OBJECTIVEThis study was aimed to investigate the regulatory effect of protein disulfide isomerase (PDI) on platelet GPIbα ectodomain shedding.

METHODSThe washed platelets were obtained from healthy volunteers. Platelets were incubated with PDI inhibitor bacitracin before stimulation with PMA (Phorbol-12-myristate-13-acetate), dibucaine and collagen. The N-terminal domain of GPIbα in supernatant was detected by Western blot, the GPIbα expression and the intraplatelet ROS levels were measured by flow cytometry.

RESULTSneither GC content nor GPIbα expression was changed after the washed platelets from the healthy donors were incubated only with PDI inhibitor. The washed platelets were incubated with PDI inhibitor before stimulation with different stimulin, PMA, dibucaine or collagen, and then GPIbα was cleaved and ROS levels were elevated more than that in the controls.

CONCLUSIONPDI participates in the induced GPIbα ectodomein shedding, and the effect of PDI in this process maybe depend on the change of ROS level inside platelets. These results might provide a new point of view for the platelet drug development.

Blood Platelets ; Collagen ; Flow Cytometry ; Humans ; Platelet Glycoprotein GPIb-IX Complex ; Protein Disulfide-Isomerases

OBJECTIVE: To explore the effects of Ena/VASP gene family on the expression of glycoprotein (GP) Ib-IX complex in human megakaryoblastic leukemia Dami cells. METHODS: SiRNAs targeting Ena/VASP gene family were designed and synthesized to interfere Enah, EVL and VASP gene expression. When the siRNAs were transfected into Dami cells by using Lipofectamine^TM 2000 for 48 h, the expression of GPIb-IX complex was detected by quantitative real-time PCR, Western blot and flow cytometry. RESULTS: We successfully established siVASP , siEVL and si Enah Dami cell lines. And it was found that the expression of GPIb-IX complex had no evident reduction in siEVL or siVASP Dami cells at both mRNA and protein level, while the total protein and membrane protein of GPIb-IX complex were obviously reduced when Enah was knocked down. CONCLUSION Enah could affect the expression of GPIb-IX complex in human megakaryoblastic leukemia Dami cells, but the underlying mechanism still needs to be further explored.
Humans ; Cell Line ; Platelet Glycoprotein GPIb-IX Complex/metabolism* ; Leukemia/metabolism* ; Blood Platelets/metabolism*

This study was aimed to investigate the influence of different platelet membrane glycoprotein monoclonal antibodies (McAb) which are common used in laboratories on the monoclonal antibody-specific immobilization of platelet antigens (MAIPA) technique according to the request of 14th International Society of Blood Transfusion Platelet Immunology Workshop. 30 participant laboratories were provided with 10 known human platelet antigen (HPA) antibodies, 1 normal serum, 9 different McAbs (against GPIIb/IIIa, GPIa/IIa, GPIb/IX and GPIV respectively), and the same protocol. Each participant laboratory carried out the test as the protocol to compare the results of different McAbs against the same glycoprotein and submitted the data to organizer. The results indicated that in McAbs against GPIIb/IIIa, AP2, Gi-5 and PL2-73 showed higher mean S/CO than that of others; in GPIa/IIa, MBC202.2 and 143.1 showed higher mean S/CO than that of others; in GPIb/IX, 142.11 and CLB-MB45 (CD42b) showed higher mean S/CO than that of others; as to GPIV, 131.4 showed higher mean S/CO. In conclusion, capture effects of various McAbs are different, so that different products of McAbs exert influences on the sensitivity of MAIPA. To use a panel of McAbs against the same glycoprotein may avoid the false negative results.
Antibodies, Monoclonal ; classification ; Antigens, Human Platelet ; immunology ; Humans ; Indicators and Reagents ; Platelet Glycoprotein GPIIb-IIIa Complex ; immunology ; Platelet Glycoprotein GPIb-IX Complex ; immunology ; Platelet Membrane Glycoproteins ; classification ; immunology

OBJECTIVETo investigate a specific and sensitive assay for the diagnosis of autoimmune thrombocytopenic purpura (AITP).

