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

OBJECTIVETo generate recombinant GPⅢa as an alternative source for HPA-1a antigen and combine it with Luminex xMAP beads for the detection of HPA-1a-specific alloantibody.

METHODSThe full coding region of ITGB3 gene was amplified and ligated with pcDNA3.1. The recombinant plasmid was transfected into CHO cells, and those with stable expression were screened with G418. Expressed protein was identified and coupled with Luminex xMAP beads, which were then reacted with sera samples. Subsequently, phycoerythrin-labeled anti-species IgG antibody was added to the reaction wells and the median fluorescence was determined on a Luminex-100 analyzer.

RESULTSDNA sequencing confirmed that the cloned ITGB3 gene was HPA-1aa. The recombinant GPⅢa was coupled with Luminex xMAP beads. The sensitivity of Luminex beads assay to detect HPA-1a antibody was dilution 1/32 (3.125 U/mL). The Luminex beads assay could specifically identify the HPA-1a antibody from the test sera, and the results were consistent with that of monoclonal antibody-specific immobilization of platelet antigens (MAIPA) technology. Cross-reactivity was not observed with the samples containing HLA, ABO and other HPA antibodies (HPA-3a and HPA-5b). The results illustrated that to detect HPA antibody with Luminex xMAP beads technology is feasible.

CONCLUSIONRecombinant GPⅢa was successfully obtained and used to establish a Luminex technology-based method for the detection of HPA antibodies.

Animals ; Antigens, Human Platelet ; immunology ; Autoantibodies ; immunology ; Base Sequence ; Blotting, Western ; CHO Cells ; Cricetinae ; Cricetulus ; Humans ; Immunoassay ; methods ; Integrin beta3 ; genetics ; immunology ; metabolism ; Microspheres ; Recombinant Proteins ; immunology ; metabolism ; Reproducibility of Results

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

OBJECTIVETo explore the effects of methylglyoxal on endothelia cell migration.

METHODSHuman umbilical vein endothelial cells (HUVECs) were stimulated by serial concentrations of methylglyoxal (MGO, 0, 25, 50, 100 and 200 µmol/L) for 24 h, and the cell migration was assessed by scratch wound and Transwell assay. The expression of integrin β3 in the treated cells was examined by immunoblotting, and the effect of an anti-β3 antibody, LM609, on cell migration was investigated.

RESULTSMethylglyoxal significantly inhibited HUVEC migration in a concentration-dependent manner (P<0.05). Methylglyoxal decreased the expression of integrin β3 in a time- and concentration-dependent manner (P<0.05). LM609 also significantly inhibited HUVEC migration (P<0.05).

CONCLUSIONMethylglyoxal inhibits HUVEC migration in vitro by down-regulating integrin β3 expression.

Cell Movement ; drug effects ; Cells, Cultured ; Down-Regulation ; Human Umbilical Vein Endothelial Cells ; drug effects ; metabolism ; Humans ; Integrin beta3 ; metabolism ; Pyruvaldehyde ; pharmacology

No abstract available.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; Automation ; Blood Platelets/*cytology/metabolism ; Child ; *Flow Cytometry ; Humans ; Integrin beta3/*metabolism ; Middle Aged ; Platelet Count/*instrumentation ; Receptors, IgG/*metabolism ; Young Adult

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 study the effects and underlying mechanisms of Zhuyun Recipe (ZR) on the endometrial receptivity in ovarian stimulation (OS) and blastocyst implantation dysfunction (BID) mice.

METHODSTotally 200 normal female Kunming mice were randomly divided into 6 groups, i. e., the control group (Group A), the OS group (Group B), the OS + ZR group (Group C), the BID group (Group D), the BID + ZR group (Group E), and the ZR group (Group F). The pregnant mare's serum gonadotrophin (PMSG) and human chorionic gonadotrophin (HCG) were intraperitoneally injected to mice in Group B. Mifepristone was subcutaneously injected to mice in Group D at 9:00 am on the 4th gestation day. Corresponding medications were given to mice in Group C, E, and F at 1.5 mL/100 g by gastrogavage at 8:00 am from the first to the 4th gestation day. Eight uterus samples were collected at 9:00 pm on the 4th gestation day and fixed. The expression levels of leukemia inhibitory factor (LIF) and integrin beta3 were detected using immunohistochemical assay. The pregnant mice were sacrificed at 9:30 pm on the 8th gestation day, and their uterus were taken out. The number of blastocysts was counted.

RESULTSCompared with Group A, the pregnant rate was 6.67% (1/15 cases) in Group B and 18.75% (3/16 cases) in Group D, the mean OD value of LIF was 0. 18 +/- 0.02 in Group B and 0.23 +/- 0.02 in Group D, and the mean OD value of integrin beta3 was 0.20 +/- 0.05 in Group B and 0.19 +/- 0. 02 in Group D, showing statistical difference (P < 0.01). The pregnant rate was 54.55% (12/22 cases) in Group C and 65. 22% (15/23 cases) in Group E, the mean OD value of LIF was 0.37 +/- 0. 09 in Group C and 0.39 +/- 0.02 in Group E, and the mean OD value of integrin beta3 was 0.34 +/- 0.04 in Group C and 0.38 +/- 0.08 in Group E, showing statistical difference when compared with those of Group B and Group D respectively (P < 0.05).

CONCLUSIONSOS and BID had negative effects on the endometrial receptivity and hindered the blastocyst implantation. ZR could improve the uterine receptivity and elevate the pregnant rate by up-regulating the expressions of endometrial LIF and integrin beta3.

