β2糖蛋白I的纯化及其多克隆抗体制备与初步应用
|
摘要
目的:获得高纯度人血浆β2糖蛋白I(β2GPI),制备兔抗人β2GPI多克隆抗体,为进一步研究β2GPI及其抗体在抗磷脂综合征(APS)病理机制中的作用奠定基础。
方法:1.采用HClO_4沉淀、Heparin Sepharose CL-6B亲和层析等方法纯化人血浆β2GPI;利用SDS-PAGE电泳、免疫印迹分析提纯蛋白的纯度;观察不同温度、NaCl浓度及防腐剂等条件下的β2GPI稳定性。2.以纯化的β2GPI作为抗原免疫新西兰大白兔,获得抗血清;进而利用Protein A及β2GPI亲和层析柱纯化抗血清中的总IgG及抗β2GPI抗体;采用免疫双向扩散试验(IDD)、ELISA和免疫印迹等方法进行抗体验证。3.应用人β2GPI及其多克隆抗体共同刺激单核细胞株THP-1,检测其组织因子(TF)活性表达情况;利用β2GPI亲和层析柱,初步分析β2GPI与单核细胞株THP-1表面膜联蛋白A2(Annexin A2)的结合情况。
结果:1.本法获得了高纯度目的蛋白β2GPI(＞95%)。β2GPI在低于0.8 mol/L NaCl溶液中室温及4℃保存易降解变性,但在1.6mol/L NaCl溶液中室温及4℃保存60天均未见蛋白明显降解。2.IDD及ELISA鉴定抗血清及总IgG效价分别达到1:16和1:100000;免疫印迹显示纯化的总IgG和抗β2GPI抗体与β2GPI蛋白均有特异反应条带;3.β2GPI及其多克隆抗体形成的复合物能够刺激单核细胞株THP-1表达TF活性;免疫印迹实验表明,来自THP-1细胞的Annexin A2能够与β2GPI亲和柱结合。
结论:1.通过本研究,简单方便地获得了高纯度人血浆β2GPI;在使用和保存β2GPI时要考虑到其降解的可能性,注意保存条件,必要时冻干保存。2.通过免疫动物及亲和层析方法,成功获得了兔抗人β2GPI多克隆抗体。3.应用制备的β2GPI亲和层析柱,证明了β2GPI可以结合单核细胞株THP-1表面的Annexin A2;β2GPI与其抗体复合物能够刺激THP-1细胞表达TF活性,是导致APS高凝状态的重要机理之一。
Objective:To purifyβ2-glycoprotein I(β2GPI) from normal human plasma and prepare rabbit polyclonal antibodies against humanβ2GPI for establishing the foundation of the further study on the roles of anti-β2GPI/β2GPI in the pathology of antiphospholipid syndrome(APS).
Methods:1.β2GPI was purified by Heparin Sepharose CL-6B affinity chromatography following by HClO_4 precipitation.The purity of the isolatedβ2GPI was identified by SDS-PAGE,Western blotting(WB) analysis.And it's stability was observed under different temperature, concentration of salt and antiseptics.2.The New Zealand White rabbit was immunized with purified humanβ2GPI and the anti-serum was made. The whole IgG and its anti-β2GPI antibody in the anti-serum were purified by Protein A andβ2GPI-affinity column.The specificity and sensitivity of the antibody were identified by IDD,ELISA and WB.3. The monocytic cell line THP-1 cells were treated withβ2GPI and its polyclonal antibodies,and the TF activity in the cells was detected.The binding of Annexin A2 in the THP-1 cell lysates toβ2GPI was analyzed by theβ2GPI-affinity column.
Results:1.β2GPI was harvested using above methods and its purity was confirmed more than 95%.The protein maintained in≤0.8 mol/L of NaCl might be degraded at room temperature,even though at 4℃.But it was not obviously degraded if it was maintained in 1.6 mol/L of NaCl at room temperature and 4℃.2.The reactive titers of the anti-serum and the whole IgG reached to 1:16 and 1:100000 with IDD and ELISA respectively.Both the whole IgG and its anti-β2GPI antibody could react withβ2GPI by WB.3.The complex ofβ2GPI and its antibody could induce the TF activity expressed on monocytic cell line THP-1.And the Annexin A2 in the THP-1 cell lysates could bind toβ2GPI-affinity column.
Conclusions:1.Humanβ2GPI with high purity from the plasma can be obtained by our methods.It is important to protectβ2GPI from denaturazation in the process of conservation and regular use.It is the best way to dry the protein into powder for keepingβ2GPI long time.2. The polyclonal antibodies against humanβ2GPI can be successfully prepared by the immunization of rabbit withβ2GPI and the purification by chromatography.3.β2GPI can bind to the Annexin A2 existed in the monocytes,which has been analyzed byβ2GPI-affinity column.β2GPI and its antibodies can enhance the TF activity on monocytes,which is contributed to the mechanism of hypercoagulability in APS.
