGA2 gene C807T polymorphism was significantly different. The relative risk suffering from ischemic stroke of the T allele carrier was 1.455 times as that of the C allele carrier (OR=1.455, 95%CI: 1.134-1.866). The level of plasma lipid in the T allele carriers was significantly higher than that in the C allele carriers (P< 0.05).

CONCLUSIONThe ITGA2 gene C807T polymorphism was associated with ischemic stroke, the 807 T allele may be a genetic risk factor for ischemic stroke. The ITGA2 gene C807T polymorphism may affect ischemic stroke through plasma lipid and lipoprotein levels.

Brain Ischemia ; blood ; genetics ; metabolism ; Cholesterol, LDL ; genetics ; metabolism ; Female ; Genetic Predisposition to Disease ; Humans ; Integrin alpha2 ; genetics ; metabolism ; Integrin beta3 ; genetics ; metabolism ; Lipid Metabolism ; genetics ; Lipids ; blood ; Male ; Middle Aged ; Polymorphism, Genetic ; Polymorphism, Restriction Fragment Length ; Polymorphism, Single Nucleotide

OBJECTIVE: To establish 293T cell lines stably expressing Calpain-cleavage related α3 cytoplasmic tail mutants, and to explore the effect of amino acid motifs in integrin β3 cytoplasmic tail on αⅡbβ3-mediated cell function. METHODS: 293T cell lines stably co-expressing human wild type integrin αⅡb and full length β3 or mutant β3, including β3-ΔNITY (β3 cytoplasmic tail NITY motif deleted), β3-Δ754 (β3 cytoplasmic tail TNITYRGT motif deleted) and β3-Δ759 (β3 cytoplasmic tail RGT motif deleted) were established. Spreading and adhesion of these stable cell lines on immobilized fibrinogen were tested. RESULTS: 293T-αⅡbβ3ΔNITY, 293T-αⅡbβ3Δ754, 293T-αⅡbβ3Δ759 and 293T-αⅡbβ3 cell lines were successfully established. Compared with the 293T cells, 293T-αⅡbβ3 cells which expressed full β3, possessed well adhesion and spread ability on immobilized fibrinogen, suggesting it can be as a surrogate for platelet. Compared with 293T-αⅡbβ3 cells, the 293T-αⅡbβ3ΔNITY cells showed a partial impairment of adhesion and spreadability on immobilized fibrinogen. while the 293T-αⅡbβ3Δ754 cells and 293T-αⅡbβ3Δ759 cells failed to adhere or spread on immobilized fibrinogen. CONCLUSION To the cell spreading function mediated by integrin β3, RGT motif is vital, while NITY can be dispensable. These established 293T cell lines stably expressing different β3 mutants provide a solid basis for a further analysis of mass spectrometry.
Amino Acid Motifs ; Animals ; CHO Cells ; Cell Adhesion ; Cricetinae ; Cricetulus ; HEK293 Cells ; Humans ; Integrin beta3 ; genetics ; metabolism ; Platelet Glycoprotein GPIIb-IIIa Complex ; genetics ; metabolism ; Signal Transduction

The membrane proximal α helix of integrin β subunit cytoplasmic tails plays an important functional role by interacting with various intracellular proteins, namely talin, α-actinin or skelemin. This study was designed to investigate the functional role of 5 highly conserved charged amino acids (R(724), K(725), E(726), E(731), E(733)) within this α helix by site-directed mutagenesis. The result showed that CHO cells expressing the αIIbβ3E726Q mutant had the most prominent phenotype and characterized by defective cell spreading on immobilized fibrinogen. In addition, this E726Q mutation induced membrane blebbing in cells adherent on fibrinogen, and this blebbing could be inhibited by the myosin light chain ATPase inhibitor blebbistatin. It is concluded that the membrane proximal α-helix of integrin β3 subunit is important in linking the phospholipid membrane to the submembraneous actin cortex.
Animals ; CHO Cells ; Cell Surface Extensions ; Cricetinae ; Cricetulus ; Heterocyclic Compounds, 4 or More Rings ; chemistry ; Integrin beta3 ; genetics ; Mutagenesis, Site-Directed ; Mutation ; Protein Structure, Tertiary

This study was purpose to establish the transgenic mouse models of the truncated platelet integrin β3 by retrovirus-infected hematopoietic stem cells (HSCs) transplantation and to provide the basis for further study of the role of integrin β3 cytoplasmic domain in platelet bi-directional signaling pathways. Wild-type β3, β3-Δ759 (R(760) GT(762) truncated β3) and β3-Δ754 (T(755) NITYRGT(762) truncated β3) cDNAs were subcloned into MSCV MigR1 retroviral vector bearing a GFP gene and packaged into infective retrovirus with BOSC23 cell strain. The bone marrow HSCs of the β3 deficient mice were infected by the retroviruses, and transplanted into lethally-irradiated wild type C57BL/6 mice. GFP positive rate and surface β3 expression of the recipients' platelets at 6 to 8 weeks after transplantation were detected by flow cytometry to evaluate the transgenic efficiency. The results showed that four kinds of transgenic mouse models including vector, wild-type β3, β3-Δ759 and β3-Δ754 were established successfully. GFP positive rates of transgenic mouse platelets ranged from 18% to 66% and the β3 expression of transgenic mouse reached heterozygote (β3(+/-) level of mouse). It is concluded that establishment of transgenic mouse models mediated by retrovirus-infected HSCs transplantation is a feasible, fast, and high throughput transgenic approach and laid a solid foundation for further research on the role of integrin β3 cytoplasmic domain for bi-directional signaling of platelets in vivo, and for the gene therapy of platelet disorders.
Animals ; Blood Platelets ; metabolism ; Genetic Vectors ; Hematopoietic Stem Cell Transplantation ; Integrin beta3 ; metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Retroviridae ; genetics

OBJECTIVETo investigate mRNA expression of integrin beta3 in gastric carcinoma, its correlation with microvascular density (MVD), growth-pattern, invasion, metastasis and prognosis.

METHODSIn situ hybridization and immunohistochemistry techniques were used to study the expression of integrin beta3 mRNA and CD34 protein in 105 gastric carcinoma specimens.

RESULTSIn situ hybridization revealed a 30% (6/20) positive rate of integrin beta3 mRNA in non-tumor gastric mucosa, which was significantly lower than that of the gastric cancer group (61.0%, 64/105, chi(2) = 8.85, P = 0.037); In infiltrating type cases (70.2%, 40/57), stage III - IV (72.1%, 44/61), lymphatic metastasis (68.6%, 48/70) and distant metastasis (85.7%, 36/42), the positive expression rates were significantly higher than those of the expanding type (chi(2) = 14.97, P = 0.002), stage I - II (chi(2) = 15.21, P = 0.015), non-lymphatic metastasis (chi(2) = 17.89, P = 0.025) and non-distant metastasis (chi(2) = 20.22, P = 0.005; chi(2) = 21.35, P = 0.035); its mean MVD was also significantly higher than that of the expanding type (t = 10.105, P = 0.001), stage I approximately II (t = 5.961, P = 0.001), non-lymphatic metastasis (t = 3.819, P = 0.01)and non-distant metastasis (t = 10.578, P = 0.001; t = 7.882, P = 0.001), respectively; The mean MVD (41.02 +/- 8.55)/0.72 mm(2) of the integrin beta3 mRNA positive expression group was significantly higher than (25.26 +/- 11.25)/0.72 mm(2) of the negative expression group. There was a positive relation between MVD and integrin beta3 mRNA expression in the tumor (r(s) = 0.316, P = 0.001). The mean survival time in cases with positive integrin beta3 mRNA expression or MVD value >or= 39.5 was significantly shorter than that in cases with negative expression or MVD value < 39.5.

CONCLUSIONSIntegrin beta3 expression correlates with enhanced tumor angiogenesis and may play a role in the invasion and nodal metastasis of gastric carcinoma, therefore it may serve as a prognostic marker of gastric cancer.

Adult ; Aged ; Disease Progression ; Female ; Humans ; Integrin beta3 ; genetics ; Male ; Middle Aged ; Neovascularization, Pathologic ; etiology ; Prognosis ; RNA, Messenger ; analysis ; Stomach Neoplasms ; blood supply ; metabolism ; pathology ; Survival Rate

OBJECTIVETo study the platelet morphology and function of an inherited macrothrombocytopenia disorder with abnormal large granules.