Animals ; Drugs, Chinese Herbal ; pharmacology ; Embryo Implantation ; drug effects ; Endometrium ; drug effects ; physiology ; Female ; Integrin beta3 ; metabolism ; Leukemia Inhibitory Factor ; metabolism ; Mice ; Mice, Inbred Strains ; Ovulation Induction ; Pregnancy

OBJECTIVETo investigate the effects of Er'zhi Tiangui Granule (, ETG) on sequential expressions of integrinβ3 and its ligand osteopontin in the mouse endometrium during controlled ovarian hyperstimulation (COH) and implantation period.

METHODSSeventy-five Mature female Kunming mice were randomly divided into 3 groups, a normal control group, a model group, and a treatment group administrated with ETG for 10 days, 25 in each group. After mated with male mice, every 5 mice were sacrified in each group at the 0, 2nd, 4th, 6th, and 8th days to take their endometrium. In-situ hybridization was used to detect the expressions of integrinβ3 and osteopontin in the endometrium.

RESULTSmRNA expressions of integrinβ3 and osteopontin in the endometrium during implantation period showed similar time sequence rules in the treatment group to those in the normal control group; the peak values of them were a little lower in the treatment group than the normal control without significant differences. In the model group, integrinβ3 mRNA expression was higher at the 2nd day, obviously lower at the 4th and 6th days, and insignificantly lower at the 8th day; and osteopontin expression was remarkably lower at the 4th, 6th, and 8th days, compared with the normal control and the treatment groups (P<0.05, P<0.01).

CONCLUSIONSCOH might influence the sequential expressions of integrinβ3 and its ligand osteopontin, bring forward the integrinβ3 expression peak, impact on the cooperation of integrinβ3 and osteopontin, so as to damage the endometrial receptivity. ETG could regulate the sequential expressions of integrinβ3 and its ligand osteopontin to improve the mouse endometrial receptivity during COH.

Animals ; Dosage Forms ; Drug Evaluation, Preclinical ; Drugs, Chinese Herbal ; administration & dosage ; pharmacology ; Endometrium ; drug effects ; metabolism ; Female ; Gene Expression Regulation ; drug effects ; Integrin beta3 ; genetics ; metabolism ; Ligands ; Male ; Mice ; Osteopontin ; genetics ; metabolism ; Ovulation Induction ; veterinary

OBJECTIVETo explore the effects of integrin β3 pathway on the proliferation, migration and extracellular matrix deposition of pulmonary arterial smooth muscle cells (PASMCs) induced by connective tissue growth factor (CTGF).

METHODSPASMCs of SD Rats were cultured in M199 culture system in vitro and the 3rd-7th passages of PASMCs were used in the experiments. The cells were randomly divided into three groups: (1) CONTROL GROUP: culture system contained no any stimulation factor; (2) CTGF group: culture system was added into 50 ng/ml CTGF; (3) CTGF+ anti-integrin β3 antibody group:culture system was added with 50 ng/ml CTGF and 10 mg/L anti-integrin β3 antibody. The PASMCs were cultured with 50 ng/ml CTGF and anti-integrin β3 antibody (0, 5, 10, 15, 20 mg/L) for 24, 48, 72 and 96 h, the proliferation of PASMCs was detected by WST-1 Cell Proliferation Assay Kit. The migration of PASMCs was observed by Transwell cell test under the phase contrast microscope. RT-PCR assay was applied to detect the mRNA expression of collagenI-α1, collagen III-α1 and fibronectin-1 gene of PASMCs. The expression of fibronectin protein was examined by Western blotting and immunohistochemistry.

RESULTSThe results of WST-1 test showed that the anti-integrin β3 antibody inhibited significantly the proliferation of PASMCs induced by CTGF (P < 0.05), among which the inhibition rate of anti-integrin β3 antibody (10 mg/L) was the most significant. Transwell test results showed that CTGF group of PASMCs migration numbers (25 ± 1.57) were higher than that of the control group (11 ± 2.08, P < 0.01); PASMCs migration numbers of CTGF+ integrin β3 antibody group (17 ± 4.16) were less than that of the CTGF group (P < 0.05). Compared with the control group, the mRNA expression of collagen typeI-α1 (4.28 ± 0.33), collagen typeIII-α1 (4.41 ± 0.35), fibronectin-1 (4.05 ± 0.33) of PASMCs was increased in CTGF group, with a time-dependence (P < 0.01); Compared with the CTGF group, the mRNA expression of collagen typeI-α1 (3.38 ± 0.30), collagen typeIII-α1 (3.40 ± 0.30), fibronectin-1 (3.12 ± 0.29) of PASMCs was reduced in CTGF+ anti-integrin β3 antibody group (P < 0.05), which was higher than that of the control group (P < 0.05); Western blot and immunohistochemical tests showed that compared with the control group, CTGF group could stimulate the expression of fibronectin protein of PASMCs (P < 0.01); the anti-integrin β3 antibody could inhibit the expression of fibronectin protein induced by CTGF(P < 0.01), which was more remarkable than that in the control group (P < 0.01).

CONCLUSIONIntegrin β3 pathway can mediate CTGF-induced proliferation, migration and extracellular matrix deposition of PASMCs, CTGF-integrin β3 signaling pathway may play an important role in pulmonary vascular remodeling.

Animals ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Connective Tissue Growth Factor ; pharmacology ; Extracellular Matrix ; metabolism ; Integrin beta3 ; metabolism ; Male ; Muscle Cells ; cytology ; metabolism ; Muscle, Smooth, Vascular ; cytology ; metabolism ; Pulmonary Artery ; cytology ; metabolism ; Rats ; Rats, Sprague-Dawley ; Signal Transduction

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