METHODSGlycoprotein specific autoantibodies in platelet eluate and plasma were detected by a modified monoclonal antibody immobilization of platelet antigens assay (MAIPA).

RESULTSThe overall positive rate of specific autoantibodies against platelet GPIIb/IIIa and GPIb/IX in plasma was 38.89% and in eluated platelet membrane was 68.52%. The difference between them was significant (corrected chi(2) = 19.39, P < 0.005). The proportion of positive MAIPA results between primary AITP and secondary AITP was not significantly different. There was a significant inverse correlation between antibody level and platelet count.

CONCLUSIONDetection of eluated GP-specific autoantibodies by MAIPA is highly specific and much more sensitive as compared with the measurement of their plasma counterparts in the diagnosing and therapeutic monitoring of AITP.

Adolescent ; Adult ; Autoantibodies ; blood ; Blood Platelets ; immunology ; Female ; Humans ; Male ; Middle Aged ; Platelet Glycoprotein GPIIb-IIIa Complex ; immunology ; Platelet Glycoprotein GPIb-IX Complex ; immunology ; Purpura, Thrombocytopenic, Idiopathic ; immunology

Platelet plays an important role in bleeding and thrombotic diseases. Humanized anti-platelet antibodies have great clinical effects in treatment of ITP and preventing thrombosis. The important role of platelet in bleeding and thrombotic diseases, the present status of development on study of humanized anti-platelet antibody and its application in treatment of bleeding and thrombotic diseases were summarized in this review.
Animals ; Antibodies, Monoclonal ; biosynthesis ; immunology ; therapeutic use ; Autoantibodies ; immunology ; Blood Platelets ; immunology ; Humans ; Platelet Glycoprotein GPIIb-IIIa Complex ; biosynthesis ; immunology ; Platelet Glycoprotein GPIb-IX Complex ; immunology

OBJECTIVETo observe the effect platelet antigen modification by mPEG-SPA with different molecular masses.

METHODSPlatelet CD42a was modified by 5 kD and 20 kD mPEG-SPA, respectively, and the fluorescence intensity of CD42a was detect by flow cytometry and the three-dimensional structure of CD42a simulated to analyze the distribution of lysine in CD42a molecule.

RESULTSAfter platelet CD42a modification by 5 kD and 20 kD mPEG-SPA, the fluorescence intensity of CD42a decreased sharply by 85.54% and 88.65%, respectively, and multiple lysine regions were identified on the surface of CD42a molecule.

CONCLUSIONBoth 5 kD and 20 kD mPEG-SPA allow useful modification of platelet CD42a, but 20 kD mPEG-SPA is more advantageous than 5 kD mPEG-SPA.

Blood Platelets ; chemistry ; Humans ; Molecular Weight ; Platelet Glycoprotein GPIb-IX Complex ; chemistry ; Polyethylene Glycols ; chemistry ; Succinimides ; chemistry

OBJECTIVETo identify a mutation G2113-->A in the glycoprotein (GP)IX gene associated with Bernard-Soulier syndrome (BSS) and to investigate BSS pathogenesis.

METHODSAllele-specific restriction enzyme was used to analyze the samples of patient, her mother, her brother and 40 healthy volunteers. Site-directed mutagenesis was performed to construct a expression vector PD-IXG2113A harboring the mutation G2113-->A. Chinese hamster ovary (CHO) cells were transiently cotransfected with plasmids harboring the entire coding region of GPIbalpha, GPIbeta and GPIX or mutant GPIX, respectively. Expression of GPIbalpha and GPIX in transfected CHO cells were analysed with flow cytometer. GPIbalpha and GPIX in the cytoplasma of transfected CHO cells were analysed by immunostaining and Western blotting.

RESULTSThe patient was found to be homozygosity of the substitution, her mother and her brother be heterozygous. Expressions of GPIbalpha and GPIX in mutant CHO cells were remarkably reduced, but abundant in the cytoplasma.

CONCLUSIONThe mutation of Ala139(GCC)-->Thr(ACC) in the GPIX did not affect synthesis and assembly of GPIb/IX complex but influence its anchoring and expression on the cell surface, which was responsible for BSS.

Adult ; Animals ; Bernard-Soulier Syndrome ; genetics ; Blotting, Western ; CHO Cells ; Cricetinae ; Female ; Humans ; Mutation ; Platelet Glycoprotein GPIb-IX Complex ; genetics

Age Factors ; Cardiopulmonary Bypass ; Child ; Child, Preschool ; Humans ; Infant ; Infant, Newborn ; P-Selectin ; blood ; Platelet Glycoprotein GPIb-IX Complex ; analysis

The structure and function of the glycoprotein (GP) Ib-IX-V complex has been extensively investigated over the decades due to its vital role in platelet activation. For the lack of nucleus in platelets, researchers usually need to study the GPIb-IX-V complex by transfecting wild type or mutant GPIb-IX-V plasmids into other mammalian cell lines, such as CHO or HEK 293T. Therefore, whether the characteristics of the GPIb-IX-V complex in these cell lines can truly represent that in platelets is pivotal to determine whether these cell lines are appropriate for GPIb-IX-V complex studies. In order to determine the most appropriate cell line to study the GPIb-IX-V complex, the surface expression level of the complex in different cell lines was detected and whether difference among cell lines will affect expression of the complex was explored in the present study. The different combinations of the GPIb-IX-V subunits were transfected into cell lines from different species or different tissues, such as CHO, HEK293T and HeLa, and the surface expression levels of the complex were detected by flow cytometry. The results indicated that in both transiently and stably transfected CHO cells, surface expression of GPV depended on the presence of the GPIb-IX complex, which is consistent with that in human platelets. In contrast, GPV could be efficiently expressed on surface in HEK 293T cells even in the absence of GPIb-IX, although the inter-subunit dependence within the GPIb-IX complex is still similar to that in CHO cells or human platelets. Further studies in HeLa, MES13 and HUVEC cell lines revealed that GPV could be efficiently expressed on the surface by itself in HeLa and MES13 cells, but not in HUVEC, suggesting different behaviors of the GPIb-IX-V complex in difference cell lines. It is concluded that this study provides some guidance and advice to future GPIb-IX-V complex studies, especially to the choice of suitable cell line. HEK 293T cell line, for example, is likely to provide misleading results since it could not represent the fact in human platelets, thus is not the optimal choice for the GPIb-IX-V complex, particularly the GPV subunit.
Animals ; CHO Cells ; Cricetinae ; Cricetulus ; HEK293 Cells ; metabolism ; Humans ; Platelet Glycoprotein GPIb-IX Complex ; genetics ; metabolism ; Transfection

The aim of this study was to construct Chinese Hamster Ovary (CHO) cell models expressing recombinant wild-type GPIb-IX and mutant GPIb-IX complex, so as to provide the platform to study the related physiologic functions of GPIb-IX. The plasmids were extracted from E.coli expressing wild-type or deletion mutant GPIbalpha and were identified by digestion with EcoR I. Three plasmids containing GPIbalpha, GPIbbeta, and GPIX genes were co-transfected into CHO cells, and then the expression of GPIb-IX complex was detected by immune coprecipitation, Western blot and flow cytometry. The results showed that the expression of GPIb-IX complex could be detected in the lysate and on the surface of CHO cells at 48 hours after transfection. In conclusion, CHO cell models expressing recombinant wild-type or mutation GPIb-IX complex has been successfully constructed.
Animals ; CHO Cells ; Cricetinae ; Cricetulus ; Mutation ; Plasmids ; Platelet Glycoprotein GPIb-IX Complex ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics