OBJECTIVE: To construct a mouse model of Glanzmann's thrombasthenia (GT) with ITGA2B c.2659 C>T (p.Q887X) nonsense mutation by CRISPR/Cas9 technology, and then further explore the expression and function of glycoprotein αIIbβ3 on the surface of platelet membrane. METHODS: The donor oligonucleotide and gRNA vector were designed and synthesized according to the ITGA2B gene sequence. The gRNA and Cas9 mRNA were injected into fertilized eggs with donor oligonucleotide and then sent back to the oviduct of surrogate mouse. Positive F0 mice were confirmed by PCR genotyping and sequence analysis after birth. The F1 generation of heterozygous GT mice were obtained by PCR and sequencing from F0 bred with WT mice, and then homozygous GT mice and WT mice were obtained by mating with each other. The phenotype of the model was then further verified by detecting tail hemorrhage time, saphenous vein bleeding time, platelet aggregation, expression and function of αIIbβ3 on the surface of platelet. RESULTS: The bleeding time of GT mice was significantly longer than that of WT mice (P<0.01). Induced by collagen, thrombin, and adenosine diphosphate (ADP), platelet aggregation in GT mice was significantly inhibited (P<0.01, P<0.01, P<0.05). Flow cytometry analysis showed that the expression of αIIbβ3 on the platelet surface of GT mice decreased significantly compared with WT mice (P<0.01), and binding amounts of activated platelets to fibrinogen were significantly reduced after thrombin stimulation (P<0.01). The spreading area of platelet on fibrinogen in GT mice was significantly smaller than that in WT mice (P<0.05). CONCLUSION A GT mouse model with ITGA2B c.2659 C>T (p.Q887X) nonsense mutation has been established successfully by CRISPR/Cas9 technology. The aggregation function of platelet in this model is defective, which is consistent with GT performance.
Animals ; CRISPR-Cas Systems ; Codon, Nonsense ; Disease Models, Animal ; Fibrinogen/genetics* ; Humans ; Integrin alpha2/genetics* ; Mice ; Oligonucleotides ; Platelet Glycoprotein GPIIb-IIIa Complex/genetics* ; RNA, Guide ; Thrombasthenia/genetics* ; Thrombin/genetics*

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

OBJECTIVEMutation screening was performed in a pedigree of Glanzmann's thrombasthenia (GT) and prenatal diagnosis was performed.

METHODSIn this study, reverse transcription-PCR-sequencing and PCR-sequencing, as well as restriction fragment length polymorphism(RFLP) and A/T-cloned-sequencing, were used to screen the ITGA2B and ITGB3 mutation in a pedigree with Glanzmann's thrombasthenia in the RNA and DNA level. Prenatal diagnosis was performed for this pedigree.

RESULTSDeletion of 99 bps was found in the cDNA of the patient in the pedigree, leading to deletion of 33 codons (from codon 160 to 192). After genomic analysis, the patient was found to be a compound heterozygote of c.374C to G mutation and intron 4(IVS-4) + 5 G to C mutation. The two mutations were inherited from the parents. IVS-4 + 5 G to C mutation was a point mutation in the splice site, while c.374C to G mutation was out of the splice site. But both of them resulted in the same splice pattern in RNA. The two mutations were novel mutations which have not been reported in Human Gene Mutation Database (HGMD) and the mutation data base of Glanzmann's thrombasthenia. The results of ITGB3 gene screening is normal in the proband and his parents.

CONCLUSIONTwo novel mutation, c.374C to G and IVS-4 + 5 G to C were found in this study, which might be the cause of GT in the pedigree.

Base Sequence ; Child, Preschool ; Female ; Gene Order ; Genetic Testing ; Genotype ; Humans ; Male ; Molecular Sequence Data ; Mutation ; genetics ; Pedigree ; Platelet Glycoprotein GPIIb-IIIa Complex ; chemistry ; genetics ; Pregnancy ; Prenatal Diagnosis ; Protein Conformation ; Thrombasthenia ; diagnosis ; genetics

