RESUMEN
OBJECTIVE: Results of previous studies suggest that anti-beta2-glycoprotein I (anti-beta2GPI) antibodies in complex with beta2GPI activate platelets in a dysregulated manner, potentially contributing to the prothrombotic tendency associated with the antiphospholipid syndrome (APS). We undertook this study to investigate the possible contribution of the GPIb-IX-V receptor to platelet activation mediated by the anti-beta2GPI antibody-beta2GPI complex. METHODS: In vitro methods were used in the present study. The interaction between beta2GPI and the GPIbalpha subunit of the GPIb-IX-V receptor was delineated using direct binding and competitive inhibition assays. The interaction between the anti-beta2GPI antibody-beta2GPI complex and platelets was studied using a novel method in which the Fc portion of the antibody was immobilized using protein A coated onto a microtiter plate. Platelet activation was assessed by two methods; one involved measuring thromboxane B2 production and the other involved assessment of the activation of the phosphatidylinositol 3-kinase/Akt/glycogen synthase kinase 3beta intracellular signaling pathway. The contribution of the GPIbalpha receptor to platelet activation induced by the anti-beta2GPI antibody-beta2GPI complex was assessed by observing the influence of 2 anti-GPIbalpha antibodies (AK2 and SZ2) directed against distinct epitopes. RESULTS: This study showed that beta(2)GPI could bind to the GPIbalpha receptor. The anti-beta2GPI antibody-beta2GPI complex was able to activate platelets, and this effect was inhibited by anti-GPIbalpha antibody directed against epitope Leu-36-Gln-59, but not by anti-GPIbalpha antibody directed against residues Tyr-276-Glu-282. CONCLUSION: Our findings show that inappropriate platelet activation by the anti-beta2GPI antibody-beta2GPI complex via the GPIbalpha receptor may contribute to the prothrombotic tendency associated with APS.
Asunto(s)
Plaquetas/inmunología , Glicoproteínas/inmunología , Activación Plaquetaria/inmunología , Complejo GPIb-IX de Glicoproteína Plaquetaria/inmunología , Anticuerpos Bloqueadores/farmacología , Autoanticuerpos/inmunología , Sitios de Unión de Anticuerpos/inmunología , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Glicoproteínas/metabolismo , Humanos , Fosfatidilinositol 3-Quinasas/metabolismo , Activación Plaquetaria/efectos de los fármacos , Complejo GPIb-IX de Glicoproteína Plaquetaria/metabolismo , Tromboxano B2/metabolismo , beta 2 Glicoproteína IRESUMEN
The fifth domain (DV) of beta2-glycoprotein I (beta2GPI) is important for binding a number of ligands including phospholipids and factor XI (FXI). Beta2GPI is proteolytically cleaved in DV by plasmin but not by thrombin, VIIa, tissue plasminogen activator, or uPA. Following proteolytic cleavage of DV by plasmin, beta2GPI retains binding to FXI but not to phospholipids. Native beta2GPI, but not cleaved beta2GPI, inhibits activation of FXI by thrombin and factor XIIa, attenuating a positive feedback mechanism for additional thrombin generation. In this report, we have defined the FXI/FXIa binding site on beta2GPI using site-directed mutagenesis. We show that the positively charged residues Lys284, Lys286, and Lys287 in DV are essential for the interaction of beta2GPI with FXI/FXIa. We also demonstrate that FXIa proteolytically cleaves beta2GPI at Lys317-Thr318 in DV. Thus, FXIa cleavage of beta2GPI in vivo during thrombus formation may accelerate FXI activation by decreasing the inhibitory effect of beta2GPI.
Asunto(s)
Factor XI/metabolismo , Factor XIa/metabolismo , Glicoproteínas/química , Glicoproteínas/metabolismo , Lisina/metabolismo , Procesamiento Proteico-Postraduccional , Treonina/metabolismo , Sitios de Unión , Fibrinolisina/metabolismo , Glicoproteínas/genética , Humanos , Mutación/genética , Péptido Hidrolasas/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Trombina/metabolismo , Factores de Tiempo , Activador de Tejido Plasminógeno/metabolismo , beta 2 Glicoproteína IRESUMEN
Autoantibodies to beta2-glycoprotein I (beta2GPI) are believed to be the primary cause of coagulation abnormalities in patients with antiphospholipid syndrome (APS). Clinical features include a range of life-threatening thrombotic events and microangiopathies affecting multiple organ systems. Current standard of care relies on long-term, high-intensity anticoagulation and is associated with a high risk for serious bleeding events. The relation between autoantibodies and the pathophysiology of APS is not clearly understood, but numerous in vitro studies have characterized the effects of antiphospholipid autoantibodies on various components of the coagulation cascade, including tissue factor and the protein C pathway. The fine specificity of autoantibodies to beta2GPI is a subject of considerable debate; however, a body of evidence may offer resolution by integrating concepts of antibody affinity and assay sensitivity with carefully designed molecular studies. An investigational new therapy for APS is based on the approach that pathogenic antibodies may be reduced via depletion of circulating autoantibodies and induction of immune tolerance at the B-cell level. Preliminary results from a phase I/II clinical trial with LJP 1082, a B-cell toleragen, indicate the drug was well tolerated and may warrant further development for reduction of thrombotic events in patients with APS.
Asunto(s)
Síndrome Antifosfolípido/tratamiento farmacológico , Inmunosupresores/uso terapéutico , Síndrome Antifosfolípido/etiología , Autoanticuerpos/efectos adversos , Autoanticuerpos/inmunología , Ensayos Clínicos Fase I como Asunto , Ensayos Clínicos Fase II como Asunto , Glicoproteínas/efectos adversos , Glicoproteínas/inmunología , Glicoproteínas/uso terapéutico , Humanos , Inmunosupresores/inmunología , Métodos , Modelos Biológicos , Compuestos Orgánicos , Polietilenglicoles/uso terapéutico , beta 2 Glicoproteína IRESUMEN
Autoantibodies in antiphospholipid syndrome react predominantly with the plasma protein beta2-glycoprotein I (beta2GPI). Work by a large number of investigators has led to considerable progress in detecting and understanding beta2GPI reactivity with autoantibodies. Characterization of B cell epitopes on beta2GPI has benefited from an appreciation of its interactions with anionic phospholipids and a variety of microplate surfaces. In particular, autoantibodies to beta2GPI are of sufficiently low affinity to require high concentrations of antigen for detectable reactivity. Moreover, some microplate surfaces do not support the proper orientation of beta2GPI to allow display of epitopes in a manner accessible to autoantibodies. These concepts have helped to explain previous notions that exposure of cryptic beta2GPI epitopes may require interactions with anionic surfaces. Finally, we review evidence identifying a dominant B cell epitope that is partially defined by residues Gly40 and Arg43 on the amino terminal domain of beta2GPI.
Asunto(s)
Autoanticuerpos/inmunología , Epítopos de Linfocito B/inmunología , Glicoproteínas/inmunología , Modelos Inmunológicos , Animales , Afinidad de Anticuerpos , Síndrome Antifosfolípido/inmunología , Síndrome Antifosfolípido/fisiopatología , Humanos , beta 2 Glicoproteína IRESUMEN
Understanding so-called anticardiolipin autoantibodies has been the focus of significant interest due to their accepted role in the pathology of antiphospholipid syndrome (APS). How these antibodies contribute to thrombosis, stroke and recurrent fetal loss is not clearly understood. A precise determination of the antigenic epitope(s) recognized by anticardiolipin autoantibodies will contribute to an understanding of their role in this complex disease; however, there have been many conflicting reports regarding these epitope(s), and many of the apparent contradictions arise from the assay systems that have been used. In this review, we attempt to highlight what we believe are the salient points on this issue. We present further evidence supporting our hypothesis that most of these autoantibodies recognize epitope(s) located on domain 1 (DI) of beta2-glycoprotein 1 (beta2GPI).
Asunto(s)
Anticuerpos Anticardiolipina/análisis , Anticuerpos Anticardiolipina/inmunología , Animales , Anticuerpos Anticardiolipina/sangre , Afinidad de Anticuerpos/inmunología , Antígenos/inmunología , Síndrome Antifosfolípido/sangre , Síndrome Antifosfolípido/inmunología , Glicoproteínas/análisis , Glicoproteínas/metabolismo , Humanos , beta 2 Glicoproteína IRESUMEN
Activation of factor XI (FXI) by thrombin in vivo plays a role in coagulation by providing an important positive feedback mechanism for additional thrombin generation. FXI is activated in vitro by thrombin, or FXIIa in the presence of dextran sulfate. In this report, we investigated the effect of beta(2)-glycoprotein I (beta(2)GPI) on the activation of FXI. beta(2)GPI bound FXI in vitro and inhibited its activation to FXIa by thrombin and FXIIa. The affinity of the interaction between beta(2)GPI and FXI was equivalent to the interaction between FXI and high molecular weight kininogen. Inhibition of FXI activation occurred with lower concentrations of beta(2)GPI than found in human plasma. Proteolytic clipping of beta(2)GPI by plasmin abolished its inhibition of FXI activation. The results suggest a mechanism of regulation whereby physiological concentrations of beta(2)GPI may attenuate thrombin generation in vivo by inhibition of FXI activation. Plasmin cleavage of beta(2)GPI provides a negative feedback that counteracts its inhibition of FXI activation.
Asunto(s)
Factor XII/metabolismo , Factor XI/metabolismo , Glicoproteínas/metabolismo , Trombina/metabolismo , Síndrome Antifosfolípido/metabolismo , Sulfato de Dextran/metabolismo , Glicoproteínas/genética , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , beta 2 Glicoproteína IRESUMEN
Poly(ethylene glycol) (PEG) was incorporated into multivalent conjugates of the N-terminal domain of beta(2)GPI (domain 1). PEG was incorporated to reduce the rate of elimination of the conjugates from plasma and to putatively improve their efficacy as toleragens for the suppression of anti-beta(2)GPI antibodies and the treatment of antiphospholipid syndrome (APS). Three structurally distinct types of multivalent platforms were constructed by incorporating PEG into the platform structures in different ways. The amount of PEG incorporated ranged from about 5000 g per mole to about 30000 g per mole. The platforms were functionalized with either four or eight aminooxy groups. The conjugates were prepared by forming oxime linkages between the aminooxy groups and N-terminally glyoxylated domain 1 polypeptide. The plasma half-life of each conjugate, labeled with (125)I, was measured in both mice and rats. The half-lives of the conjugates ranged from less than 10 min to about 1 h in mice, and from less than 3 h to about 19 h in rats. The ability of five tetravalent conjugates to suppress anti-domain 1 antibodies in immunized rats was also measured. Incorporation of PEG in the conjugates significantly reduced the doses required for suppression, and the amount of reduction correlated with the amount of PEG incorporated.
Asunto(s)
Glicoproteínas/química , Inmunoconjugados/química , Terapia de Inmunosupresión , Polietilenglicoles/química , Animales , Formación de Anticuerpos , Autoanticuerpos/inmunología , Femenino , Glicoproteínas/inmunología , Glicoproteínas/farmacología , Tolerancia Inmunológica , Inmunoconjugados/farmacocinética , Inmunoconjugados/farmacología , Radioisótopos de Indio , Masculino , Ratones , Ratones Endogámicos , Estructura Molecular , Peso Molecular , Polietilenglicoles/farmacocinética , Polietilenglicoles/farmacología , Ratas , Ratas Sprague-Dawley , beta 2 Glicoproteína IRESUMEN
This study investigated the use of well-defined bioconjugate molecules to suppress antigen-specific B cell responses to domain I (DI) of human beta(2)-glycoprotein I (beta(2)GPI) in rats. DI is the dominant target of pathogenic autoimmune antibodies in patients with antiphospholipid syndrome (APS), a disease characterized by antibody-mediated thromboembolic events. Rats primed with DI conjugated to keyhole limpet hemocyanin (DI-KLH) were rendered tolerant to subsequent antigen challenge by treatment with multivalent conjugates of DI. Antibodies to DI were suppressed 89-96% with intravenous doses of 500 micro g, and reductions were paralleled by decreases in splenic antigen-specific antibody-forming cells (AFC). Suppression was achieved with a variety of conjugates having two to four copies of DI and circulating half-lives of 2.6-8.7 h. Antibodies to KLH were not suppressed, indicating the specificity of the approach. These results establish the basis for further development of therapeutic B cell toleragens to suppress pathogenic antibodies in APS and other autoimmune diseases.
Asunto(s)
Síndrome Antifosfolípido/inmunología , Autoanticuerpos/inmunología , Linfocitos B/inmunología , Glicoproteínas/inmunología , Terapia de Inmunosupresión/métodos , Animales , Anticuerpos/sangre , Anticuerpos Antifosfolípidos/inmunología , Formación de Anticuerpos/inmunología , Síndrome Antifosfolípido/sangre , Disponibilidad Biológica , Ensayo de Inmunoadsorción Enzimática , Femenino , Glicoproteínas/química , Glicoproteínas/farmacología , Hemocianinas/química , Hemocianinas/inmunología , Humanos , Inmunoconjugados/química , Inmunoconjugados/inmunología , Inmunoconjugados/farmacocinética , Inyecciones Intraperitoneales , Inyecciones Intravenosas , Modelos Moleculares , Polietilenglicoles/química , Ratas , Ratas Endogámicas Lew , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/farmacocinética , Bazo/inmunología , Vacunación , beta 2 Glicoproteína IRESUMEN
Autoantibodies against beta(2)-glycoprotein I (beta(2)GPI) appear to be a critical feature of the antiphospholipid syndrome (APS). As determined using domain deletion mutants, human autoantibodies bind to the first of five domains present in beta(2)GPI. In this study the fine detail of the domain I epitope has been examined using 10 selected mutants of whole beta(2)GPI containing single point mutations in the first domain. The binding to beta(2)GPI was significantly affected by a number of single point mutations in domain I, particularly by mutations in the region of aa 40-43. Molecular modeling predicted these mutations to affect the surface shape and electrostatic charge of a facet of domain I. Mutation K19E also had an effect, albeit one less severe and involving fewer patients. Similar results were obtained in two different laboratories using affinity-purified anti-beta(2)GPI in a competitive inhibition ELISA and with whole serum in a direct binding ELISA. This study confirms that anti-beta(2)GPI autoantibodies bind to domain I, and that the charged surface patch defined by residues 40-43 contributes to a dominant target epitope.
Asunto(s)
Anticuerpos Antifosfolípidos/metabolismo , Sitios de Unión de Anticuerpos , Epítopos/inmunología , Glicoproteínas/genética , Glicoproteínas/inmunología , Mutación Puntual , Sustitución de Aminoácidos/genética , Anticuerpos Antifosfolípidos/sangre , Anticuerpos Monoclonales/sangre , Anticuerpos Monoclonales/metabolismo , Síndrome Antifosfolípido/inmunología , Arginina/genética , Sitios de Unión de Anticuerpos/genética , Unión Competitiva/genética , Unión Competitiva/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Epítopos/análisis , Epítopos/metabolismo , Glicina/genética , Glicoproteínas/biosíntesis , Glicoproteínas/aislamiento & purificación , Glicoproteínas/metabolismo , Humanos , Modelos Moleculares , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/aislamiento & purificación , Fragmentos de Péptidos/metabolismo , Estructura Terciaria de Proteína/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Electricidad Estática , beta 2 Glicoproteína IRESUMEN
(Beta2-glycoprotein I (beta2GPI) is a plasma protein that plays an important role in the antigenic specificity of antiphospholipid autoantibodies (aPL). These antibodies are associated with an increased risk for thrombosis and recurrent foetal loss in humans. Crystallographic analysis of beta2GPI showed that its five complement control protein (CCP) or 'sushi' domains are arranged in an elongated, fish-hook shape; yet the domain-specific location of epitopes recognized by these autoantibodies has remained the subject of considerable controversy. Investigators have used different forms of recombinant beta2GPI and different ELISA methods to obtain conflicting results. One group mapped autoimmune epitopes to domain I using deletion mutants of beta2GPI in a competitive inhibition ELISA on NUNC Maxisorp microplates. Another group mapped epitopes to domain IV using beta2GPI with mutations in domain IV in a direct binding ELISA on polyoxygenated microplates. In an effort to resolve these discrepancies, a collaboration between the groups compared wildtype beta2GPI with domain IV mutants in both types of ELISA. Autoantibodies bound very poorly to domain IV mutants coated on polyoxygenated plates, yet they bound very well to the same mutants coated on NUNC Maxisorp plates. The amount of protein adsorbed on to both types of plates was similar. In the competitive inhibition ELISA, no difference could be detected between wildtype beta2GPI and domain IV mutants. These results strongly suggest that the orientation of beta2GPI on the microplate, and not necessarily the lateral density, plays the predominant role in the binding of autoantibodies.