RESUMEN
UNLABELLED: ESSENTIALS: Antithrombin III (AT)ß binds heparin with higher affinity than ATα. A conformation-specific antibody against ATß, TPP2009, was made to investigate ATß in hemostasis. TPP2009 bound specifically to heparin-ATß and greatly reduced the anticoagulant effect of AT. This antibody was effective in elucidating the importance of ATß in hemostasis. BACKGROUND: Antithrombin III (AT)ß is an isoform of AT that lacks the post-translational carbohydrate modification at Asn135. This isoform binds heparin with greater affinity than ATα, and has been shown to target antithrombotic function to the extracellular vascular endothelial injury site. OBJECTIVES: To characterize a conformation-specific antibody against ATß and begin to investigate the role of ATß in maintaining hemostasis. METHODS: Surface plasmon resonance (SPR), antigen binding and functional assays were conducted to characterize the mode of action of antibodies generated against heparin-bound ATß (ATß*H) by the use of phage display. RESULTS: SPR and binding studies showed that one of the antibodies, TPP2009, bound specifically to ATß*H and glycosaminoglycan-associated ATß on endothelial cells. In diluted prothrombin and activated factor X (FXa)-induced clotting assays, TPP2009 dose-dependently reduced the anticoagulant effect of heparin in non-hemophilic and FVIII-deficient human plasma, with half-maximal effective concentrations (EC50 ) of 10.5 nm and 4.7 nm, respectively. In AT-deficient human plasma, TPP2009 dose-dependently inhibited the effects of exogenously added ATß and heparin. In purified systems with ATß and pentasaccharide, TPP2009 restored > 91% of FXa activity. TPP2009 dose-dependently reversed the effects of heparin in rabbit (EC50 , 25.7 nm) and cynomolgus monkey (EC50 , 21.5 nm) plasma, but not in mouse plasma. TPP2009 was also effective in partially restoring FXa activity in rabbit and cynomolgus monkey plasma treated with FVIII function-neutralizing antibodies. CONCLUSIONS: TPP2009 specifically targets a unique conformational epitope on ATß*H and blocks ATß-mediated anticoagulation. It effectively promotes coagulation in plasma, indicating the importance of ATß in hemostasis.
Asunto(s)
Anticuerpos/farmacología , Antitrombina III/metabolismo , Coagulación Sanguínea/efectos de los fármacos , Coagulantes/farmacología , Animales , Anticuerpos/inmunología , Anticuerpos/metabolismo , Especificidad de Anticuerpos , Antitrombina III/química , Antitrombina III/inmunología , Sitios de Unión de Anticuerpos , Pruebas de Coagulación Sanguínea , Línea Celular , Técnicas de Visualización de Superficie Celular , Coagulantes/inmunología , Coagulantes/metabolismo , Relación Dosis-Respuesta a Droga , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Mapeo Epitopo , Humanos , Unión Proteica , Estructura Secundaria de Proteína , Conejos , Relación Estructura-Actividad , Resonancia por Plasmón de Superficie , Factores de TiempoRESUMEN
BACKGROUND: For many enzymes, substrate specificity is directed by secondary binding sites (exosites) that are remote from the active site. Peptide inhibition studies of protein-protein interactions are useful to identify exosite functions. OBJECTIVE: To develop an approach to manipulate these exosites using ligand-directed covalent modification of the enzyme. METHOD: To demonstrate this strategy, we have engineered an exosite-deficient variant of human plasma-derived thrombin (FIIa) . Desulfato-hirugen (Hir(55-65)) analogs were synthesized with a fluorescent label, photocrosslinker, and an optional cleavable linker conjugated to the N-terminus of the peptide, specifically fluorescein-benzoyl-phenylalanyl-(Fl-bF-)glycyl-Hir(55-65), Fl-bF-mercaptopropionyl-Hir(55-65) and Fl-bF-lactyl-Hir(55-65) were synthesized. Each analog was bound and photocrosslinked to FIIa, and the resulting covalent complex was purified. RESULTS: This modified enzyme, FIIa-Hir(55-65), hydrolyzed small substrates as efficiently as native FIIa, but was significantly inhibited in fibrinogen clotting and in thrombomodulin-mediated PC activation, implying that the active site was unaffected by labeling but exosite I was blocked. In addition, this approach was used to transfer a fluorescein label from the exosite I binding peptide Hir(55-65) to a site proximal to but not obstructing exosite I. The activity of this fluorescently labeled FIIa (Fl-FIIa) could be inhibited by unlabeled Hir(55-65), suggesting that exosite I is unmodified. Importantly, this interaction could be followed spectroscopically by fluorescence, demonstrating that the exosite I proximal probe can be used to monitor specific ligand binding interactions. CONCLUSION: Our results show that exosites of clotting factors (e.g. thrombin) can be specifically inhibited and labeled with fluorescent reporters. This novel technology may have broad applicability for studies of protein-protein interactions that regulate coagulation.
Asunto(s)
Ligandos , Trombina/química , Sitios de Unión , Reactivos de Enlaces Cruzados/farmacología , Colorantes Fluorescentes/farmacología , Humanos , Luz , Modelos Químicos , Péptidos/química , Unión Proteica , Estructura Terciaria de Proteína , Sefarosa/química , Espectrometría de Fluorescencia/métodos , Especificidad por SustratoRESUMEN
The dysfunctional mutant R352W-protein C was found in two patients with venous thrombosis. The mutant R352A-protein C was constructed to define the contribution of charge/size of the residue at 352 on protein C (chymotrypsin numbering 187). Compared with wild type-protein C, R352W-protein C showed no difference in activation by thrombin-thrombomodulin or alpha-thrombin. However, R352W-activated protein C (APC) anticoagulant activity (aPTT assay) was reduced to approximately 65%. Although the catalytic efficiency of R352W-APC towards the oligopeptide substrate S-2366 was unperturbed, factor Va and R506Q-factor Va were not efficiently inactivated by R352W-APC compared with wild type-APC. R352A-APC showed reduced anticoagulant activity and reduced efficiency in factor Va inactivation and in factor VIIIa-inactivation in the presence of protein S. These observations suggest that the dysfunction of R352W-APC in factor Va inactivation may be one of the mechanisms leading to venous thrombosis in affected patients and that R352 plays an important role in the physiological functioning of APC.
Asunto(s)
Factor Va/metabolismo , Proteína C/genética , Proteínas Recombinantes/genética , Sustitución de Aminoácidos , Anticoagulantes/farmacología , Compuestos Cromogénicos/metabolismo , Relación Dosis-Respuesta a Droga , Factor V/efectos de los fármacos , Factor VIIIa/efectos de los fármacos , Factor Va/efectos de los fármacos , Humanos , Cinética , Mutagénesis Sitio-Dirigida , Oligopéptidos/metabolismo , Tiempo de Tromboplastina Parcial , Proteína C/efectos de los fármacos , Proteína C/farmacología , Proteína S/farmacología , Ácido Pirrolidona Carboxílico/análogos & derivados , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/farmacología , Trombina/farmacologíaRESUMEN
The first epidermal growth factor-like module of human plasma protein S (EGF1, residues 76-116) was chemically synthesized and tested for its ability to inhibit the anticoagulant cofactor activity of protein S for the anticoagulant protease, activated protein C (APC). EGF1 completely inhibited the stimulation of APC activity by protein S in plasma coagulation assays, with 50% inhibition at approx. 1 microM+ EGF1, suggesting direct binding of EGF1 to APC. To investigate a direct interaction between EGF1 and APC, fluorescence resonance energy transfer (FRET) experiments were employed. APC labelled in the active site with fluorescein as the donor, and phospholipid vesicles containing octadecylrhodamine as the acceptor, showed that EGF1 association with APC caused an increase in energy transfer consistent with a relocation of the active site of APC from 94 A (9.4 nm) to 85 A above the phospholipid surface (assuming kappa(2)=2/3). An identical increase in energy transfer between the APC active site-bound fluorescein and phospholipid-bound rhodamine was obtained upon association of protein S or protein S-C4b-binding protein complex with APC. The latter suggests the presence of a ternary complex of protein S-C4b-binding protein with APC on the phospholipid surface. To confirm a direct interaction of EGF1 with APC, rhodamine was covalently attached to the alpha-N-terminus of EGF1, and binding of the labelled EGF1 to APC was directly demonstrated using FRET. The data suggested a separation between the active site of APC and the N-terminus of EGF1 of 76 A (kappa(2)=2/3), placing the APC-bound protein S-EGF1 close to, but above, the phospholipid surface and near the two EGF domains of APC. Thus we provide direct evidence for binding of protein S-EGF1 to APC and show that it induces a conformational change in APC.
Asunto(s)
Factor de Crecimiento Epidérmico/metabolismo , Proteína C/química , Proteína C/metabolismo , Proteína S/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , Rodaminas/químicaRESUMEN
The effect of replacing the gamma-carboxyglutamic acid domain of activated protein C (APC) with that of prothrombin on the topography of the membrane-bound enzyme was examined using fluorescence resonance energy transfer. The average distance of closest approach (assuming kappa2 = 2/3) between a fluorescein in the active site of the chimera and octadecylrhodamine at the membrane surface was 89 A, compared with 94 A for wild-type APC. The gamma-carboxyglutamic acid domain substitution therefore lowered and/or reoriented the active site, repositioning it close to the 84 A observed for the APC. protein S complex. Protein S enhances wild-type APC cleavage of factor Va at Arg306, but the inactivation rate of factor Va Leiden by the chimera alone is essentially equal to that by wild-type APC plus protein S. These data suggest that the activities of the chimera and of the APC.protein S complex are equivalent because the active site of the chimeric protein is already positioned near the optimal location above the membrane surface to cleave Arg306. Thus, one mechanism by which protein S regulates APC activity is by relocating its active site to the proper position above the membrane surface to optimize factor Va cleavage.
Asunto(s)
Proteína C/metabolismo , Proteína S/metabolismo , Sitios de Unión , Cromatografía en Gel , Transferencia de Energía , Fluoresceína , Fluorescencia , Humanos , Membranas Artificiales , Fosfolípidos/metabolismo , Espectrometría de FluorescenciaRESUMEN
The location of the active site of membrane-bound activated protein C (APC) relative to the phospholipid surface was determined both in the presence and absence of its cofactor, protein S, using fluorescence resonance energy transfer (FRET). APC was chemically modified to create the FRET donor species, Fl-FPR-APC, with a fluorescein dye (Fl) covalently attached to the active site via a D-Phe-Pro-Arg (FPR) tether and located in the active site near S4. FRET was observed when Fl-FPR-APC was titrated in the presence of Ca2+ ions with phosphatidylcholine/phosphatidylserine (4:1) vesicles containing the FRET acceptor, octadecylrhodamine (OR). Assuming a random orientation of transition dipoles (kappa2 = 2/3), the average distance of closest approach between the fluorescein in the active site of the membrane-bound APC and the OR at the membrane surface is 94 A. The same calcium-dependent distance was obtained for both small and large unilamellar vesicles and for vesicles that contained phosphatidylethanolamine. The active site of membrane-bound APC is therefore located far above the phospholipid surface. Upon addition of protein S, the efficiency of Fl-FPR-APC to OR energy transfer increased due to a protein S-dependent rotational and/or translational movement of the APC protease domain relative to the surface. If this movement were solely translational, then the average height of the fluorescein in the membrane-bound APC.protein S complex would be 84 A above the surface. The extent of Fl-FPR-APC to OR energy transfer was unaltered by the addition of thrombin-inactivated protein S. The protein S effect was also specific for APC, since the addition of protein S to similarly-labeled derivatives of factor Xa, factor IXa, or factor VIIa did not alter the locations of their active sites. This direct measurement demonstrates that the binding of the protein S cofactor to its cognate enzyme elicits a relocation of the active site of APC relative to the membrane surface and thereby provides a structural explanation for the recently observed protein S-dependent change in the site of factor Va cleavage by APC.