Yang He

Soochow University (PRC), Wu-hsien, Jiangsu Sheng, China

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Publications (9)12.6 Total impact

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    ABSTRACT: Autoantibodies against platelet glycoproteins (GPs) play an important role in immune thrombocytopenic purpura (ITP). This study was to develop an improved flow cytometric immunobead array (FCIA) assay to detect platelet autoantibodies in ITP patient plasma.
    09/2014;
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    ABSTRACT: Uncoordinated-5 homolog B receptor (UNC5B) was first found to mediate neural chemorepulsive effects by binding to its ligand netrin-1 in the nervous system. Newer evidence indicated that UNC5B also has functions outside the nervous system. In this study, we report on the generation of a monoclonal antibody specific to the outer-membrane immunoglobulin-like domains of UNC5B using the hybridoma technique. Western blot, immunofluorescence, and flow cytometry analyses showed that the antibody specifically bound to UNC5B protein. Interestingly, the antibody blocked the Netrin-1-induced inhibitory effect on the mobility of melanoma A375 cells by wound healing assay and transwell migration assay, whereas it had no effects on cell proliferation measured by CCK-8 assay. Thus, the functional antibody may provide a useful tool for the study of UNC5B expression profiles and functions outside the nervous system.
    Monoclonal antibodies in immunodiagnosis and immunotherapy. 08/2014; 33(4):280-6.
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    ABSTRACT: Matriptase-2 is a hepatic membrane serine protease that regulates iron homeostasis. Defects in matriptase-2 cause iron deficiency anemia. In cells, matriptase-2 is synthesized as a zymogen. To date, how matriptase-2 expression and activation are regulated remains poorly understood. Here we expressed human matriptase-2 in HEK293 and hepatic BEL-7402, SMMC-7721 and QGY-7703 cells. By labeling cell surface proteins and Western analysis, we examined matriptase-2 cell surface expression, zymogen activation and ectodomain shedding. Our results show that matriptase-2 was activated on the cell surface but not intracellularly. Activated matriptase-2 underwent ectodomain shedding, producing soluble fragments in the conditioned medium. By testing inactive mutants, R576A and S762A, we found that matriptase-2 activation and shedding were mediated by its own catalytic activity and that one-chain form of matriptase-2 had little activity in ectodomain shedding. We made additional matriptase-2 mutants, N136Q, N184Q, N216Q, N338Q, N433Q, N453Q and N518Q, in which each of the predicted N-glycosylation sites was mutated. All these mutants were expressed on the cell surface. However, mutants N216Q, N453Q and N518Q, but not the other mutants, had impaired zymogen activation and ectodomain shedding. Our results indicate that N-glycans at specific sites are critical for matriptase-2 activation. Together, these data provide new insights into the cell surface expression, zymogen activation, and ectodomain shedding of matriptase-2.
    The Journal of biological chemistry. 05/2014;
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    ABSTRACT: The interactions between collagen, von Willebrand factor (VWF), and glycoprotein Ib (GPIb) are crucial for hemostasis and thrombosis. This axis represents a promising target for the development of new antithrombotic agents. In this study, we investigate the in vivo antithrombotic efficacy of an anti-VWF monoclonal antibody SZ-123 and its potential underlying mechanisms. Cyclic flow reductions (CFRs), an indicator of arterial thrombosis, were measured in the femoral artery of anesthetized Rhesus monkeys before and after intravenous administration of SZ-123. Ex vivo VWF binding to collagen, platelet agglutination, platelet count, and template bleeding time were used as measurements of antithrombotic activity. In addition, plasma VWF and SZ-123 levels, and VWF occupancy were measured by ELISA. Administration of 0.1, 0.3, and 0.6mg/kg SZ-123 resulted in 45.3%, 78.2%, and 100% reductions in CFRs, respectively. When 0.3 and 0.6mg/kg SZ-123 were administered, 100% of VWF was occupied by the antibody. Moreover, 100% ex vivo inhibition of VWF-collagen binding and 60-95% inhibition of platelet agglutination were observed from 15min to 1h. None of the doses resulted in significant prolongation of bleeding time. In vitro experiments revealed that SZ-123 not only blocks the collagen-VWF A3 interaction but also indirectly inhibits VWF A1 binding to GPIbα induced by ristocetin. Thus, we demonstrate that SZ-123 prevents in vivo arterial thrombus formation under high shear conditions by inhibiting VWF A3-collagen and VWF A1-platelet interactions and does not significantly prolong bleeding time.
    Biochemical pharmacology 01/2013; · 4.25 Impact Factor
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    ABSTRACT: 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.
    Clinica chimica acta; international journal of clinical chemistry 10/2012; · 2.54 Impact Factor
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    ABSTRACT: The purpose of this study was to examine whether adoptive transfer with in vitro expanded CD4+CD25+ regulatory T cells (Tregs) could prevent immune response-mediated spontaneous abortion in mice. Female CBA/J mice were mated with male Balb/c as the control with normal pregnancy or with DBA/2J mice as a model of spontaneous abortion. The CBA/J mice mated with DBA/2J were treated intravenously with freshly isolated or in vitro expanded Tregs on day 1 or 4 of pregnancy, respectively. The numbers of surviving and reabsorbed fetuses in the different groups of mice were counted on day 14 of pregnancy, and the concentrations of cytokines in individual sera and the supernatants of cultured Tregs were measured by ELISA. Adoptive transfer with freshly isolated Tregs only slightly reduced the fetal resorption rate, which was not significantly different from that of the mice without Treg treatment, regardless of treatment at early stage and implementation of pregnancy. In contrast, adoptive transfer with in vitro expanded Tregs significantly reduced the fetal resorption rates, particularly for treatment at early stage of pregnancy (P<0.05). Furthermore, adoptive transfer with in vitro expanded Tregs at early stage of pregnancy significantly increased the levels of serum IL-10, TGF-β1, and the ratios of IL-10 to IFN-γ. Our data clearly indicated that adoptive transfer with in vitro expanded Tregs at early stage of pregnancy protected fetuses from spontaneous abortion by re-establishing immune tolerance in mice.
    European journal of obstetrics, gynecology, and reproductive biology 01/2012; 161(2):177-81. · 1.97 Impact Factor
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    ABSTRACT: von Willebrand factor (VWF) serves as a molecular bridge between the constituents of the subendothelium, such as collagen, and receptors of the platelet membrane, primarily GPIb. We have previously reported two monoclonal antibodies (mAbs), SZ-123 and SZ-125, which specifically bind the VWF A3 domain and block the interaction of VWF with collagen type III and ristocetin- or botrocetin-induced platelet aggregation. Here, we identified the epitopes recognized by SZ-123 and SZ-125 using matrix-assisted laser desorption ionization mass spectrometry in combination with proteolysis protection assays. Our results demonstrated that SZ-123 recognizes a discontinuous epitope, involving the residues (989)AHLLSLVDVMQR(1000) and (1017)YLTSEMHGARPGASK(1031) of VWF. SZ-125 recognizes a linear epitope, encompassing the sequence (1001)EGGPSQIGDALGFAVR(1016). Immunoassays further indicated that the synthetic peptide, NH2-EGGPSQIGDALGFAVR-COOH, is sufficient for the binding to SZ-125. These results provide insight into the mechanistic basis for the inhibition of VWF binding to collagen by SZ-123/SZ-125.
    International journal of hematology 08/2011; 94(3):241-7. · 1.17 Impact Factor
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    ABSTRACT: The aim of this study was to investigate the potential role of ADAMTS13 analysis in the early recognition and management of thrombotic thrombocytopenic purpura (TTP) in pregnant women. Five cases of TTP were evaluated retrospectively. Clinical and laboratory findings, von Willebrand factor (vWF)-cleaving metalloprotease (ADAMTS13) activity and maternal and neonatal outcome were recorded and analysed. Five cases were all nulliparous. ADAMTS13 assay was performed and the enzyme activity was less than 5% of the normal controls in three cases. Gene mutation in the 9th exon resulting in amino acid exchange 349Arg→Cys in ADAMTS13 was identified in one patient. After treatment including transfusion of fresh-frozen plasma (n = 5), packed red blood cells (n = 5), platelet transfusions (n = 2) and/or continued renal replacement therapy (CRRT) (n = 1) and plasma exchange (n = 2), three patients were alive, one died on postpartum day 6 in hospital without plasma exchange and one of familial TTP died three months after discharge. With increasing awareness, extra-attention must be paid to patients with thrombotic microangiopathy and to measurement of ADAMTS13 activity for early diagnosis. Although severe ADAMTS13 deficiency may be helpful for TTP, it may not be sensitive enough to identify all TTP patients. Therefore, despite ADAMTS13 result positive or negative, prompt aggressive management should include early termination of pregnancy, plasma transfusion and/or plasma exchange.
    Australian and New Zealand Journal of Obstetrics and Gynaecology 12/2010; 50(6):519-22. · 1.30 Impact Factor
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    ABSTRACT: Platelet membrane glycoproteins (GPs) are critical for normal platelet adhesion, activation and aggregation. To define the abnormalities in surface GP expression on circulating platelets and provide a better biomarker of bleeding and thrombotic disorders, we have developed a accurate, time-saving and high-throughput biotin-avidin enzyme-linked immunosorbent assay (BA-ELISA) with the monoclonal antibodies (mAbs), 7E3 against the complex of GPIIb and GPIIIa (GPIIb/IIIa), SZ-51 against P-selectin, and SZ-2 against GPIb, respectively. The levels of P-selectin and GPIIb/IIIa were measured in patients with acute myocardial infarction (AMI), intracerebral hemorrhage (ICH), or diabetes mellitus (DM)) and healthy subjects. Inhibition of GP expression was evaluated with SZ-21, an inhibitory mAb against GPIIIa and aspirin, respectively. The sensitivity of BA-ELISA is high enough to detect platelet count as low as 3.13 x 10(9)/L in platelet-rich plasma (PRP). Both the inter-assay and intra-assay coefficient variation are less than 10%. Adenosine diphosphate (ADP)-induced or non-ADP-induced expression of P-selectin and GPIIb/IIIa was significantly higher in AMI, ICH or DM than that in controls (P < 0.01 for each). Either SZ-21 or aspirin can inhibit the ADP-induced expression of P-selectin and GPIIb/IIIa. Importantly, a high correlation was detected between BA-ELISA and flow cytometry methods. These observations indicate that BA-ELISA is a sensitive and high-throughput assay for evaluating platelet GP expression. The newly developed BA-ELISA can be popularized in community hospitals, because it does not require sophisticated equipments and reagents. This method is suitable for screening inhibitors of platelet activation and has a potential in use for diagnostic purposes.
    The Tohoku Journal of Experimental Medicine 01/2010; 222(1):83-8. · 1.37 Impact Factor