[The Effect of VWF Propeptide on VWF Mutant in D1 Domain].

OBJECTIVE: To investigate whether co-transfection of wild-type VWFpp with VWF mutant in D1 region is able to correct VWF defects in biosynthesis and secretion. METHODS: Four VWF mutant plasmids were single transfected into HEK 293 cells, or co-transfected into HEK 293 cells with the wild type VWFpp plasmids. The VWF in supernatant and lysate of transfected cells were analyzed by ELISA, vertical VWF multimer electrophoresis. The retention of VWF in endoplasmic reticulum of transfected cells were detected by immunofluorescence confocal microscope. RESULTS: In the vertical VWF multimer analysis, with co-expressing VWF mutant and VWFpp, the VWF multimer bands disappeared, and the VWF antigen in both supernatant and lysate of cells decreased, compared with the single expression of VWF mutant. Although the intracellular levels of VWF antigens decreased after co-expression, the retention rate of VWF mutant decreased in endoplasmic reticulum. CONCLUSION: VWFpp can reduce the retention of VWF in endoplasmic reticulum, assists the transport of VWF between subcellular organelles. However, VWFpp inhibits the biosynthesis and secretion of VWF about the mutant in D1 domain.

[Genetic and Clinical Characteristics of A Family with Combined PROC and PROS1 Genetic Variants].

OBJECTIVE: To test the anticoagulation functions, perform the genetic diagnosis and analyze the clinical characteristics in a family with combined heterozygous genetic variants of PROC and PROS1. METHODS: Peripheral blood was collected from all the family members. Hematological phenotypes and activity of anticoagulant factors were analyzed. Target genes were amplified by PCR from DNA isolated from peripheral blood, and then were analyzed by Sanger DNA sequencing. RESULTS: Many members in the family displayed the combined genetic variants in protein C and protein S, and six family members accompanied by deep venous thrombosis (DVT). The influences of genetic and secondary factors on the incidence of venous thrombosis in the family members were analyzed. The results showed that in this family, carriers of combined protein C and protein S gene defects had a higher incidence of VTE, but acquired factors still played a key role in the eventual thrombotic symptoms. CONCLUSION: Venous thromboembolism (VTE) is a multifactorial disease, the combined genetic heterozygous mutations of protein C and S is an important genetic factor, and the clinical phenotype show a high heterogenicity, the secondary factors contribute to the VTE incidence.

The Litsea genome and the evolution of the laurel family.

The laurel family within the Magnoliids has attracted attentions owing to its scents, variable inflorescences, and controversial phylogenetic position. Here, we present a chromosome-level assembly of the Litsea cubeba genome, together with low-coverage genomic and transcriptomic data for many other Lauraceae. Phylogenomic analyses show phylogenetic discordance at the position of Magnoliids, suggesting incomplete lineage sorting during the divergence of monocots, eudicots, and Magnoliids. An ancient whole-genome duplication (WGD) event occurred just before the divergence of Laurales and Magnoliales; subsequently, independent WGDs occurred almost simultaneously in the three Lauralean lineages. The phylogenetic relationships within Lauraceae correspond to the divergence of inflorescences, as evidenced by the phylogeny of FUWA, a conserved gene involved in determining panicle architecture in Lauraceae. Monoterpene synthases responsible for production of specific volatile compounds in Lauraceae are functionally verified. Our work sheds light on the evolution of the Lauraceae, the genetic basis for floral evolution and specific scents.

[Establishment of Flow Cytometric Immunobead Array Assay for Quantitation of Platelet-Specific Antibodies and Its Application].

OBJECTIVE: To establish a flow cytometric immunobead array assay (FCIA) to quantify platelet antibodies and to explore its application in the diagnosis and treatment of ITP. METHODS: The guantitative standard curve was established by binding the human IgG of known concentration on antibody-coated microbeads; at the same time, the platelet-specific antigen and antibody complex was captured and levels of platelet antibodies were detected using the microbeads coated by 5 kinds of antibodies against platelets suca as GPIX (SZ1), GPⅠb (SZ2), GpⅢa (SZ21), GPⅡb (SZ22) and p-selection (SZ51). The fluorescence signal detected by flow cytometry were transformed into the conentration of platelet antibodies in samples through the quantitative standard curve, thereby establishing the method for quantititive detection of platelet-specific antibodies in plasm samples (FCIA), moreover the property, efficiency and clinical application of establishod FCIA method were evaluated. RESULTS: The FCIA could detect 5 kinds of antibodies against GPIX, GPⅠb, GpⅢa, GPⅡb and ß-selection within a broad range of 33.29-1280 ng/ml, 45.17-1280 ng/ml, 42.07-1280 ng/ml, 46.40-1280 ng/ml, 42.48-1280 ng/ml and 42.48-1280 ng/ml respectively, and their recovery rates were 115.23%, 112.58%, 117.47%, 107.64% and 112.67% respectively. The intra-assay coefficient of variation (CV) for anti- GPIX, -GPⅠb, -GpⅢa, -GPⅡb and p-selection antibodies was 3.54%, 3.63%, 4.66%, 6.43% and 6.67% respectively, and the inter-assay CV for above mentioned antibodies were 10.89%, 7.57%, 10.34%, 6.95% and 10.72% respectively. The detection showed that the levels of 5 kinds of platelet-specific antibodies in ITP group all were higher than those in non-ITP and healthy control groups (P＜0.01). The sensitivity, specificity and accuracy of quantitatively detecting 5 kinds of antibodies for diagnosis of ITP by FCIA were 68.29%, 84.98% and 78.95% respectively, while the sensitivity, specificity and accuracy of detecting 5 kinds of antibodies by modified indirect MAIPA were 41.46%, 90.41% and 72.81% respectively. CONCLUSION: The established quantitative FCIA for detection of antibodies provides a powerful tool for diaghosis and evaluation of therapeutic efficacy and prognosis of ITP patients.

[Development of Hybridoma Cell Lines Secreting Monoclonal Antibodies against ADAMTS13].

OBJECTIVE: To establish the hybridoma cell lines secreting anti-ADAMTS13-T7 monoclonal antibodies (MA6) and to construct the MAb directed against different domains of ADAMTS13-T7. METHODS: The trucated type protein ADAMTS13 of eukaryote-expressed recombinant ADAMTS13-T7 was constructed and was purified by using Ni-NTA agarose. Then the purified ADAMTS13 was used to immunize the BALB/c mice; the antiserum titer of ADAMTS13 protein of immunized mice was deteceted by ELISA. The spleen was taken from mice for construcing the single cell suspension, then the single cell suspension was mixed with myeloma cells SP2/0 at 10:1 ratio for cell fusion, and the elution culture of hybridoma cells was carried out; after 2 weeks, the wells in which clones were well grown were selected for detection, then the positive clonal wells were expansively cultured and the detection again was performed. RESULTS: The purified ADAMTS13-T7 protein was gained. The ELISA showed that the antiserum titer in mice immumized by ADAMTS13-T7 protein was 1:20000. The results of fusion culture by hybridoma techmique showed that 30 hyridoma cell lines secreting MAb against recombinant ADAMTS13 were established. CONCLUSION: The hybridoma cell lines secreting MAb against recombinant ADAMTS13 have been successfully established by fusion culture, which provide more powerful tools for further screening the MAb with certain functional activity and studying the structure and function of ADAMTS13.

[Correlation of Plasma Co-stimulatory Molecules B7-H2 and B7-H3 with Platelet Auto-antibodies in Patients with Immune Thrombocytopenic Purpura].

OBJECTIVE: To investigate whether the plasma level of platelet auto- antibodies in ITP patients is related to that of co-stimulatory molecules sB7-H2 and sB7-H3. METHODS: A total of 61 ITP patients and 25 healthy controls from the First Affiliated Hospital of Soochow University from June 2012 to August 2013 were enrolled in this study. The expression levels of platelet auto-antibodies against 5 glycoproteins (GPIX, GP Ib, GP IIIa, GPIIb and P-selectin) in plasma were detected by flow cytometric immuno-beads array, and the expression of soluable co-stimulatory molecules sB7-H2 and sB7-H3 was measured by ELISA. RESULTS: The plasma levels of 5 auto-antibodies against platelet membrance glycoproteins significantly increased in ITP patiens (P < 0.01). Compared with healthy controls, sB7-H2 levels increased (P < 0.05), while the sB7-H3 level did not significantly change (r = 0.13, P > 0.05). However, the correlation analysis showed that sB7-H3 negatively correlated with platelet P-selectin auto-antibody (r = -0.46, P < 0.05), and sB7-H2 and sB7-H3 significantly reduced in ITP patients with positive P-selectin auto-antibody (P < 0.01). In ITP patients, platelet counts negatively correlated with sB7-H2 (r = -0.3907, P < 0.01), but did not correlate with sB7-H3. CONCLUSION: Soluble costimulatory molecule sB7-H2 elevates in ITP patients, and the level of sB7-H3 is associated with auto-antibodies against P-selectin, suggesting that costimulatory molecules B7-H2 and B7-H3 may be involved in the pathogenesis of immune regulation abnormality in ITP.

[Evaluation of a new method and instrument for detection platelet aggregation function and its clinical application].

This study was purpose to evaluate a new method and instrument for detecting platelet aggregation function, establish the reference intervals for PL-11 platelet analyzer, and evaluate its clinical application. The evaluation was based on the guidelines of Clinical and Laboratory Standards Institute (CLSI or NCCLS) and Clinical Laboratory Improvement Amendment 88. Intravenous blood samples anticoagulated with sodium citrate were detected by PL-11 platelet analyzer. The reference intervals were defined after statistic analysis. The clinical diagnostic significance of the PL-11 platelet analyzer was evaluated by testing the change rate of platelet maximum aggregation rate (MAR) of acute cerebral infarction (ACI) patients in the department of Neurology who took clopidogrel 7 d before and after. The result showed that all the parameters meet the standard of CLIA'88. The platelet MAR of 247 healthy volunteers which was induced by PLR-06, PLR-07, PLR-09 and PLR-10, was detected by the PL-11 platelet analyzer, respectively. The MAR is 58.8 ± 10.1 (%), 61.2 ± 11.8 (%), 51 ± 10.2 (%), 53.1 ± 9.2 (%), respectively. The MAR of ACI patients is significantly lower than that after taking clopidogrel. It is concluded that the PL-11 platelet analyzer is an ideal platelet function detector for early warning and diagnosis of thromboembolic disease, which is worthy to be extended and applied.

Detection of autoantibodies against platelet glycoproteins in patients with immune thrombocytopenic purpura by flow cytometric immunobead array.

BACKGROUND: The goal of this study is to develop a flow cytometric immunobead array (FCIA) assay to detect platelet autoantibodies commonly present in bleeding patients with immune thrombocytopenic purpura (ITP). METHODS: Polystyrene microbeads coated with antibodies against human platelet glycoproteins (GPs) IX (SZ1), Ib (SZ2), IIIa (SZ21), IIb (SZ22), and P-selectin (SZ51) were incubated with platelet lysate from 50 ITP patients and 86 controls. The platelet antigen-autoantibody complexes were detected by flow cytometry using an FITC-labeled antibody. The results were compared with that of a monoclonal antibody immobilization of platelet antigen (MAIPA) assay. RESULTS: By FCIA, platelet autoantibodies against GPIb, GPIIb, GPIIIa, GPIX and P-selectin were detected in ITP patients. Mean fluorescent intensity values with antibodies SZ1, SZ2, SZ21, SZ22 and SZ51 were all higher in ITP patients than controls (p values<0.01). In ROC analysis, values of the area under the curve were 0.89, 0.82, 0.93, 0.94 and 0.95, respectively. In ITP diagnosis, the FCIA assay with these five antibodies had better sensitivity and accuracy than the MAIPA assay (96% vs. 44% in sensitivity; 80.9% vs. 64.7% in accuracy, p<0.01). CONCLUSION: FCIA assays with multiple antibodies against platelet GPs may be used to improve the diagnosis of ITP in hospitals.