METHODSPlatelet size and structure were investigated by both light microscopy and electron microscopy. The platelet membrane expression of GP I b, GP II b, GPIII a, P-selectin and CD63 were analyzed by using respective monoclonal antibodies. Platelet 5-hydroxy-tryptamine was measured with spectrophotofluorometer.

RESULTSBoth the patient and her father had large granules in their platelets, with exocytosis being easily observed. The expressions of GP I b, GP II b and GP II a on the platelets were in normal range, while P-selectin and CD63 were somewhat increased. The abnormal large granules were not the alpha granules, lysosomes or dense bodies.

CONCLUSIONBoth morphological and functional abnormalities of the platelets from the patient are clearly distinguishable from other hereditary giant platelet disorders. It would probably represent a novel platelet disorder.

Adult ; Blood Platelets ; metabolism ; ultrastructure ; Female ; Humans ; Integrin beta3 ; biosynthesis ; Microscopy, Immunoelectron ; Platelet Glycoprotein GPIb-IX Complex ; biosynthesis ; Platelet Membrane Glycoprotein IIb ; biosynthesis ; Thrombocytopenia ; genetics ; pathology

OBJECTIVE: To explore the molecular mechanism of Glanzmann thrombasthenia (GT). METHODS: All 45 exons of alphaIIb and beta3 subunit genes as well as their splicing sites were amplified by polymerase chain reaction(PCR) with 40 primer pairs, and then the PCR products were used to screen the gene mutation by single strand conformation polymorphism-polyacrylamide gel electrophoresis (SSCP-PAGE). The mutation was further confirmed by direct DNA sequencing. RESULTS: A DNA band alterated migration was detected after SSCP-PAGE. DNA sequencing showed that a base deletion within the band at the site of 540 in GPIIb gene(540A) was found. CONCLUSION The frame-shift mutation caused by the deletion of 540A in GPIIb gene is a novel mutation which is a genetic defect in patients with GT.
Base Sequence ; Child, Preschool ; Exons ; genetics ; Frameshift Mutation ; genetics ; Gene Deletion ; Humans ; Integrin beta3 ; genetics ; Male ; Molecular Sequence Data ; Platelet Membrane Glycoprotein IIb ; genetics ; Sequence Analysis, DNA ; Thrombasthenia ; genetics

OBJECTIVETo explore the function of a novel nonsense mutation c.1476G>A of ITGB3 gene using an in vitro expression system.

METHODSAn eukaryotic expression vector containing ITGB3 c.1476G>A cDNA was generated by site-directed mutagenesis and transformed into E.coli. Plasmid DNA was extracted and sequenced to confirm the target mutations. Wild-type and mutant recombination plasmids were transfected into Chinese hamster ovarian cancer (CHO) cells by nonliposome method, and the stable expression cells were harvested by G418 screening. The ITGB3 gene mRNA transcription and GPIIIa expression level in CHO cells were detected with real-time quantitative PCR, Western blotting and flow cytometry, respectively.

RESULTSThe eukaryotic expression vectors of wild ITGB3 cDNA and c.1476G>A mutant were successfully constructed. CHO cells with stable expression were obtained after transfection and screening. Compared with the wild-type transfected cells, the amount of CD61 antigen expression was 37% and mRNA transcription level was only 6% in the mutant-transfected cells. Full length GPIIIa protein was found only in the stably wild-type-transfected cells, but not in mutant-transfected cells by Western blotting analysis.

CONCLUSIONThe ITGB3 c.1476G>A mutation can decrease the transcription level and further affect GPIIIa synthesis and CD61 antigen expression.

Animals ; Base Sequence ; Blood Platelets ; cytology ; metabolism ; CHO Cells ; Cloning, Molecular ; Codon, Nonsense ; genetics ; Cricetinae ; Cricetulus ; Humans ; Integrin beta3 ; genetics ; metabolism ; Molecular Sequence Data ; Plasmids ; genetics ; metabolism ; Point Mutation