OBJECTIVE: To investigate the damaging of human umbilical vein endothelial cells (HUVEC) induced by antiplatelet integrin β3 antibodies in vitro. METHODS: The serum from 36 chronic ITP patients were collected, flow cytometry and monoclonal antibody specific immobilization of platelet antigen (MAIPA) assay were used to collect antiplatelet integrin β3 antibodies from the serum of the patients. After HUVEC were treated by ITP patient serum (PS) containing anti-integrin β3 antibodies, the cell damage was detected by Lactate dehydrogenase (LDH) assay, cell apoptosis was detected by flow cytometry, the expression of apoptosis-related gene Bax was detected by Reverse transcription-Quantitative real-time PCR (RT-qPCR), and expression of Apoptosis-related signaling pathway protein Akt and related protein Bax were detected by Western blot. HUVEC were treated by PS combined with Akt activator SC79, the cells damage were detected by LDH assay, apoptosis of the cells were detected by flow cytometry, the expression of apoptosis-related gene Bax was detected by RT-qPCR. RESULTS: Among 36 cases of serum from the chronic ITP patients, 5 patients' serum containing anti-integrin β3 antibodies were collected. After HUVEC was treated by PS, the viability of LDH was significant increased（P<0.05）, so as for the apoptosis of the cells（P<0.05）, the expression of gene and protein of Bax was increased up-regulated（P<0.05）, the protein expression of pAkt was down-regulated（P<0.05）. Comparing with HUVEC cultured with PS alone, the viability of LDH of HUVEC treated by PS combined with SC79 was significantly reduced（P<0.05）, so as for the apoptosis of the cells（P<0.05）, and gene expression of Bax was significantly decreased（P<0.05）. CONCLUSION Anti-integrin β3 serum can cause the damage and apoptosis of HUVEC through Akt signaling pathway，the apoptotic effects of anti-integrin β3 antibodies to HUVEC was effectively reversed by SC79.
Apoptosis ; Cells, Cultured ; Human Umbilical Vein Endothelial Cells ; Humans ; Integrin beta3 ; Signal Transduction

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

OBJECTIVE: To comparatively investigate the expression of several integrins in specimens of human bone metastases and degenerative bone tissue. METHODS: Degenerative cancellous tissue was obtained from a sample of human degenerative spine. Thirteen human specimens were obtained from metastatic spine tumors, whose primary cancer was colon cancer (n=3), hepatocellular cancer (n=3), lung cancer (n=4), and breast cancer (n=3). The expression of vimentin and integrins alphav, beta1, and beta3 was assessed in metastatic and degenerative specimens by immunohistochemistry and real-time reverse transcription-polymerase chain reaction (qRT-PCR). RESULTS: Immunohistochemical staining showed that vimentin and integrin alphav was broadly expressed in all tissues examined. By contrast, integrin beta1 was weakly expressed only in 38.4% (5/13) of tissues. Integrin beta3 was consistently negative in all cases examined. qRT-PCR analysis showed that vimentin gene expression was higher in all metastatic specimens, as compared to degenerative bone. The gene expression of integrin alphav in breast specimen was significantly higher than others (p=0.045). The gene expression of integrin beta1 was also higher in all metastatic specimens than in degenerative bone tissue. The gene expression of integrin beta3 was variable. CONCLUSION: Spinal metastatic tumors have mesenchymal characteristics such as increased expression of vimentin. The increased expression of integrin alphav and beta1 in spine metastatic tumors suggests that adhesive molecules such as integrin may have implications for the prevention of spine metastasis.
Adhesives ; Antigens, CD29 ; Bone and Bones ; Breast ; Breast Neoplasms ; Colonic Neoplasms ; Gene Expression ; Humans ; Immunohistochemistry ; Integrin alphaV ; Integrin beta3 ; Integrins* ; Liver Neoplasms ; Lung Neoplasms ; Neoplasm Metastasis ; Spine* ; Vimentin*

UNLABELLEDOBJLECTIVE: To investigate the effect of integrin β3 cytoplasmic NITY motif on αIIbβ3-mediated cell functions.

METHODSStable Chinese hamster ovary (CHO) cell lines that co-express human wild type integrin αIIb and wild type β3 or mutant β3ΔNITY (β3 deleting cytoplasmic NITY motif) were established. Expression of αIIb and β3 were tested by Western blot and flow cytometry in CHO cell lines. Spreading and adhesion of stable cell lines on immobilized fibrinogen were examined. The co-immunoprecipitation was used to detect protein interactions.

RESULTSCHO-αIIbβ3, CHO-αIIbβ3ΔNITY cells were successfully established. The CHO cells transfected with wild type αIIbβ3 had the ability of adhesion and spreading. Compared with CHO-αIIbβ3 cells, CHO-αIIbβ3ΔNITY cells showed an impaired capacity of adhesion but no significant difference was observed in spreading of adhered cells. The co-immunoprecipitation showed that kindlin-2 associated with wild type integrin αIIbβ3. The β3ΔNITY mutation substantially reduced kindlin-2 association.

CONCLUSIONDeletion of NITY motif causes an impaired ability of adhesion. The deletion mutation can suppress kindlin-2 binding to integrin β3, thereby partially inhibit the integrin β3 signaling.

Animals ; CHO Cells ; Cricetinae ; Cricetulus ; Fibrinogen ; Humans ; Integrin alpha2 ; Integrin beta3 ; Platelet Glycoprotein GPIIb-IIIa Complex ; Protein Structure, Tertiary ; Signal Transduction

OBJECTIVETo study the effect of interaction of the talin rod domain integrin binding site 2 with integrin β3 on platelet signal transduction.

METHODSA peptide that mimics the membrane proximal α helix 6 residues R724 KEFAK729 of the integrin β3 cytoplasmic tails was designed and synthesized, to which the myristoylation was covalently linked to the N-terminal of the peptide enabling membrane penetration. The effects of myr-RKEFAK peptide on the typical platelet outside-in signaling ovent (stable adhesion and spreading on immobilized fibrinogen, aggregation, fibrin clot retraction) and inside-out signaling events (soluble fibrinogen binding) were tested.

RESULTSmyr-RKEFAK peptide dose-dependently inhibited platelet stable adhesion and spreading on immobilized fibrinogen, irreversible aggregation, as well as fibrin clot retraction, but not soluble fibrinogen binding and reversible phase of platelet aggregation.

CONCLUSIONThe cell-penetrating peptide myr-RKEFAK causes an inhibitory effect on integrin β3 outside-in signaling-regulated platelets functions, but did not affect inside-out signaling-regulated platelets functions.

Blood Platelets ; Fibrinogen ; Humans ; Integrin beta3 ; Peptides ; Platelet Adhesiveness ; Platelet Aggregation ; Signal Transduction

OBJECTIVETo detect platelet anti-HPA-1a and -1b antibodies using recombinant GPIIIa fragments coupled to Luminex beads.

METHODSThe sensitivity of 2 techniques, monoclonal antibody specific immobilization of platelet antigen (MAIPA) and Luminex bead assay, was compared using 12 twofold-serial dilutions (from neat to 1 in 2048) of an anti-HPA-1a WHO international standard. The specificity of Luminex assay to identify anti-HPA-1a and -1b antibodies was assessed using 8 negative or positive controls and 36 blinded samples provided by WHO Platelet Workshop.

RESULTSThe sensitivity of MAIPA and Luminex bead assay to detect anti-HPA-1a was dilution 1/64 (i.e. 1.56 IU/ml) and far more than dilution 1/2048 (i.e. 0.049 IU/mL), respectively. The Luminex bead assay could specifically identify negative and positive controls of anti-HPA-1a and -1b. All results of 36 blinded samples by Luminex assay were accordant to reference results except one sample which contained high concentration antithetical antibody and resulted in false positive of anti-HPA-1b. Cross-reactivity was also not observed with the samples containing HLA, ABO or other platelet antibodies.

CONCLUSIONThe Luminex beads coupled with recombinant GPIIIa fragments can be used to detect HPA-1 system antibodies with sufficient sensitivity and specificity, that is suitable for the detection of platelet alloantibodies in clinical alloimmune thrombocytopenia.

Antibodies, Monoclonal ; Antigens, Human Platelet ; immunology ; Blood Platelets ; Humans ; Integrin beta3 ; chemistry ; Isoantibodies ; blood ; Purpura, Thrombocytopenic, Idiopathic ; diagnosis ; Recombinant Proteins ; chemistry ; Sensitivity and Specificity

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