This study was purposed to investigate the role of integrin beta3 cytoplasmic domain in signal transduction mediated by integrin alphaIIbbeta3 and to explore the effect of integrin beta3 on signal transduction and specificity in condition without alphaIIb subunit. The fusion protein (Tac/beta3) was stably expressed in CHO cell line expressing GPIbIX, integrin alphaIIbbeta3 (IbIX/IIbIIIa-CHO cell line) by combining extracellular and transmembrane domains (Tac) of IL-2 receptor with integrin beta3 cytoplasmic domain (beta3) for formation of fusion protein (Tac/beta3). Then a series of tests were performed, including spreading and stable adhesion of IbIX/IIbIIIa-CHO cell line in solid phase fibrinogen (Fg), fibrin clot restriction and soluble fibrinogen binding, which represent outside-in and inside-out signal transduction events. The results showed that the bidirectional signal transduction mediated by alphaIIbbeta3 in IbIX/IIbIIIa-CHO/Tac-762 cells stably expressing Tac/beta3 was seriously inhibited. It is concluded that the Tac/beta3 can play a significant role in IbIX/IIbIIIa-CHO/Tac-762 cells through a dominant negative mode, the independent presence of beta3 subunit cytoplasmic domain can regulate the bidirectional signal transduction mediated by integrin alphaIIbbeta3.
Animals ; CHO Cells ; Cricetinae ; Cricetulus ; Platelet Glycoprotein GPIIb-IIIa Complex ; genetics ; metabolism ; Receptors, Interleukin-2 ; genetics ; Recombinant Fusion Proteins ; genetics ; metabolism ; Signal Transduction

OBJECTIVETo comparatively study the expressive conditions of platelet activation related factors (GP I b, GP II b- III a and GMP-140) in healthy subjects and patients with coronary heart disease (CHD) of blood-stasis (BS) or non-blood-stasis (non-BS) syndrome, and to analyze the relationship between the activities of various glycoproteins and the polymorphism of genes.

METHODSWith case control design adopted, patients with the CHD (40 of BS, 37 of non-BS) and 39 healthy subjects for control, all fitting to the inclusion criteria, were selected in this study. The number of affected coronary branches was recorded by the contrast examination. The mean fluorescence intensity (MFI) of GP I b, GP II b- III a, and GMP-140 (CD42b, CD61, CD62p) in patients and healthy persons was measured with flow cytometry, the polymorphism of HPA-3 gene was detected by Taqman probe technique and that of HPA-2 gene was determined by gene sequencing.

RESULTSMFI of CD61 and CD62p was higher in the CHD patients than in the healthy control, which was also higher in patients of BS syndrome than in patients of non-BS syndrome (P<0.05); MFI of CD42b was lower in the CHD patients than in the healthy control (P<0.05), but showing insignificant difference between BS and non-BS syndrome (P>0.05); at the same time, no significant difference of all the above-mentioned three MFI could be found in patients with various numbers of affected coronary branches, neither in patients with different genotypes at GP II b HPA-3 and GP I b HPA-2 polymorphism loci (P>0.05).

CONCLUSION(1) The activities of GP II b- III a and GMP-140 were obviously increased in the genesis and developing process of CHD and CHD of BS syndrome, and so they could be taken as one of the objective indexes for microscopic diagnosis of BS syndrome. (2) The level of GP I b was lower in CHD patients than in healthy persons, but it was not a sensitive indicator for BS syndrome of CHD. (3) Levels of GP II b- III a, GP I b and GMP-140 were not related with the number of affected coronary branches in CHD patients. (4) The changes in amino-acids expression induced by the two loci brought no significant influence on GP I b and GP II b- III a activities.

Coronary Disease ; blood ; genetics ; Female ; Humans ; Male ; Medicine, Chinese Traditional ; Middle Aged ; P-Selectin ; blood ; genetics ; Platelet Activating Factor ; analysis ; genetics ; Platelet Glycoprotein GPIIb-IIIa Complex ; analysis ; genetics ; Platelet Glycoprotein GPIb-IX Complex ; analysis ; genetics

This study was aimed to explore the possibility of rAAV2 vector-mediating gene therapy for Glanzmann' s thrombasthenia. The rAAV2-GPIIb/IIIa vector was constructed. The GPIIb/IIIa gene expression in mammal cell were examined by different methods, such as: detection of mRNA expression in BHK-21 cells after 24 hours of infection (MOI = 1 x 10(5) v.g/cell) was performed by RT-PCR; the relation between MOI and quantity of GPII6/IIIa gene expression was detected by FACS after 48 hours of infection; GPIIb/IIIa protein expression in BHK-21 cells after 48 hours of infection (MOI = 10(5) v x g/cell) was assayed by Western blot, GPIIb/IIIa protein expression on cell surface was detected by immunofluorescence, and the biological function of expressing product was determined by PAC-1 conjunct experiments. The results showed that GPIIb/IIIa gene expression in mRNA level could be detected in BHK-21 cells after 24 hours of infection at MOI = 1 x 10(5) v x g/cell and the GPIIb/IIIa gene expression in protein level could be detected in BHK-21 cells after 48 hours of infection at MOI = 1 x 10(5) v x g/cell. In certain range, quantity of GPIIb/IIIa gene expression increased with MOI, but overdose of MOI decreased quantity of GPIIb/IIIa gene expression. Activated product of GPIIb/IIIa gene expression could combined with PAC-I, and possesed normal biological function. In conclusion, rAAV2 vactor can effectively mediate GPIIb and GPIIIa gene expressing in mammal cells, and the products of these genes exhibit biological function. This result may provide a basis for application of rAAV2 vector in Glanzmann's thrombasthenia gene therapy in furture.
Dependovirus ; genetics ; metabolism ; Genetic Vectors ; genetics ; Humans ; Platelet Glycoprotein GPIIb-IIIa Complex ; biosynthesis ; genetics ; Platelet Membrane Glycoprotein IIb ; genetics ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Thrombasthenia ; metabolism ; therapy

OBJECTIVETo study the effect of glycoprotein (GP) alpha II bA2334C mutation on the biosynthesis and expression of alpha II bbeta3 complex.

METHODSThe GP alpha II bA2334C eukaryotic expression plasmid pc3.1-2334M2b was constructed. Chinese hamster ovary (CHO) cells were transfected with the plasmid with or without integrin beta3 expression plasmid pc3.1-3a. The whole expression of alpha II bA2334C was confirmed by Western blot and the membrane expression was analyzed by flow cytometry. A newly constructed alpha II bA2334C GFP fusion protein expressing plasmid was used to determine its subcellular localization by laser confocal scanning microscopy.

RESULTSExpression of the mutant protein, alpha II bA2334C, in the transfected CHO cells was confirmed by Western blot with a lower rate of the mature type than the wild type control. The expression on membrane was only 25% of the normal. Subcellular localization analysis showed that alpha II bA2334C GFP was able to be expressed in CHO cells and could be transported from endoplasmic reticulum to Golgi apparatus.

CONCLUSIONSThe mutant alpha II bA2334C can be synthesized in CHO cells and form alpha II bbeta3 complex. However, only a small fraction of the premature alpha II bA2334C can be transported to Golgi apparatus and transformed to mature alpha II b. The possible pathogenesis of this type II thrombasthenia may be that the misfolded alpha II bA2334C is partially degraded in the endoplasmic reticulum causing lower expression of alpha II bbeta3 complex on the membrane and resulting in impared function of platelets than normal alpha II b.

Animals ; Biological Transport ; Blotting, Western ; CHO Cells ; Cricetinae ; Cricetulus ; Female ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Liposomes ; Microscopy, Confocal ; Middle Aged ; Mutation ; Plasmids ; genetics ; Platelet Glycoprotein GPIIb-IIIa Complex ; genetics ; metabolism ; Transfection

To investigate the effect of GFP fused to C terminal of integrin alpha(IIb) on the biosynthesis and expression of alpha(IIb) beta(3) compound, the alpha(IIb) GFP expression plamid, named palpha(IIb) GFP, the cDNA of alpha(IIb) was constructed from p3.1-2b and fused to pEGFP-N1 in frame. When the sequence of palpha(IIb) GFP was confirmed by sequencing it was transferred to Chinese Hamster Ovary (CHO) cells with or without p3.1-3a expressing integrin beta(3). Then the expression of alpha(IIb) GFP fusion protein was confirmed by Western blot and then its subcellular localization was determined with laser confocal scanning microscopy. The results showed that the target gene was cloned into recombinant vector by restriction analysis and sequencing. Overexpression of the fusion protein in the transfected CHO cells was identified with Western blot. Subcellular localization analysis confirmed that alpha(IIb) GFP was expressed in CHO cells and could be transferred from endoplasmic reticulum to Golgi apparatus. It is concluded that the eukaryotic expression plasmid containing alpha(IIb) GFP fusion gene is successfully constructed. GFP fused to the cytoplasmic tail of integrin alpha(IIb) allows the normal expression of alpha(IIb) beta(3) in CHO cells.
Animals ; Blotting, Western ; CHO Cells ; Cricetinae ; Cricetulus ; Endoplasmic Reticulum ; metabolism ; Golgi Apparatus ; metabolism ; Green Fluorescent Proteins ; genetics ; metabolism ; Microscopy, Confocal ; Platelet Glycoprotein GPIIb-IIIa Complex ; genetics ; metabolism ; Recombinant Fusion Proteins ; genetics ; metabolism ; Transfection

OBJECTIVETo investigate the role of cytoplasmic domain of integrin alpha IIb in platelet signal transduction.

METHODSBinding capacity of integrin alpha IIb(R995A) to antibody platelet activation complex-1 (PAC-1) and pp125 focal adhesion kinase (FAK) phosphorylation of cells were detected by flow cytometry, immune precipitation, and Western blotting.

RESULTSWithout activation, wild-type alpha IIb beta3 Chinese hamster ovary (CHO) cells failed to bind to PAC-1, but mutant chimera alpha IIb(R995A)beta3 CHO cells were able to bind with PAC-1. Furthermore, phosphorylation of pp125 (FAK) in wild-type alpha IIb beta3 CHO cells occured only when cells were adhered to fibrinogen, but could not be detected in bovine serum albumin suspension. However in the mutant chimera group, it could be detected in both conditions.

CONCLUSIONThe mutation in integrin alpha IIb(R995A) alters its affinity state as a receptor, thus also mediating cytoplasmic signal transduction leading to the phosphorylation of pp125 (FAK) without ligand binding.

Animals ; Blood Platelets ; metabolism ; CHO Cells ; Cell Adhesion ; Cricetinae ; Cricetulus ; Cytoplasm ; metabolism ; Dual Specificity Phosphatase 2 ; Focal Adhesion Kinase 1 ; Focal Adhesion Protein-Tyrosine Kinases ; Humans ; Phosphorylation ; Platelet Glycoprotein GPIIb-IIIa Complex ; genetics ; metabolism ; physiology ; Point Mutation ; Protein Phosphatase 2 ; Protein Tyrosine Phosphatases ; metabolism ; Protein-Tyrosine Kinases ; metabolism ; Signal Transduction ; Transfection

OBJECTIVETo gain an insight into the relations between human leukocyte antigen-DRB1 (HLA-DRB1) alleles and idiopathic thrombocytopenic purpura (ITP) in children.

METHODSPolymerase chain reaction-sequence specific oligonucleotide (PCR-SSO) was used to identify DRB1 alleles of 42 children with ITP. Among them, 36 were identified for anti-GPIIb/IIIa and anti-GPIb/Ix autoantibody by modified monoclonal antibody specific immobilization of platelet antigens.

RESULTSCompared with health controls, the frequency of HLA-DRB1*17 significantly increased (P<0.05, relative risk=2.76, etiologic factor=0.1064) and the frequency of HLA-DRB1*1202 significantly decreased (P<0.025, relative risk=0.20, prophylactic factor=0.7616) in children with ITP. In comparison with patients of good response to steroids and IVIgG therapy, the frequency of HLA DRB1*11 significantly increased (Chi-square=6.091, P<0.025) in patients with a poor response, furthermore, the most of HLA-DRB1*11 positive patients were female teen-agers. Twenty-seven patients (75%) had anti GPIIb/IIIa and seventeen (47.22%) had anti_GPIb/Ix autoantibodies. The positivities of both anti_GP IIb/IIIa (P=0.02) and anti-GPIb/Ix (P=0.01) were associated with HLA-D RB1*02. However, the positivity of autoantibodies between refractory and non-refractory patients showed no significant difference.

CONCLUSIONThe allele of HLA-DRB1*17 seems to predict susceptibility of ITP in children, while HLA-DRB1*1202 appears to be protective to ITP. The allele of HLA DRB1*11 plays an important role in resistance to steroid and IgG therapy in children with ITP. It seems that the response to the antigenic epitope of GPIIb/IIIa and GPIb/Ix is restricted by HLA-DRB1*02, while the presence of the antibodies could not predict prognosis. In conclusion, the above preliminary findings indicate that genetic factors influence the clinical course of ITP, but the exact mechanism needs to be investigated further.

Adolescent ; Alleles ; Autoantibodies ; blood ; Child ; Child, Preschool ; DNA ; genetics ; Drug Resistance ; genetics ; Female ; Gene Frequency ; Genotype ; HLA-DR Antigens ; genetics ; HLA-DRB1 Chains ; Humans ; Immunoglobulin G ; therapeutic use ; Male ; Platelet Glycoprotein GPIIb-IIIa Complex ; immunology ; Platelet Glycoprotein GPIb-IX Complex ; immunology ; Platelet Membrane Glycoproteins ; Purpura, Thrombocytopenic, Idiopathic ; blood ; drug therapy ; genetics ; Steroids ; therapeutic use