方法:1.采用HClO_4沉淀、Heparin Sepharose CL-6B亲和层析等方法纯化人血浆β2GPI;利用SDS-PAGE电泳、免疫印迹分析提纯蛋白的纯度;观察不同温度、NaCl浓度及防腐剂等条件下的β2GPI稳定性。2.以纯化的β2GPI作为抗原免疫新西兰大白兔,获得抗血清;进而利用Protein A及β2GPI亲和层析柱纯化抗血清中的总IgG及抗β2GPI抗体;采用免疫双向扩散试验(IDD)、ELISA和免疫印迹等方法进行抗体验证。3.应用人β2GPI及其多克隆抗体共同刺激单核细胞株THP-1,检测其组织因子(TF)活性表达情况;利用β2GPI亲和层析柱,初步分析β2GPI与单核细胞株THP-1表面膜联蛋白A2(Annexin A2)的结合情况。
结果:1.本法获得了高纯度目的蛋白β2GPI(＞95%)。β2GPI在低于0.8 mol/L NaCl溶液中室温及4℃保存易降解变性,但在1.6mol/L NaCl溶液中室温及4℃保存60天均未见蛋白明显降解。2.IDD及ELISA鉴定抗血清及总IgG效价分别达到1:16和1:100000;免疫印迹显示纯化的总IgG和抗β2GPI抗体与β2GPI蛋白均有特异反应条带;3.β2GPI及其多克隆抗体形成的复合物能够刺激单核细胞株THP-1表达TF活性;免疫印迹实验表明,来自THP-1细胞的Annexin A2能够与β2GPI亲和柱结合。
结论:1.通过本研究,简单方便地获得了高纯度人血浆β2GPI;在使用和保存β2GPI时要考虑到其降解的可能性,注意保存条件,必要时冻干保存。2.通过免疫动物及亲和层析方法,成功获得了兔抗人β2GPI多克隆抗体。3.应用制备的β2GPI亲和层析柱,证明了β2GPI可以结合单核细胞株THP-1表面的Annexin A2;β2GPI与其抗体复合物能够刺激THP-1细胞表达TF活性,是导致APS高凝状态的重要机理之一。
Objective:To purifyβ2-glycoprotein I(β2GPI) from normal human plasma and prepare rabbit polyclonal antibodies against humanβ2GPI for establishing the foundation of the further study on the roles of anti-β2GPI/β2GPI in the pathology of antiphospholipid syndrome(APS).
Methods:1.β2GPI was purified by Heparin Sepharose CL-6B affinity chromatography following by HClO_4 precipitation.The purity of the isolatedβ2GPI was identified by SDS-PAGE,Western blotting(WB) analysis.And it's stability was observed under different temperature, concentration of salt and antiseptics.2.The New Zealand White rabbit was immunized with purified humanβ2GPI and the anti-serum was made. The whole IgG and its anti-β2GPI antibody in the anti-serum were purified by Protein A andβ2GPI-affinity column.The specificity and sensitivity of the antibody were identified by IDD,ELISA and WB.3. The monocytic cell line THP-1 cells were treated withβ2GPI and its polyclonal antibodies,and the TF activity in the cells was detected.The binding of Annexin A2 in the THP-1 cell lysates toβ2GPI was analyzed by theβ2GPI-affinity column.
Results:1.β2GPI was harvested using above methods and its purity was confirmed more than 95%.The protein maintained in≤0.8 mol/L of NaCl might be degraded at room temperature,even though at 4℃.But it was not obviously degraded if it was maintained in 1.6 mol/L of NaCl at room temperature and 4℃.2.The reactive titers of the anti-serum and the whole IgG reached to 1:16 and 1:100000 with IDD and ELISA respectively.Both the whole IgG and its anti-β2GPI antibody could react withβ2GPI by WB.3.The complex ofβ2GPI and its antibody could induce the TF activity expressed on monocytic cell line THP-1.And the Annexin A2 in the THP-1 cell lysates could bind toβ2GPI-affinity column.
Conclusions:1.Humanβ2GPI with high purity from the plasma can be obtained by our methods.It is important to protectβ2GPI from denaturazation in the process of conservation and regular use.It is the best way to dry the protein into powder for keepingβ2GPI long time.2. The polyclonal antibodies against humanβ2GPI can be successfully prepared by the immunization of rabbit withβ2GPI and the purification by chromatography.3.β2GPI can bind to the Annexin A2 existed in the monocytes,which has been analyzed byβ2GPI-affinity column.β2GPI and its antibodies can enhance the TF activity on monocytes,which is contributed to the mechanism of hypercoagulability in APS.
引文
1.Hughes GR.Thrombosis,abortion,cerebral disease,and the lupus anticoagulant.Br Med J.1983;287:1088-1089.
2.Miyakis S,Lockshin MD,Atsumi T,et al.International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS).J Thromb Haemost.2006;4:295-306.
3.Urbanus RT,Derksen RH,de Groot PG.Current insight into diagnostics and pathophysiology of the antiphospolipid syndrome.Blood Rev.2008;22:93-105.
4.Okkels H,Rasmussen TE,Sanghera DK,et al.Structure of the human beta2-glycoprotein I (apolipoprotein H)gene.Eur J Biochem.1999;259:435-440.
5.Chamley LW,Allen JL,Johnson PM.Synthesis of beta 2-glycoprotein I by the human placenta.Placenta.1997;18:403-410.
6.Averna M,Paravizzini G,Marino G,et al.Liver is not the unique site of synthesis of beta 2-glycoprotein I:evidence for an intestinal localization.Int J Clin Lab Res.1997;27:207-212.
7.Kamboh MI,Ferrell RE,Sepehmia B.Genetic studies of human apolipoproteins.Ⅳ.Structural heterogeneity of apolipoprotein H.Am J Hum Genet.1988;4:452-457.
8.Gries A,Nimpf J,Wurm H,et al.Characterization of isoelectric subspecies of asialo-beta 2-glycoprotein I.Biochem J.1989;260:531-534.
9.Wurm H.Beta2-glycoprotein I interactions with phospholipid vesicles.Int J Biochem.1984;16:511-515.
10.Lee NS,Brewer HB,Osborne JC.Beta 2-glycoprotein I.Molecule properties of an unusual apolipoprotein,apolipoprotein H.J Biol Chem.1983;258:4765-4770.
11.Lozier J,Takahashi N,Putnam FW,et al.Complete amino acid sequence of human plasma beta 2-gylcoprotein I.Proc Natl Acad Sci USA.1984;81:3640-3644.
12.de Groot PG,Bouma B,Lutters BC,et al.Structure—Function Studies on beta2-glycoprotein I.J Autoimmun.2000;15:87-89.
13.Hammel M,Kriechbaum M,Gries A,et al.Solution structure of human and bovine beta2-glycoprotein I revealed by small angle X-ray scattering.J Mol Biol.2002;321:85-97.
14.Steinkasserer A,Estaller C,Weiss EH,et al.Complete nucleotide and deduced amino acid sequence of human β2-glycoporotein I.Biochem J.1991;277:387-391.
15.Schwarzenbacher R,Zeth K,Diederichs K,et al.Crystal structure of human β2-glycoprotein Ⅰ:implications for phospholipid binding and the antiphospholipid syndrome.EMBO.1999;18:6228-6239.
16.Sheng Y,Herzog H,Krilis SA.Cloning and characterization of the gene encoding the mouse β2-glycoprotein I.Genomics.1997;41:128-30.
17.Blank M,Shoenfeld Y.Beta2-glycoprotein I,infections,antiphospholipid syndrome and therapeutic considerations.Clin Immunol.2004;112:190-199.
18.Wurm H,Beubler E,Polz E,et al.Studies on the possible function of β2-glycoprotein I:influence in the triglyceride metabolism in rat.Metabolism.1982;31:484-486.
19.Brighton TA,Hogg PJ,Dai YP,et al.Beta2-gylcoprotein I in thrombosis:evidence for a role as a natural anticoagulant.Br J Haematol.1996;93:185-194.
20.Koike T,Ichikawa K,Atsumi T,et al.Beta2-glycoprotein I,anti-beta2-glycoprotein I interaction.Autoimmun.2000;15:97-100
21.Chonn A,Semple SC,Cullis PR.Beta2-glycoprotein I is a major protein associated with very rapidly clearance liposomes in vivo,suggesting a significant role in the immune clearance of “non-self”particles.J Boil Chem.1995;270:25845-25849.
22.Lopez-Lira F,Rosales Leon L,Monroy Martinez V et al.The role of β2-glycoprotein I in the activation of plasminogen.Biochim Biophys Acta.2006;64:815-823.
23.Ohkura N,Hagihara Y,Yoshimura T,et al.Plasmin can reduce the function of human β2-glycoprotein I by cleaving domain V into a nicked form.Blood.1998;91:4173-4179.
24.Shi T,Giannakopoulos B,Iverson GM,et al.Domain V of β2-glycoprotein I binds factor Xl/XIa and is cleaved at Lys317-Thr318.J Biol Chem.2005;280:907-912.
25.Yasuda S,Atsumi T,Ieko M,et al.Nicked p2-glycoprotein I:a marker of cerebral infarct and a novel role in the negative feedback pathway of extrinsic fibrinolysis.Blood.2004;103:3766-3772.
26.Rahgozar S,Giannakopoulos B,Yan X,et al.P2GPI protects thrombin from inhibition by heparin cofactor Ⅱ:potentiation of this effect in the presence of anti-p2-GPI autoantibodies.Arthritis Rheum.2008;58:1146-1155.
27.Beecken WD,Enql T,Ringel EM,et al.An endogenous inhibitor of angiogenesis derived from a transitional cell carcinoma:Clipped beta2-glycoprotein I.Ann of Surg Oncol.2006;13:1241-1251.
28.Yu P,Passam FH,Yu DM,et al.Beta2-glycoprotein I inhibits vascular endothelial growth factor and basic fibroblast growth factor induced angiogenesis through its amino terminal domain.J Thromb Haemost.2008;6:1215-123.
29.de Planque MR,Kruijtzer JA,Liskamp RM,et al.Different membrane anchoring positions of tryptophan and lysine in synthetic transmembrane α-helical peptides.J Biol Chem.1999;274:20839-20846.
30.Hunt J and Krilis S.The fifth domain of p2-glycoprotein I contains a phospholipid binding site (Cys281~Cys288)and a region recognized by anticardiolipin antibodies.J Immunol.1994;152:653-659.
31.Mehdi H,Naqvi A,Kamboh MI.A hydrophobic sequence at position 313～316 (Leu-Ala-Phe-Trp)in the fifth domain of apolipoprotein H is crucial for cardiolipin binding.Eur J Biochem.2000;267:1770-1776.
32.Hunt JE,Simpson RJ,Krilis SA.Identification of a region of beta 2-glycoprotein I critical for lipid binding and anticardiolipin antibody cofactor activity.Proc Natl Acad Sci USA.1993;90:2141-2145.
33.Hamdan R,Maiti SN,Schroit AJ.Interaction of p2-glycoprotein I with Phosphatidylserine containing membranes:ligand dependent conformational alterations initiate bivalent binding.Biochemistry.2007;46:10612-10620.
34.Giannakopoulos B,Passam F,Rahgozar S,et al.Current concepts on the pathogenesis of the antiphospholipid syndrome.Blood.2007;109:422-430.
35.de Laat B,Wu XX,van Lummel M,et al.Correlation between antiphospholipid antibodies that recognize domain I of P2-glycoprotein I and a reduction in the anticoagulant activity of annexin A5.Blood.2007;109:1490-1494.
36.Kuwana M,Matsuura E,Kobayashi K,et al.Binding of P2GPI to anionic phospholipids facilitates processing and presentation of a cryptic epitope that activates pathogenic autoreactive T cells.Blood.2005;105:1552-1557.
37.Meroni PL,Raschi E,Tostoni C,et al.Statins prerent endothelial cell activation induced by antiphospholipid (anti-P2-glycoprotein I)antibodies:effect on the proadhesive and proinflammotory phenotype.Arthitis Rheum.2001;44:2870-2878.
38.Zhou H,Wolberg AS,Roubey RA.Characterization of monocyte tissue factor activity induced by IgG antiphospholipid antibodies and inhibition by dilazep.Blood.2004;104:2353-2358.
39.Matsuura E,Igarashi Y,Yasuda T,et al.Anticardiohpin antibodies recognize beta 2-glycoprotein I structure altered by interacting with an oxygen modified solid phase surface.J Exp Med.1994;179:457-462.
40.Chamley LW,Duncalf AM,Konarkowska B,et al.Conformationally altered beta 2-glycoprotein I is the antigen for anticardiohpin autoantibodies.Clin Exp Immunol.1999;115:571-576.
41.Roubey RA,Eisenberg RA,Harper MF,et al.“Anticardiohpin”autoantibodies recognize beta 2-glycoprotein I in the absence of phospholipid:importance of Ag density and bivalent binding.J Immunol.1995;154:954-960.
42.Iverson GM,Matsuura E,Victoria EJ,et al.The orientation of (32GPI on the plate is important for the binding of anti-P2GPI autoantibodies by ELISA.J Autoimmun.2002;18:289-297.
43.Rand JH,Wu XX,Lapinski R,et al.Detection of antibody mediated reduction of annexin A5 anticoagulant activity in plasmas of patients with the antiphospholipid syndrome.Blood.2004;104:2783-2790.
44.de Laat B,Derksen RH,Urbanus RT,et al.IgG antibodies that recognize epitope Gly40~Arg43 in domain I of beta2-glycoprotein I cause LAC,and their presence correlates strongly with thrombosis.Blood.2005;105:1540-1545.
45.Galli M,Luciani D,Bertolini G,et al.Anti-beta2-glycoprotein I,antiprothrombin antibodies,and the risk of thrombosis in the antiphospholipid syndrome.Blood.2003;102:2717-2723.
46.Giannakopoulos B,Passam F,Ioannou Y,et al.How we diagnose the antiphospholipid syndrome.Blood.2009;113:985-994.
47.Nojima J,Kuratsune H,Suehisa E,et al.Association between the prevalence of antibodies to beta 2-glycoprotein I,prothrombin,protein C,protein S,and annexin V in patients with systemic lupus erythematosus and thrombotic and thrombocytopenic complications.Clin Chem.2001;47:1008-1015.
48.Lutters BC,Derksen RH,Tekelenburg WL,et al.Dimers of beta 2-glycoprotein I increase platelet deposition to collagen via interaction with phospholipids and the apolipoprotein E receptor 2'.J Biol Chem.2003;278:33831-33838.
49.Zhang J,McCrae KR.Annexin A2 mediates endothelial cell activation by antiphospholipid/anti-beta2-glycoprotein I antibodies.Blood.2005;105:1964-1969.
50.Forastiero R,Martinuzzo M,Carreras LO,et al.Anti-beta2-glycoprotein I antibodies and platelet activation in patients with antiphospholipid antibodies:association with increased excretion of platelet derived thromboxane urinary metabolites.Thromb Haemost.1998;79:42-45.
51.Jankowski M,Vreys I,Wittevrongel C,et al.Thrombogenicity of beta 2-glycoprotein I dependent antiphospholipid antibodies in a photochemically induced thrombosis model in the hamster.Blood.2003;101:157-162.
52.van Lummel M,Pennings MT,Derksen RH,et al.The binding site in p2-glycoprotein I for ApoER2' on platelets is located in domain V.J Biol Chem.2005;280:36729-36736.
53.Shi T,Giannakopoulos B,Yan X,et al.Anti-beta2-glycoprotein I antibodies in complex with beta2-glycoprotein I can activate platelets in a dysregulated manner via glycoprotein Ib-IX-V.Arthritis Rheum.2006;54:2558-2567.
54.Yap CL,Anderson KE,Hughan SC,et al.Essential role for phosphoinositide 3-kinase in shear dependent signaling between platelet glycoprotein Ib/V/LX and integrin a2bp3.Blood.2002;99:151-158.
55.Pennings MT,Derksen RH,van Lummel M,et al.Platelet adhesion to dimeric beta 2-glycoprotein I under conditions of flow is mediated by at least two receptors:glycoprotein Ib alpha and apolipoprotein E receptor 2'.J Thromb Haemost.2007;5:369-77.
56.Hulstein JJ,Lenting PJ,de Laat B,et al.β2-glycoprotein I inhibits von Willebrand factor dependent platelet adhesion and aggregation.Blood.2007;110:1483-1491.
57.Roubey RA.Tissue factor pathway and the antiphospholipid syndrome.J Autoimmun.2000;15:217-220.
58.Cuadrado MJ,Lopez-Pedrera C,Khamashta MA,et al.Thrombosis in primary antiphospholipid syndrome:a pivotal role for monocyte tissue factor expression.Arthritis Rheum.1997;40:834-841.
59.Dobado-Berrios PM,Lopez-Pedrera C,Velasco F,et al.Increased levels of tissue factor mRNA in mononuclear blood cells of patients with primary antiphospholipid syndrome.Thromb Haemost.1999;82:1578-1582.
60.Reverter JC,Tassies D,Font J,et al.Effects of human monoclonal anticardiolipin antibodies on platelet function and on tissue factor expression on monocytes.Arthritis Rheum.1998;41:1420-1427.
61.Dignat-George F,Camoin-Jau L,Sabatier F,et al.Endothelial microparticles:a potential contribution to the thrombotic complications of the antiphospholipid syndrome.Thromb Haemost.2004;91:667-673.
62.Ma K,Simantov R,Zhang JC,et al.High affinity binding of beta2-glycoprotein I to human endothelial cells is mediated by annexin Ⅱ.J Biol Chem.2000;275:15541-15548.
63.Zhou H,Ling S,Yu Y,et al.Involvement of annexin A2 in anti-beta2-GP1/beta2-GPI induced tissue factor expression on monocytes.Cell Res.2007;17:737-739.
64.Raschi E,Testoni C,Bosisio D,et al.Role of the MyD88 transduction signaling pathway in endothelial cell activation by antiphospholipid antibodies.Blood.2003;101:3495-3500.
65.Meroni PL,Raschi E,Testoni C,et al.Innate immunity in the antiphospholipid syndrome:role of toll-like receptors in endothelial cell activation by antiphospholipid antibodies.Autoimmun Rev.2004;3:510-515.
66.Buttari B,Profumo E,Mattei V,et al.Oxidized beta2-glycoprotein I induces human dendritic cell maturation and promotes a T helper type I response.Blood.2005;106:3880-3887.
67.Mehdi H,Kaplan MJ,Anlar FY,et al.Hepatitis B virus surface antigen binds to apolipoprotein H.J Virol.1994;68:2415-2424.
68.Mehdi H,Yang X,Peeples ME et al.An altered form of apolipoprotein H binds hepatitis B virus surface antigen most efficiently.Virology.1996;217:58-66.
69.de Groot PG,van Lummel M,Pennings MT,et al.Beta 2-glycoprotein I and LDL receptor family members.Thromb Res.2004;114:455-459.
70.Nykjaer A,Dragun D,Walther D,et al.An endocytotic pathway essential for renal uptake and activation of the steroid 25-(OH)vitamin D3.Cell.1999;96:507-515.
71.Hammes A,Andreassen TK,Spoelgen R,et al.Role of endocytosis in cellular uptake of sex steroids.Cell.2005;122:751-762.
72.Caronti B,Pittoni V,Palladini G,et al.Anti-beta2-glycoprotein Ⅰ antibodies bind to central nervous system.J Neurol Sci.1998;156:211-219.
73.Hasununa Y,Matsuura E,Makia Z.Involvement of beta2-glycoprotein Ⅰ and anticardiolipid antibodies in oxidatively modified low density lipoprotein uptake by macrophages.Clin Exp Immunol.1997;107:569-573.
74.Kajiwara T,Yasuda T,Matsuura E.Intracellular trafficking of β2-glycoprotein Ⅰ complexes with lipid vesicles in macrophages:Implications on the development of antiphospholipid syndrome.J Autoimmun.2007;29:164-173.
75.Ostertag MV,Liu X,Henderson V,et al.A peptide that mimics the Ⅴth region of beta2-glycoprotein Ⅰ reverses antiphospholipid mediated thrombosis in mice.Lupus.2006;15:358-365.
76.黄宏亮,周红,王婷,等.β2糖蛋白Ⅰ纯化及稳定性分析.江苏大学学报.2008;18:344-347.
77.王婷,周红,俞颖,等.抗β2糖蛋白Ⅰ抗体诱导血液单核细表达组织因子活性的机制探讨.江苏大学学报.2006:16:1-4.
2.Miyakis S,Lockshin MD,Atsumi T,et al.International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS).J Thromb Haemost.2006;4:295-306.
3.Urbanus RT,Derksen RH,de Groot PG.Current insight into diagnostics and pathophysiology of the antiphospolipid syndrome.Blood Rev.2008;22:93-105.
4.Okkels H,Rasmussen TE,Sanghera DK,et al.Structure of the human beta2-glycoprotein I (apolipoprotein H)gene.Eur J Biochem.1999;259:435-440.
5.Chamley LW,Allen JL,Johnson PM.Synthesis of beta 2-glycoprotein I by the human placenta.Placenta.1997;18:403-410.
6.Averna M,Paravizzini G,Marino G,et al.Liver is not the unique site of synthesis of beta 2-glycoprotein I:evidence for an intestinal localization.Int J Clin Lab Res.1997;27:207-212.
7.Kamboh MI,Ferrell RE,Sepehmia B.Genetic studies of human apolipoproteins.Ⅳ.Structural heterogeneity of apolipoprotein H.Am J Hum Genet.1988;4:452-457.
8.Gries A,Nimpf J,Wurm H,et al.Characterization of isoelectric subspecies of asialo-beta 2-glycoprotein I.Biochem J.1989;260:531-534.
9.Wurm H.Beta2-glycoprotein I interactions with phospholipid vesicles.Int J Biochem.1984;16:511-515.
10.Lee NS,Brewer HB,Osborne JC.Beta 2-glycoprotein I.Molecule properties of an unusual apolipoprotein,apolipoprotein H.J Biol Chem.1983;258:4765-4770.
11.Lozier J,Takahashi N,Putnam FW,et al.Complete amino acid sequence of human plasma beta 2-gylcoprotein I.Proc Natl Acad Sci USA.1984;81:3640-3644.
12.de Groot PG,Bouma B,Lutters BC,et al.Structure—Function Studies on beta2-glycoprotein I.J Autoimmun.2000;15:87-89.
13.Hammel M,Kriechbaum M,Gries A,et al.Solution structure of human and bovine beta2-glycoprotein I revealed by small angle X-ray scattering.J Mol Biol.2002;321:85-97.
14.Steinkasserer A,Estaller C,Weiss EH,et al.Complete nucleotide and deduced amino acid sequence of human β2-glycoporotein I.Biochem J.1991;277:387-391.
15.Schwarzenbacher R,Zeth K,Diederichs K,et al.Crystal structure of human β2-glycoprotein Ⅰ:implications for phospholipid binding and the antiphospholipid syndrome.EMBO.1999;18:6228-6239.
16.Sheng Y,Herzog H,Krilis SA.Cloning and characterization of the gene encoding the mouse β2-glycoprotein I.Genomics.1997;41:128-30.
17.Blank M,Shoenfeld Y.Beta2-glycoprotein I,infections,antiphospholipid syndrome and therapeutic considerations.Clin Immunol.2004;112:190-199.
18.Wurm H,Beubler E,Polz E,et al.Studies on the possible function of β2-glycoprotein I:influence in the triglyceride metabolism in rat.Metabolism.1982;31:484-486.
19.Brighton TA,Hogg PJ,Dai YP,et al.Beta2-gylcoprotein I in thrombosis:evidence for a role as a natural anticoagulant.Br J Haematol.1996;93:185-194.
20.Koike T,Ichikawa K,Atsumi T,et al.Beta2-glycoprotein I,anti-beta2-glycoprotein I interaction.Autoimmun.2000;15:97-100
21.Chonn A,Semple SC,Cullis PR.Beta2-glycoprotein I is a major protein associated with very rapidly clearance liposomes in vivo,suggesting a significant role in the immune clearance of “non-self”particles.J Boil Chem.1995;270:25845-25849.
22.Lopez-Lira F,Rosales Leon L,Monroy Martinez V et al.The role of β2-glycoprotein I in the activation of plasminogen.Biochim Biophys Acta.2006;64:815-823.
23.Ohkura N,Hagihara Y,Yoshimura T,et al.Plasmin can reduce the function of human β2-glycoprotein I by cleaving domain V into a nicked form.Blood.1998;91:4173-4179.
24.Shi T,Giannakopoulos B,Iverson GM,et al.Domain V of β2-glycoprotein I binds factor Xl/XIa and is cleaved at Lys317-Thr318.J Biol Chem.2005;280:907-912.
25.Yasuda S,Atsumi T,Ieko M,et al.Nicked p2-glycoprotein I:a marker of cerebral infarct and a novel role in the negative feedback pathway of extrinsic fibrinolysis.Blood.2004;103:3766-3772.
26.Rahgozar S,Giannakopoulos B,Yan X,et al.P2GPI protects thrombin from inhibition by heparin cofactor Ⅱ:potentiation of this effect in the presence of anti-p2-GPI autoantibodies.Arthritis Rheum.2008;58:1146-1155.
27.Beecken WD,Enql T,Ringel EM,et al.An endogenous inhibitor of angiogenesis derived from a transitional cell carcinoma:Clipped beta2-glycoprotein I.Ann of Surg Oncol.2006;13:1241-1251.
28.Yu P,Passam FH,Yu DM,et al.Beta2-glycoprotein I inhibits vascular endothelial growth factor and basic fibroblast growth factor induced angiogenesis through its amino terminal domain.J Thromb Haemost.2008;6:1215-123.
29.de Planque MR,Kruijtzer JA,Liskamp RM,et al.Different membrane anchoring positions of tryptophan and lysine in synthetic transmembrane α-helical peptides.J Biol Chem.1999;274:20839-20846.
30.Hunt J and Krilis S.The fifth domain of p2-glycoprotein I contains a phospholipid binding site (Cys281~Cys288)and a region recognized by anticardiolipin antibodies.J Immunol.1994;152:653-659.
31.Mehdi H,Naqvi A,Kamboh MI.A hydrophobic sequence at position 313～316 (Leu-Ala-Phe-Trp)in the fifth domain of apolipoprotein H is crucial for cardiolipin binding.Eur J Biochem.2000;267:1770-1776.
32.Hunt JE,Simpson RJ,Krilis SA.Identification of a region of beta 2-glycoprotein I critical for lipid binding and anticardiolipin antibody cofactor activity.Proc Natl Acad Sci USA.1993;90:2141-2145.
33.Hamdan R,Maiti SN,Schroit AJ.Interaction of p2-glycoprotein I with Phosphatidylserine containing membranes:ligand dependent conformational alterations initiate bivalent binding.Biochemistry.2007;46:10612-10620.
34.Giannakopoulos B,Passam F,Rahgozar S,et al.Current concepts on the pathogenesis of the antiphospholipid syndrome.Blood.2007;109:422-430.
35.de Laat B,Wu XX,van Lummel M,et al.Correlation between antiphospholipid antibodies that recognize domain I of P2-glycoprotein I and a reduction in the anticoagulant activity of annexin A5.Blood.2007;109:1490-1494.
36.Kuwana M,Matsuura E,Kobayashi K,et al.Binding of P2GPI to anionic phospholipids facilitates processing and presentation of a cryptic epitope that activates pathogenic autoreactive T cells.Blood.2005;105:1552-1557.
37.Meroni PL,Raschi E,Tostoni C,et al.Statins prerent endothelial cell activation induced by antiphospholipid (anti-P2-glycoprotein I)antibodies:effect on the proadhesive and proinflammotory phenotype.Arthitis Rheum.2001;44:2870-2878.
38.Zhou H,Wolberg AS,Roubey RA.Characterization of monocyte tissue factor activity induced by IgG antiphospholipid antibodies and inhibition by dilazep.Blood.2004;104:2353-2358.
39.Matsuura E,Igarashi Y,Yasuda T,et al.Anticardiohpin antibodies recognize beta 2-glycoprotein I structure altered by interacting with an oxygen modified solid phase surface.J Exp Med.1994;179:457-462.
40.Chamley LW,Duncalf AM,Konarkowska B,et al.Conformationally altered beta 2-glycoprotein I is the antigen for anticardiohpin autoantibodies.Clin Exp Immunol.1999;115:571-576.
41.Roubey RA,Eisenberg RA,Harper MF,et al.“Anticardiohpin”autoantibodies recognize beta 2-glycoprotein I in the absence of phospholipid:importance of Ag density and bivalent binding.J Immunol.1995;154:954-960.
42.Iverson GM,Matsuura E,Victoria EJ,et al.The orientation of (32GPI on the plate is important for the binding of anti-P2GPI autoantibodies by ELISA.J Autoimmun.2002;18:289-297.
43.Rand JH,Wu XX,Lapinski R,et al.Detection of antibody mediated reduction of annexin A5 anticoagulant activity in plasmas of patients with the antiphospholipid syndrome.Blood.2004;104:2783-2790.
44.de Laat B,Derksen RH,Urbanus RT,et al.IgG antibodies that recognize epitope Gly40~Arg43 in domain I of beta2-glycoprotein I cause LAC,and their presence correlates strongly with thrombosis.Blood.2005;105:1540-1545.
45.Galli M,Luciani D,Bertolini G,et al.Anti-beta2-glycoprotein I,antiprothrombin antibodies,and the risk of thrombosis in the antiphospholipid syndrome.Blood.2003;102:2717-2723.
46.Giannakopoulos B,Passam F,Ioannou Y,et al.How we diagnose the antiphospholipid syndrome.Blood.2009;113:985-994.
47.Nojima J,Kuratsune H,Suehisa E,et al.Association between the prevalence of antibodies to beta 2-glycoprotein I,prothrombin,protein C,protein S,and annexin V in patients with systemic lupus erythematosus and thrombotic and thrombocytopenic complications.Clin Chem.2001;47:1008-1015.
48.Lutters BC,Derksen RH,Tekelenburg WL,et al.Dimers of beta 2-glycoprotein I increase platelet deposition to collagen via interaction with phospholipids and the apolipoprotein E receptor 2'.J Biol Chem.2003;278:33831-33838.
49.Zhang J,McCrae KR.Annexin A2 mediates endothelial cell activation by antiphospholipid/anti-beta2-glycoprotein I antibodies.Blood.2005;105:1964-1969.
50.Forastiero R,Martinuzzo M,Carreras LO,et al.Anti-beta2-glycoprotein I antibodies and platelet activation in patients with antiphospholipid antibodies:association with increased excretion of platelet derived thromboxane urinary metabolites.Thromb Haemost.1998;79:42-45.
51.Jankowski M,Vreys I,Wittevrongel C,et al.Thrombogenicity of beta 2-glycoprotein I dependent antiphospholipid antibodies in a photochemically induced thrombosis model in the hamster.Blood.2003;101:157-162.
52.van Lummel M,Pennings MT,Derksen RH,et al.The binding site in p2-glycoprotein I for ApoER2' on platelets is located in domain V.J Biol Chem.2005;280:36729-36736.
53.Shi T,Giannakopoulos B,Yan X,et al.Anti-beta2-glycoprotein I antibodies in complex with beta2-glycoprotein I can activate platelets in a dysregulated manner via glycoprotein Ib-IX-V.Arthritis Rheum.2006;54:2558-2567.
54.Yap CL,Anderson KE,Hughan SC,et al.Essential role for phosphoinositide 3-kinase in shear dependent signaling between platelet glycoprotein Ib/V/LX and integrin a2bp3.Blood.2002;99:151-158.
55.Pennings MT,Derksen RH,van Lummel M,et al.Platelet adhesion to dimeric beta 2-glycoprotein I under conditions of flow is mediated by at least two receptors:glycoprotein Ib alpha and apolipoprotein E receptor 2'.J Thromb Haemost.2007;5:369-77.
56.Hulstein JJ,Lenting PJ,de Laat B,et al.β2-glycoprotein I inhibits von Willebrand factor dependent platelet adhesion and aggregation.Blood.2007;110:1483-1491.
57.Roubey RA.Tissue factor pathway and the antiphospholipid syndrome.J Autoimmun.2000;15:217-220.
58.Cuadrado MJ,Lopez-Pedrera C,Khamashta MA,et al.Thrombosis in primary antiphospholipid syndrome:a pivotal role for monocyte tissue factor expression.Arthritis Rheum.1997;40:834-841.
59.Dobado-Berrios PM,Lopez-Pedrera C,Velasco F,et al.Increased levels of tissue factor mRNA in mononuclear blood cells of patients with primary antiphospholipid syndrome.Thromb Haemost.1999;82:1578-1582.
60.Reverter JC,Tassies D,Font J,et al.Effects of human monoclonal anticardiolipin antibodies on platelet function and on tissue factor expression on monocytes.Arthritis Rheum.1998;41:1420-1427.
61.Dignat-George F,Camoin-Jau L,Sabatier F,et al.Endothelial microparticles:a potential contribution to the thrombotic complications of the antiphospholipid syndrome.Thromb Haemost.2004;91:667-673.
62.Ma K,Simantov R,Zhang JC,et al.High affinity binding of beta2-glycoprotein I to human endothelial cells is mediated by annexin Ⅱ.J Biol Chem.2000;275:15541-15548.
63.Zhou H,Ling S,Yu Y,et al.Involvement of annexin A2 in anti-beta2-GP1/beta2-GPI induced tissue factor expression on monocytes.Cell Res.2007;17:737-739.
64.Raschi E,Testoni C,Bosisio D,et al.Role of the MyD88 transduction signaling pathway in endothelial cell activation by antiphospholipid antibodies.Blood.2003;101:3495-3500.
65.Meroni PL,Raschi E,Testoni C,et al.Innate immunity in the antiphospholipid syndrome:role of toll-like receptors in endothelial cell activation by antiphospholipid antibodies.Autoimmun Rev.2004;3:510-515.
66.Buttari B,Profumo E,Mattei V,et al.Oxidized beta2-glycoprotein I induces human dendritic cell maturation and promotes a T helper type I response.Blood.2005;106:3880-3887.
67.Mehdi H,Kaplan MJ,Anlar FY,et al.Hepatitis B virus surface antigen binds to apolipoprotein H.J Virol.1994;68:2415-2424.
68.Mehdi H,Yang X,Peeples ME et al.An altered form of apolipoprotein H binds hepatitis B virus surface antigen most efficiently.Virology.1996;217:58-66.
69.de Groot PG,van Lummel M,Pennings MT,et al.Beta 2-glycoprotein I and LDL receptor family members.Thromb Res.2004;114:455-459.
70.Nykjaer A,Dragun D,Walther D,et al.An endocytotic pathway essential for renal uptake and activation of the steroid 25-(OH)vitamin D3.Cell.1999;96:507-515.
71.Hammes A,Andreassen TK,Spoelgen R,et al.Role of endocytosis in cellular uptake of sex steroids.Cell.2005;122:751-762.
72.Caronti B,Pittoni V,Palladini G,et al.Anti-beta2-glycoprotein Ⅰ antibodies bind to central nervous system.J Neurol Sci.1998;156:211-219.
73.Hasununa Y,Matsuura E,Makia Z.Involvement of beta2-glycoprotein Ⅰ and anticardiolipid antibodies in oxidatively modified low density lipoprotein uptake by macrophages.Clin Exp Immunol.1997;107:569-573.
74.Kajiwara T,Yasuda T,Matsuura E.Intracellular trafficking of β2-glycoprotein Ⅰ complexes with lipid vesicles in macrophages:Implications on the development of antiphospholipid syndrome.J Autoimmun.2007;29:164-173.
75.Ostertag MV,Liu X,Henderson V,et al.A peptide that mimics the Ⅴth region of beta2-glycoprotein Ⅰ reverses antiphospholipid mediated thrombosis in mice.Lupus.2006;15:358-365.
76.黄宏亮,周红,王婷,等.β2糖蛋白Ⅰ纯化及稳定性分析.江苏大学学报.2008;18:344-347.
77.王婷,周红,俞颖,等.抗β2糖蛋白Ⅰ抗体诱导血液单核细表达组织因子活性的机制探讨.江苏大学学报.2006:16:1-4.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |