The Effect of Ena/VASP Family on the Expression of GPIb-IX Complex in Human Megakaryoblastic Leukemia Dami Cells / 中国实验血液学杂志

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 LipofectamineTM 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.

Regulatory Effect of Protein Disulfide Isomerase on Platelet GPIbα Ectodomain Shedding / 中国实验血液学杂志

<p><b>OBJECTIVE</b>This study was aimed to investigate the regulatory effect of protein disulfide isomerase (PDI) on platelet GPIbα ectodomain shedding.</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>neither 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.</p><p><b>CONCLUSION</b>PDI 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.</p>

Identificación por nuestro grupo de una nueva mutación en el gen GP1BA (P.W246L) asociada al fenotipo PT-VWD. Sexta mutación reportada internacionalmente / Identification by our group of a novel mutation in the GP1BA gene (P.W246L) responsible for PT-VWD. The 6th mutation internationally reported

La enfermedad de von Willebrand tipo plaquetario (PT-VWD) y tipo 2B (2B-VWD) son trastornos hemorrágicos raros, caracterizados por agregación plaquetaria a bajas concentraciones de ristocetina (RIPA). El diagnóstico diferencial no es fácil y representa un desafío. Hasta el presente, sólo se habían reportado cinco mutaciones en el gen GP1BA relacionadas con este desorden. Describimos aquí la sexta mutación relacionada con PT-VWD, en un paciente con sintomatología hemorrágica severa, macro-trombocitopenia, leve agregación plaquetaria espontánea, RIPA positivo a 0,3 y 0,4 mg/mL, VWF:RCo/VWF: Ag<0,2 y estudios discriminatorios positivos para PT-VWD. VWFpp/VWF: Ag resultó normal a diferencia del 2B-VWD que en algunas oportunidades resulta afectado. El exón 28 del gen VWF del paciente y su madre no reveló mutaciones. Identificamos una sustitución G>T en el nucleótido 3805 en el gen GP1BA del paciente, resultando en un cambio de Trp a Leu en el residuo 246 (p.W246L), en la región de la GPIBa que une al VWF. Esta mutación no se identificó en su madre ni en 100 controles sanos. Es considerada como dañina por análisis in sílico. Consideramos que esta sustitución es responsable del fenotipo PT-VWD del paciente. Dada la ausencia de la misma en los 100 normales estudiados, no se considera un polimorfismo

Platelet-type von Willebrand disease (PT-VWD) and type 2B von Willebrand disease (2B-VWD) are rare bleeding disorders characterized by increased ristocetin-induced platelet aggregation (RIPA) at low concentrations. Diagnosis of either condition is not easy and the differential diagnosis is especially challenging. Five mutations in the GP1BA gene related to PT-VWD and near 50 patients are currently reported worldwide. We herein describe a patient with severe bleeding symptoms, macro thrombocytopenia, mild spontaneous platelet aggregation, positive RIPA at 0.3 and 0.4 mg/mL, VWF: RCo/VWF: Ag <0.2, normal VWFpp/VWF: Ag ratio, and RIPA mixing tests and cryoprecipitate challenge positive for PT-VWD. GP1BA gene was studied in the patient, his mother, and 100 healthy control subjects. We identified a substitution G>T at nucleotide 3805 in the patient's GP1BA gene, resulting in a Trp to Leu amino acid change at residue 246 (p.W246L), within the VWF binding region. This mutation was absent in his unaffected mother and also in the 100 controls, and was predicted as damaging by in silico analysis. The residue is located in a strongly conserved position in the phylogenetic tree. These findings argue in favor of considering this substitution does not represent a polymorphism, and is therefore responsible for the PT-VWD phenotype of the patient

Validación de una técnica de actividad de factor von Willebrand que utiliza glicoproteína Ib mutada / Validation of a method based on a mutant glycoprotein Ib for von Willebrand factor activity measure / Validação de uma técnica da atividade do fator de von Willebrand que usa mutante de glicoproteína Ib

La medida de actividad del factor von Willebrand (vWF) es importante para el diagnóstico de la enfermedad de von Willebrand (vWD). El equipo Innovance®vWF Ac (Siemens Healthcare Diagnostics) utiliza un método inmunoturbidimétrico diseñado para coagulómetros Sysmex CA (Siemens). El objetivo del estudio fue desarrollar y validar la metodología del reactivo Innovance®vWF Ac para el coagulómetro ACL TOP 700 (Instrumentation Laboratory, IL) y realizar un estudio comparativo de su desempeño frente a la de la técnica vWF Activity (vWF Act, IL) y a la del cofactor de ristocetina (vWF:RCo) por agregación de transmisión de luz. Se desarrolló, calibró y validó la técnica mediante estudios de verificación de linealidad y precisión total para dos niveles de control. Se midió la actividad del vWF por los métodos Innovance®vWF Ac y vWF Act en 82 muestras consecutivas dentro del rango reportable. Para comparar métodos se utilizó la regresión de Deming. El análisis inicial de los resultados de Innovance®vWF Ac y vWF Act demostró que no eran estadísticamente comparables. Se observó un Bias negativo para Innovance®vWF Ac en muestras con vWF Act>150%. Excluyendo las mismas, se demostró que los resultados de ambas pruebas eran comparables, al igual que los de Innovance®vWF Ac y vWF:RCo en un subgrupo de 27 muestras. La adaptación del método Innovance®vWF Ac en el ACL TOP 700 fue exitosa, presentó linealidad y precisión acorde con los requerimientos de calidad del laboratorio, brindando resultados comparables a los de vWF Act y vWF:RCo en el rango de 5-150%, útil para el diagnóstico de vWD.

Measurement of vonWillebrand factor (vWF) activity is important for the diagnosis of von Willebrand disease (vWD). Innovance®vWF Ac assay was designed to Sysmex CA (Siemens) coagulometer. The aim of the study was to develop and validate a method for Innovance®vWF Ac in the ACL TOP 700 (Instrumentation Laboratory) coagulometer and to compare its performance against vWF Activity assay (Instrumentation Laboratory, vWF Act) and Ristocetin cofactor assay by light transmission aggregation (vWF:RCo). The method was designed, calibrated and validated through verification of linearity and total precision studies at two levels of control plasmas. vWF activity was measured by Innovance®vWF Ac and vWF Act in 82 consecutive samples with vWF Act values within the reportable range. For method comparison, Deming regression was used. The initial analysis of results showed that they were not statistically comparable with a negative Bias with Innovance®vWF Ac in samples with vWF Act>150%. Excluding these samples, Deming regression curve showed that both tests gave statistically comparable results, and also did Innovance®vWF Ac and vWF:RCo in a subset of 27 samples. The adaptation of the Innovance®vWF Ac method on the ACL TOP 700 coagulometer was successful, it met the quality requirements of the laboratory for linearity and accuracy, providing comparable results to vWF Act and vWF:RCo in the of 5-150% range, useful for vWD diagnosis.

Medir a atividade do fator von Willebrand (vWF) é importante para o diagnóstico da doença de von Willebrand (vWD). A equipe Innovance®vWF Ac (Siemens Healthcare Diagnostics) utiliza um método imunoturbidimétrico projetado para coagulômetros Sysmex CA (Siemens). O objetivo do estudo foi desenvolver e validar a metodologia do reagente Innovance®vWF Ac para o coagulômetro ACL TOP 700 (laboratório de instrumentação, IL) e realizar um estudo comparativo do seu desempenho perante a técnica vWF Activity (vWF Act, IL) e a do cofator de ristocetina (vWF:RCo) por agregação de transmissão de luz. A técnica foi projetada, calibrada e validada através de testes de verificação da linearidade e total precisão para dois níveis de controle. Foi mensurada a atividade do vWF pelos métodos Innovance®vWF Ac e vWF Act em 82 amostras consecutivas dentro do intervalo reportável. Para comparar métodos foi utilizada a regressão de Deming. A análise inicial dos resultados da Innovance®vWF Ac e vWF Act demonstrou que eles não eram estatisticamente comparáveis. Observou-se um viés negativo para Innovance®vWF Ac em amostras com vWF Act> 150%. Excluindo as mesmas, foi demonstrado que os resultados de ambos os testes eram comparáveis, do mesmo modo que os Innovance®vWF Ac e vWF:RCo num subgrupo de 27 amostras. A adaptação do método Innovance®vWF Ac no ACL TOP 700 foi bem-sucedida, apresentou linearidade e precisão conforme os requisitos de qualidade do laboratório, fornecendo resultados comparáveis aos vWF Act e vWF:RCo na faixa de 5-150%, útil para o diagnóstico da vWD.

Novel Combination Markers for Predicting Survival in Patients with Muscle Invasive Bladder Cancer: USP18 and DGCR2

We performed gene expression profiling in bladder cancer patients to identify cancer-specific survival-related genes in muscle invasive bladder cancer (MIBC) patients. Sixty-two patients with MIBC were selected as the original cohort and another 118 MIBC patients were chosen as a validation cohort. The expression of USP18, DGCR2, and ZNF699 genes were measured and we analyzed the association between gene signatures and survival. USP18 and DGCR2, were significantly correlated to cancer-specific death (P=0.020, P=0.007, respectively). Cancer-specific survival in the low USP18 or DGCR2 expression group was significantly longer than the high expression group (P=0.018, P=0.006, respectively). In multivariate Cox regression analysis, a combination of USP18 and DGCR2 mRNA expression levels were significant risk factors for cancer-specific death (HR, 2.106; CI, 1.043-4.254, P=0.038). Overall survival and cancer-specific survival rates in the low-combination group were significantly longer than those in the high-expression group (P=0.001, both). In conclusion, decreased expressions of USP18 and DGCR2 were significantly associated with longer cancer-specific survival, and also the combination of two genes was correlated to a longer survival for MIBC patients. Thus, the combination of USP18 and DGCR2 expression was shown to be a reliable prognostic marker for cancer-specific survival in MIBC.

Relationship between the expression of autoantibodies against platelet membrane glycoprotein and therapeutic effect in primary immune thrombocytopenia / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the expression of specific anti- platelet glycoprotein autoantibodies GP II b/III a, GP I b/IX and GP I a/II a in primary immune thrombocytopenia (ITP), and to evaluate the relationship between the therapeutic effect and the expression of specific anti- platelet glycoprotein antibodies GPIIb/IIIa, GPIb/IX and GPIa/IIa.</p><p><b>METHODS</b>Anti-GPIIb/IIIa, GPIb/ IX and GP I a/II a antibodies were assayed by ELISA for patients with ITP. Total 442 patients in our hospital, who were retrospectively investigated from December 2010 to November 2012, were divided into newly diagnosed ITP, persistent and chronic ITP. The expression of specific anti- platelet glycoprotein antibody in each group was measured separately. The newly diagnosed ITP patients were treated with intravenous IgG (IVIG) and corticosteroids. The relationship between the expression of specific anti- platelet glycoprotein antibodies GPIIb/IIIa, GPIb/IX and GPIa/IIa and the complete response (CR) was studied.</p><p><b>RESULTS</b>Positive rates of anti- platelet glycoprotein antibodies were 59.09%, 26.97% and 37.35% respectively in newly diagnosed ITP, persistent and chronic ITP, the difference was statistical significant (P<0.05). In newly diagnosed ITP, positive rate of antibody against GPIIb/IIIa was 38.64%, double positive rate of antibodies against both GP II b/III a and GP I a/II a was 15.91%, there was statistical significance (P<0.05) compared with that of persistent and chronic ITP. The complete response (CR) rate in newly diagnosed ITP patients with positive antibody against GP II b/III a was 80.39% after treatment with IVIG and corticosteroids. There was statistical significance compared with that in patients having no antibodies (P<0.05).</p><p><b>CONCLUSION</b>The expression of antibodies against GP II b/III a and double positive for both GP II b/III a and GP I a/II a autoantibodies increased in newly diagnosed ITP patients. Patients with anti-GP II b/III a autoantibody had good response to medication with IVIG and corticosteroids.</p>

Changes in platelet GPIbα and ADAM17 during the acute stage of atherosclerotic ischemic stroke among Chinese / 华中科技大学学报（医学）（英德文版）

Glycoprotein (GP) Ibα ectodomain shedding has important implications for thrombosis and hemostasis. A disintegrin and metalloproteinase 17 (ADAM17) was identified to play an essential role in agonist induced GPIbα shedding. The relationship of GPIbα shedding and ADAM17 in the acute stage of atherosclerotic ischemic stroke (AIS) patients has not been thoroughly studied. A total of 306 patients and 230 controls matched for age, sex, race, history of hypertension and diabetes mellitus were enrolled in the study. GPIbα, ADAM17, glycocalicin were detected by flow cytometry, Western blotting, and enzyme-linked immunosorbent assay (ELISA) respectively. Compared with the control group, the expression of GPIbα in patients with acute ischemic stroke was significantly lower (P=0.000, P<0.01). Plasma glycocalicin and ADAM17 in AIS group were higher than those in control group (P=0.699, P=0.000). Pearson's analysis showed glycocalicin bore no correlation with GPIbα in AIS patients (r=0.095, P>0.05). GPIbα and National Institute of Health Stroke Scale (NIHSS) had negative correlation (r=-0.514, P<0.01). Our findings indicate that ADAM17 may be a risk factor for ischemic stroke in Chinese and the expression of GPIbα can serve as a measure for stroke severity.

Influence of S-nitrosoglutathione on the membrane glycoprotein of frozen platelets / 中国实验血液学杂志

This study was purposed to explore the influence of S-nitrosoglutathione (GSNO) on membrane glycoprotein of frozen platelet. The levels of membrane glycoprotein on fresh liquid platelets, frozen platelets and frozen platelets with GSNO were measured by flow cytometry. The results showed that the GSNO obviously decreased platelet aggregation, the PAC-1 change in the three groups was not significant. The changes of CD42b and CD62P in fresh liquid platelet group, frozen platelet group and frozen platelets with GSNO were significant different. The change of membrane glycoprotein in above-mentioned three group was not significant. It is concluded that the GSNO inhibits platelet aggregation, maintains the function of platelets and may be used as a cryoprotectant. When frozen platelets were added with GSNO, the molecular rearrangement, structure change and other mechanism may occur in platelets.

Abnormal expression of the platelet membrane receptor glycoprotein Ib-IX-V complex in transiently transfected HEK 293T cells / 中国实验血液学杂志

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.

A novel diagnostic measure of platelet-specific antibody in immune thrombocytopenia / 中华血液学杂志

<p><b>OBJECTIVE</b>To detect the platelet glycoprotein-specific antibodies in serum of thrombocytopenia patients and evaluate its diagnostic value for immune thrombocytopenia.</p><p><b>METHOD</b>Anti-GPIIb/IIIa, GPIb/IX and GPIa/IIa antibodies were assayed by ELISA kit (PAKUTO) in patients with thrombocytopenia.</p><p><b>RESULTS</b>The sensitivity and specificity of PAKAUTO in immune thrombocytopenia were 44.0% and 95.7%, respectively. The values of positive and negative predictions were 98.0% and 26.2%, respectively. Among those PAKAUTO positive patients, positive rates of GPIIb/IIIa, GPIa/IIa and GPIb/IX were 87%, 35% and 10%, respectively. The positive rate of patients not received immune suppressive agents (58.5%) was significantly higher than those received immune suppressive agents (26.9%) (P < 0.01). The positive rate of patients with platelet count ≤ 20 × 10(9)/L (51.6%) was significantly higher than those with platelet count > 20 × 10(9)/L (27.8%) (P < 0.01). The positive rate of patients with secondary immune thrombocytopenia (66.7%) was significantly higher than those with primary immune thrombocytopenia (41.7%) (P < 0.05).</p><p><b>CONCLUSION</b>The highly specific method (PAKAUTO) could effectively differentiate immune or non-immune thrombocytopenia and be applied to diagnosis of immune thrombocytopenia.</p>

Effect of polymorphism and type II diabetes on aspirin resistance in patients with unstable coronary artery disease / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Aspirin is widely used in the secondary prevention of coronary artery diseases, including myocardial infarction, stroke, and vascular related deaths. However, the antiplatelet effect of aspirin appears to be variable and aspirin resistance (AR) is currently still controversial for Chinese patients. The aim of this study was to describe the prevalence of AR, and identify possible risk factors associated with a lack of response to aspirin treatments in patients with unstable coronary artery disease.</p><p><b>METHODS</b>Platelet function tests with arachidonic acid (ARA) and urinary 11-dehydro-thromboxane B2 (11-DH-TXB2) concentrations were performed in 262 patients with unstable coronary artery disease who had not been taking aspirin before admission. ARA induced platelet aggregation and 11-DH-TXB2 were detected to evaluate the functional and biochemical responses to aspirin before and on days 1, 4, and 10 after aspirin administration. Six-month follow-up was completed in patients who developed AR to evaluate the effect of aspirin in a long-term treatment. GP1Bα (C1018T), Pl (A1/A2), P2Y1 (A1622G), TBXA2R (T924C) were also detected to evaluate the influence of genetic variant on aspirin responsiveness.</p><p><b>RESULTS</b>A total of 8.8% of patients were indentified as AR at the first day after aspirin treatment. The level of urine 11-DH-TXB2 in the AR group was higher compared to non-AR group (P < 0.05). There was no relationship between ARA induced platelet aggregation and urinary 11-DH-TXB2 levels (r = 0.038, P = 0.412). The results of DNA sequencing showed that TBXA2R-924TT homozygotes had a significantly high rate of AR. Logistic regression demonstrated that diabetes was an independent risk factor of AR.</p><p><b>CONCLUSIONS</b>In the beginning period of administration, aspirin was not a sufficient factor that inhibits platelet aggregation. TBXA2R-924T allele was involved in AR. Diabetes was an independent risk factor of AR.</p>

The role of amino acid sequence between 551 and 565 in the cytoplasmic domain of glycoprotein (GP) I b alpha in the regulation of the VWF binding to GP I b alpha / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the role of the amino acids between 551 and 565 in the cytoplasmic domain of glycoprotein (GP) I b alpha in the VWF binding to GP I b alpha.</p><p><b>METHODS</b>The VWF binding to GP I b alpha induced by ristocetin was analyzed by flow cytometry, in three GP I b-IX-expressing Chinese hamster ovary (CHO) cell lines 1b9, delta 565 and delta 551, adhesion of above cells on VWF by flow chamber analysis at shear rate of 200 s(-1). The spread of GP I b-IX-expressing cells were stimulated with botrocetin on VWF-coated coverslips by confocal microscope.</p><p><b>RESULTS</b>The VWF binding to GP I b alpha was higher in delta 565 cells stimulated by ristocetin than in delta 551 or 1b9 cells. The number of delta 565 cells adhered on the VWF-coated-chamber was more than that of controls at shear rate of 200 s(-1). Moreover, the surface spreading areas of delta 565 cells were greater than that of the controls on VWF-coated coverslips.</p><p><b>CONCLUSIONS</b>The amino acids between 551 and 565 in the cytoplasmic domain of GP I b alpha regulates the VWF binding to GP I b alpha.</p>

Association of serotonin transporter gene linked polymorphic region polymorphism with early onset myocardial infarction and platelet membrane glycoprotein I b / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the association of serotonin transporter gene linked polymorphic region (5-HTTLPR) insertion/deletion polymorphism with early onset myocardial infarction(MI) and platelet membrane glycoprotein I b(GP I b) in Northern Han population of China.</p><p><b>METHODS</b>A total of 150 patients with early onset MI and 150 age- and sex-matched controls with negative coronary arteriography were genotyped for the 5-HTTLPR polymorphism by using a polymerase chain reaction-based technique. The percentage of positive platelet membrane GP I b and the average fluorescence intensity were quantified by flow cytometry.</p><p><b>RESULTS</b>The genotype frequencies of LL, LS and SS in the 5-HTTLPR were 32%, 47% and 21% in the MI patients, 17%, 43% and 39% in the controls respectively(P<0.01). The L allele frequency in the MI patients was significantly higher than that of the control group (56% vs 39%, P<0.01). The percentage of positive platelet membrane GP I b and the fluorescence intensity in subjects with LL homozygote were markedly lower than that of LS and SS genotypes in the MI and control groups (all P<0.01). Multivariate logistic regression analysis showed that the 5-HTTLPR LL genotype was independently related to the occurrence of early onset MI(OR was 1.961, P was 0.037).</p><p><b>CONCLUSION</b>The LL genotype of the 5-HTTLPR might be associated with the susceptibility to developing early MI in Northern Han population of China. The platelet activation is increased in individuals of LL genotype.</p>

Expression of anti-platelet glycoprotein specific antibodies and anti-HLA antibodies in idiopathic thrombocytopenic purpura / 中国实验血液学杂志

In order to investigate the expression of the anti-platelet glycoprotein specific antibodies and anti-HLA antibodies in idiopathic thrombocytopenic purpura (ITP), 45 patients with ITP were selected in this study. An easy PCR-SSP assay was used to detect single-nucleotide polymorphisms or deletion in HPA and HLA systems. The anti-platelet glycoprotein specific antibodies and anti-HLA antibodies in plasma or platelet eluate were tested with a solid phase ELISA. The results indicated that the anti-platelet glycoprotein specific antibodies were detected in plasma or platelet eluate of 45 patients, among which anti-GPIIb/IIIa/and anti-GpIb/IX were most common. Both the anti-platelet glycoprotein specific antibodies and anti-HLA antibodies were found in plasma of 11 patients. Pedigree investigation in 2 patients (case 37 and case 40) was carried out, the results showed that anti-platelet glycoprotein specific antibodies and anti-HLA antibodies detected in 2 patients closely related to incompatibility with platelet antigens and HLA antigens in parents. In conclusion, the results suggested that detection of the anti-platelet glycoprotein specific antibodies and anti-HLA antibodies in plasma or platelet eluate in combination with investigation of clinical manifestation of patients is important for diagnosis of idiopathic thrombocytopenic purpura.

Influence of different products of platelet membrane glycoprotein monoclonal antibodies used internationally on tests for monoclonal antibody-specific immobilization of platelet antigens / 中国实验血液学杂志

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.

Inhibition of protein kinase A leads to cleavage of platelet GP I balpha and downregulation of GP I b-dependent platelet aggregation / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the regulatory role of protein kinase A (PKA) in platelet surface glycoprotein (GP) I balpha expression.</p><p><b>METHODS</b>Washed platelets from healthy volunteers were incubated with PKA inhibitor. The N-terminal fragment of GP I balpha (glycocalicin, GC) in the supernatant of platelet suspensions was detected by Western blot and GP I balpha surface expression by flow cytometry. Calpain activity was determined by cytoskeletal proteins proteolysis and calpain surface expression by flow cytometry. The effect of PKA inhibitor on ristocetin-induced platelet aggregation was measured by platelet aggregometer.</p><p><b>RESULTS</b>After PKA was inhibited in washed platelets, GP I balpha was cleaved and released to the supernatant, which significantly decreased the surface expression of GP I balpha (P < 0.05). The event was suppressed by pre-treatment with various calpain inhibitors, indicating that PKA inhibitor-mediated shedding was calpain dependent. The actin-binding protein (ABP) and talin proteolysis demonstrated that calpain was activated by PKA inhibitor and expressed on the platelet membrane. Ristocetin-induced aggregation was inhibited by PKA inhibitor.</p><p><b>CONCLUSION</b>PKA inhibition results in calpain-dependent GP I balpha shedding, which thus reduces GP I balpha surface expression and GP I balpha-dependent platelet aggregation. These results might provide a view to develop new drugs for thrombotic diseases.</p>

Genetic analysis of genitourinary malformations / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To analyze the expression of genes from chromosomal region 22q11.2 and assess the association between mutation(s) of particular gene(s) from this region and malformations of the urinary system.</p><p><b>METHODS</b>Expression of rat homologs of 33 genes from above region was determined in kidney tissues derived from rats of different fetal development ages (E13, E15, E19) and adulthood with reverse transcriptase-PCR. Potential mutation(s) in candidate gene SNAP29, whose expression pattern appeared to be unique, was screened in 44 patients and 220 normal controls with PCR-single strand conformation polymorphism (SSCP). Suspected positive regions were sequenced to verify the mutations.</p><p><b>RESULTS</b>Nine genes showed no expression throughout the whole development process; 18 genes with various expression levels showed continuous expression from the beginning of development; 6 genes only expressed for a short time, among which SNAP29 was selected for mutation screening. Upon sequencing, three mutations were identified from the 44 patients, including a G to A transition (GAG to AAG) in exon 2, and two A to G transitions (AGC to GGC) in exon 3.</p><p><b>CONCLUSION</b>Through systematic analysis of the expression of genes from chromosomal region 22q11.2, the SNAP29 gene was found to have a potential role in the development of genitourinary system. Two missense mutations were identified in three patients. These included one in exon 2 (featuring cryptorchidism), and the other in exon 3 (featuring cryptorchidism and hypospadia). Neither of the mutations was found in the normal controls. The results suggested that mutation(s) of gene(s) from chromosomal region 22q11.2 may play an important role in the genesis of genitourinary malformations.</p>

Glanzmann's thrombasthenia and pregnancy

Glanzmann's thrombasthenia is rare autosomal recessive platelet disorder caused by abnormal platelet glycoprotein complex [GP IIb-IIIa] leading to excessive mucosal bleeding. Patients with Glanzmann's thrombasthenia may present with hemorrhagic symptoms including mucosal bleeding, easy bruising and post-surgical bleeding. We report a 37-year old lady, who underwent a cesarean section at term. She received recombinant factor VIIa at the time of surgery. She had uneventful post operative period and she was discharged home in good general conditions

Determination of the detachment force of platelet membrane protein GPIb/IX and von Willebrand factor / 生物医学工程学杂志

CHO cells expressing human GPIb/IX and rabbit red blood cells coated with human von Willebrand factor (VWF) were adapted to our study on the binding probability and the detachment force of GPIb/IX and VWF. With the micropipette system, the two cells were impinged under a constant force for controlled time. When the cells were pulled apart, the deformation of RBC was recorded, and the binding score and detachment force of the proteins were determined. After the two cells were impinged into 0.5 microm for 30 s, the binding probability of the two cells carrying GPIb/IX and VWF was 15.0%. Via analyzing the deformation of red blood cells, we found out the distribution of rupture forces of cells with GPIb/IX and VWF. Therefore, we infer that the continuous distribution of the detachment force is due to the stochastic effect. The most probable value of the detachment force was 10 pN.

Establishment of Chinese hamster ovary cell line expressing recombinant GPIb-IX complex / 中国实验血液学杂志

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.