RESUMEN
BACKGROUND: Factor (F)XI can be activated by proteases, including thrombin and FXIIa. The interactions of these enzymes with FXI are transient in nature and therefore difficult to study. OBJECTIVES: To identify the binding interface between thrombin and FXI and understand the dynamics underlying FXI activation. METHODS: Crosslinking mass spectrometry was used to localize the binding interface of thrombin on FXI. Molecular dynamics simulations were applied to investigate conformational changes enabling thrombin-mediated FXI activation after binding. The proposed trajectory of activation was examined with nanobody 1C10, which was previously shown to inhibit thrombin-mediated activation of FXI. RESULTS: We identified a binding interface of thrombin located on the light chain of FXI involving residue Pro520. After this initial interaction, FXI undergoes conformational changes driven by binding of thrombin to the apple 1 domain in a secondary step to allow migration toward the FXI cleavage site. The 1C10 binding site on the apple 1 domain supports this proposed trajectory of thrombin. We validated the results with known mutation sites on FXI. As Pro520 is conserved in prekallikrein (PK), we hypothesized and showed that thrombin can bind PK, even though it cannot activate PK. CONCLUSION: Our investigations show that the activation of FXI is a multistaged procedure. Thrombin first binds to Pro520 in FXI; thereafter, it migrates toward the activation site by engaging the apple 1 domain. This detailed analysis of the interaction between thrombin and FXI paves a way for future interventions for bleeding or thrombosis.
Asunto(s)
Factor XI , Simulación de Dinámica Molecular , Unión Proteica , Trombina , Trombina/metabolismo , Trombina/química , Humanos , Factor XI/metabolismo , Factor XI/química , Sitios de Unión , Multimerización de Proteína , Mutación , Conformación Proteica , Coagulación Sanguínea , Precalicreína/metabolismo , Precalicreína/química , Subunidades de Proteína/metabolismo , Activación Enzimática , Factor XIa/metabolismo , Factor XIa/químicaRESUMEN
BACKGROUND: Factor XI (FXI) is a promising target for novel anticoagulants because it shows a strong relation to thromboembolic diseases, while fulfilling a mostly supportive role in hemostasis. Anticoagulants targeting FXI could therefore reduce the risk for thrombosis, without increasing the chance of bleeding side effects. OBJECTIVES: To generate nanobodies that can interfere with FXIa mediated activation of factor IX (FIX). METHODS: Nanobodies were selected for binding to the apple 3 domain of FXI and their effects on FXI and coagulation were measured in purified protein systems as well as in plasma-based coagulation assays. Additionally, the binding epitope of selected nanobodies was assessed by hydrogen-deuterium exchange mass spectrometry. RESULTS: We have identified five nanobodies that inhibit FIX activation by FXI by competing with the FIX binding site on FXI. Interestingly, a sixth nanobody was found to target a different binding epitope in the apple 3 domain, resulting in competition with the FXI-high molecular weight kininogen (HK) interaction. CONCLUSIONS: We have characterized a nanobody targeting the FXI apple 3 domain that elucidates the binding orientation of HK on FXI. Moreover, we have produced five nanobodies that can inhibit the FXI-FIX interaction.
Asunto(s)
Factor IX , Factor XI , Quininógeno de Alto Peso Molecular , Anticuerpos de Dominio Único , Humanos , Anticoagulantes , Sitios de Unión , Deuterio , Epítopos , Factor IX/metabolismo , Factor XI/metabolismo , Quininógeno de Alto Peso Molecular/metabolismoRESUMEN
Damage and disease of nerves activates the complement system. We demonstrated that activation of the terminal pathway of the complement system leads to the formation of the membrane attack complex (MAC) and delays regeneration in the peripheral nervous system. Animals deficient in the complement component C6 showed improved recovery after neuronal trauma. Thus, inhibitors of the MAC might be of therapeutic use in neurological disease. Here, we describe the development, structure, mode of action, and properties of a novel therapeutic monoclonal antibody, CP010, against C6 that prevents formation of the MAC in vivo. The monoclonal antibody is humanized and specific for C6 and binds to an epitope in the FIM1-2 domain of human and primate C6 with sub-nanomolar affinity. Using biophysical and structural studies, we show that the anti-C6 antibody prevents the interaction between C6 and C5/C5b by blocking the C6 FIM1-2:C5 C345c axis. Systemic administration of the anti-C6 mAb caused complete depletion of free C6 in circulation in transgenic rats expressing human C6 and thereby inhibited MAC formation. The antibody prevented disease in experimental autoimmune myasthenia gravis and ameliorated relapse in chronic relapsing experimental autoimmune encephalomyelitis in human C6 transgenic rats. CP010 is a promising complement C6 inhibitor that prevents MAC formation. Systemic administration of this C6 monoclonal antibody has therapeutic potential in the treatment of neuronal disease.
RESUMEN
BACKGROUND: Thrombin-mediated activation of FXI supports clot stability and protects the clot from fibrinolysis. This generally poor activation could be enhanced to stimulate coagulation, which might serve patients experiencing bleeding, for example due to FXI deficiency. OBJECTIVES: To establish a reliable assay that can monitor FXI-thrombin binding and is suitable for high throughput screening. METHODS: A time-resolved fluorescence resonance energy transfer assay was set up to measure binding between FXI and thrombin in a dose-dependent manner. This assay was subjected to varying concentration of NaCl, MgCl2, and DMSO to test the robustness of the output signal. Moreover, the stability of the signal was tested after going through various freeze-thaw cycles. RESULTS: The assay produces a stable signal that meets the sensitivity and robustness criteria for application in high-throughput screening. Moreover, it was possible to measure modulation of the interaction with non-labelled FXI. CONCLUSIONS: We have established and validated a time-resolved fluorescence resonance energy transfer assay that can quantify the thrombin-FXI interaction. We propose that the assay is compatible with high-throughput screening. Thus, the assay could be used to screen for small molecules that interfere with the interaction on a high-throughput scale.
Asunto(s)
Deficiencia del Factor XI , Factor XI , Pruebas de Coagulación Sanguínea , Factor XI/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Humanos , Trombina/metabolismoRESUMEN
BACKGROUND: The prothrombinase complex consists of factors Xa (FXa) and Va (FVa) on an anionic phospholipid surface and converts prothrombin into thrombin. Both coagulation factors require activation before complex assembly. We recently identified TIX-5, a unique anticoagulant tick protein that specifically inhibits FXa-mediated activation of FV. Because TIX-5 inhibited thrombin generation in blood plasma, it was concluded that FV activation by FXa contributes importantly to coagulation. OBJECTIVE: We aimed to unravel the structure-function relationships of TIX-5. METHOD: We used a structure model generated based on homology with the allergen Der F7. RESULTS: Tick inhibitor of factor Xa toward FV was predicted to consist of a single rod formed by several beta sheets wrapped around a central C-terminal alpha helix. By mutagenesis we could show that two hydrophobic loops at one end of the rod mediate the phospholipid binding of TIX-5. On the other end of the rod an FV interaction region was identified on one side, whereas on the other side an EGK sequence was identified that could potentially form a pseudosubstrate of FXa. All three interaction sites were important for the anticoagulant properties of TIX-5 in a tissue factor-initiated thrombin generation assay as well as in the inhibition of FV activation by FXa in a purified system. CONCLUSION: The structure-function properties of TIX-5 are in perfect agreement with a protein that inhibits the FXa-mediated activation on a phospholipid surface. The present elucidation of the mechanism of action of TIX-5 will aid in deciphering the processes involved in the initiation phase of blood coagulation.
Asunto(s)
Anticoagulantes , Inhibidores del Factor Xa , Coagulación Sanguínea , Factor V , Factor Va , Factor Xa , Inhibidores del Factor Xa/farmacología , Humanos , Protrombina , Trombina , TromboplastinaRESUMEN
BACKGROUND: Factor XI (FXI) is a zymogen in the coagulation pathway that, once activated, promotes haemostasis by activating factor IX (FIX). Substitution studies using apple domains of the homologous protein prekallikrein have identified that FIX binds to the apple 3 domain of FXI. However, the molecular changes upon activation of FXI or binding of FIX to FXIa have remained largely unresolved. OBJECTIVES: This study aimed to gain more insight in the FXI activation mechanism by identifying the molecular differences between FXI and FXIa, and in the conformational changes in FXIa induced by binding of FIX. METHODS: Hydrogen-deuterium exchange mass spectrometry was performed on FXI, FXIa, and FXIa in complex with FIX. RESULTS: Both activation and binding to FIX induced conformational changes at the interface between the catalytic domain and the apple domains of FXI(a)-more specifically at the loops connecting the apple domains. Moreover, introduction of FIX uniquely induced a reduction of deuterium uptake in the beginning of the apple 3 domain. CONCLUSIONS: We propose that the conformational changes of the catalytic domain upon activation increase the accessibility to the apple 3 domain to enable FIX binding. Moreover, our HDX MS results support the location of the proposed FIX binding site at the beginning of the apple 3 domain and suggest a mediating role in FIX binding for both loops adjacent to the apple 3 domain.
Asunto(s)
Factor IX/metabolismo , Factor XI/metabolismo , Factor XIa/metabolismo , Hemostasis , Espectrometría de Masas de Intercambio de Hidrógeno-Deuterio , Activación Enzimática , Factor IX/química , Factor XI/química , Factor XI/genética , Factor XIa/química , Factor XIa/genética , Células HEK293 , Humanos , Modelos Moleculares , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Relación Estructura-ActividadRESUMEN
Coagulation factor XI is activated by thrombin or factor XIIa resulting in a conformational change that converts the catalytic domain into its active form and exposing exosites for factor IX on the apple domains. Although crystal structures of the zymogen factor XI and the catalytic domain of the protease are available, the structure of the apple domains and hence the interactions with the catalytic domain in factor XIa are unknown. We now used chemical footprinting to identify lysine residue containing regions that undergo a conformational change following activation of factor XI. To this end, we employed tandem mass tag in conjunction with mass spectrometry. Fifty-two unique peptides were identified, covering 37 of the 41 lysine residues present in factor XI. Two identified lysine residues that showed altered flexibility upon activation were mutated to study their contribution in factor XI stability or enzymatic activity. Lys357, part of the connecting loop between A4 and the catalytic domain, was more reactive in factor XIa but mutation of this lysine residue did not impact on factor XIa activity. Lys516 and its possible interactor Glu380 are located in the catalytic domain and are covered by the activation loop of factor XIa. Mutating Glu380 enhanced Arg369 cleavage and thrombin generation in plasma. In conclusion, we have identified novel regions that undergo a conformational change following activation. This information improves knowledge about factor XI and will contribute to development of novel inhibitors or activators for this coagulation protein.
Asunto(s)
Factor XI/química , Factor XIa/química , Lisina/química , Arginina/química , Sitios de Unión , Coagulación Sanguínea , Pruebas de Coagulación Sanguínea , Dominio Catalítico , Clonación Molecular , Cristalografía por Rayos X , Humanos , Isoleucina/química , Espectrometría de Masas , Péptidos/química , Conformación Proteica , Proteínas Recombinantes/químicaRESUMEN
BACKGROUND: Thrombin-activatable fibrinolysis inhibitor (TAFI) is a risk factor for coronary heart disease. TAFI is proteolytically activated by thrombin, the thrombin-thrombomodulin complex and plasmin. Once active, it dampens fibrinolysis and inflammation. The aim of this study was to generate TAFI-derived peptides that specifically modulate TAFI activation and activity. METHODS: Thirty-four overlapping TAFI peptides, and modifications thereof, were synthesized. The effects of these peptides on TAFI activation and TAFIa activity were determined. In addition, the binding of the peptides to thrombin were determined. RESULTS: Four peptides (peptides 2, 18, 19 and 34) inhibited TAFI activation and two peptides (peptides 14 and 24) inhibited TAFIa activity directly. Peptide 2 (Arg12-Glu28) and peptide 34 (Cys383-Val401) inhibited TAFI activation by the thrombin-thrombomodulin complex with IC50 values of 7.3 ± 1.8 and 6.1 ± 0.9 µm, respectively. However, no inhibition was observed in the absence of thrombomodulin. This suggests that the regions Arg12-Glu28 and Cys383-Val401 in TAFI are involved in thrombomodulin-mediated TAFI activation. Peptide 18 (Gly205-Ser221) and peptide 19 (Arg214-Asp232) inhibited TAFI activation by thrombin and the thrombin-thrombomodulin complex. Furthermore, these peptides bound to thrombin (KD : 1.5 ± 0.4 and 0.52 ± 0.07 µm for peptides 18 and 19, respectively), suggesting that Gly205-Asp232 of TAFI is involved in binding to thrombin. Peptide 14 (His159-His175) inhibited TAFIa activity. The inhibition was TAFIa specific, because no effect on the homologous enzyme carboxypeptidase B was observed. CONCLUSIONS: Thrombin-activatable fibrinolysis inhibitor-derived peptides show promise as new tools to modulate TAFI activation and TAFIa activity. Furthermore, these peptides revealed potential binding sites on TAFI for thrombin and the thrombin-thrombomodulin complex.
Asunto(s)
Carboxipeptidasa B2/antagonistas & inhibidores , Fragmentos de Péptidos/farmacología , Trombina/farmacología , Secuencia de Aminoácidos , Carboxipeptidasa B2/química , Activación Enzimática/efectos de los fármacos , Semivida , Humanos , Concentración 50 Inhibidora , Modelos Moleculares , Datos de Secuencia Molecular , Fragmentos de Péptidos/síntesis química , Unión Proteica , Conformación Proteica , Relación Estructura-Actividad , Resonancia por Plasmón de Superficie , Trombina/metabolismo , Trombomodulina/metabolismoRESUMEN
We investigated a small Dutch family with a bleeding diathesis, prolonged prothrombin, and activated partial thromboplastin times, in whom no classifying diagnosis was made. The 2 affected relatives had severely decreased in vitro thrombin generation, and levels of tissue factor pathway inhibitor (TFPI) were strongly increased. To identify the genetic cause of the bleeding diathesis, we performed whole exome sequencing analysis of all living relatives. We found a novel gain-of-function mutation in the F5 gene (c.C2588G), which leads to an aberrant splicing of F5 and ultimately to a short factor V protein (missing 623 amino acids from the B domain), which we called factor V Amsterdam. Factor V Amsterdam binds to TFPI, prolonging its half-life and concentration. This is the second report of an association between a shorter form of factor V and increased TFPI levels, resulting in severely reduced thrombin generation and a bleeding tendency.
Asunto(s)
Trastornos de la Coagulación Sanguínea Heredados/genética , Factor V/genética , Mutación , Empalme Alternativo , Trastornos de la Coagulación Sanguínea Heredados/sangre , ADN/genética , Exoma , Factor V/química , Factor V/metabolismo , Femenino , Humanos , Lipoproteínas/sangre , Lipoproteínas/genética , Masculino , Países Bajos , Linaje , Fragmentos de Péptidos/sangre , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Trombina/biosíntesisRESUMEN
Coagulation factor XI (FXI) is a promising target for anticoagulation, because of its major role in thrombosis and relatively minor role in haemostasis. This implies that inhibition of FXI can prevent thrombosis without causing bleeding. It was our aim to investigate the antithrombotic properties of two novel inhibitory anti-human FXI antibodies (αFXI-175 and αFXI-203). The in vitro properties of both antibodies were analysed using standard clotting assays and calibrated automated thrombography. For the in vivo model we used FXI knockout mice, in which FXI plasma levels were restored with purified human FXI. Thrombosis was induced by applying ferric chloride to the vena cava inferior, after which time to occlusion was analysed. A tail bleeding assay was used to investigate the safety of both antibodies. Using calibrated automated thrombography, both antibodies inhibited thrombin generation initiated via the intrinsic pathway. In contrast, upon tissue factor (TF)-initiated thrombin generation, αFXI-203 did not inhibit thrombin generation, while αFXI-175 inhibited thrombin generation only at low concentrations of TF. In the murine thrombosis model, the vena cava inferior remained patent for 25 minutes (min) in mice treated with αFXI-175 and for 12.5 min in αFXI-203 treated animals, which was significantly longer than in placebo-treated animals (5 min, p<0.05). Neither antibody caused severe blood loss in a tail bleeding assay. In conclusion, the two inhibitory antibodies against FXI prevented cessation of blood flow in a murine thrombosis model without inducing a bleeding tendency.
Asunto(s)
Anticuerpos Bloqueadores/aislamiento & purificación , Factor XI/metabolismo , Proteínas Recombinantes/administración & dosificación , Trombosis/tratamiento farmacológico , Animales , Anticuerpos Bloqueadores/farmacología , Coagulación Sanguínea/efectos de los fármacos , Coagulación Sanguínea/genética , Células Cultivadas , Modelos Animales de Enfermedad , Factor XI/genética , Factor XI/inmunología , Femenino , Hemostasis/efectos de los fármacos , Hemostasis/genética , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Trombosis/sangreRESUMEN
BACKGROUND: Generation of active procoagulant cofactor factor Va (FVa) and its subsequent association with the enzyme activated factor X (FXa) to form the prothrombinase complex is a pivotal initial event in blood coagulation and has been the subject of investigative effort, speculation, and controversy. The current paradigm assumes that FV activation is initiated by limited proteolysis by traces of (meizo) thrombin. METHODS AND RESULTS: Recombinant tick salivary protein TIX-5 was produced and anticoagulant properties were studied with the use of plasma, whole blood, and purified systems. Here, we report that TIX-5 specifically inhibits FXa-mediated FV activation involving the B domain of FV and show that FXa activation of FV is pivotal for plasma and blood clotting. Accordingly, tick feeding is impaired on TIX-5 immune rabbits, displaying the in vivo importance of TIX-5. CONCLUSIONS: Our data elucidate a unique molecular mechanism by which ticks inhibit the host's coagulation system. From our data, we propose a revised blood coagulation scheme in which direct FXa-mediated FV activation occurs in the initiation phase during which thrombin-mediated FV activation is restrained by fibrinogen and inhibitors.
Asunto(s)
Anticoagulantes/farmacología , Proteínas de Artrópodos/farmacología , Coagulación Sanguínea/efectos de los fármacos , Factor V/metabolismo , Factor Xa/metabolismo , Proteínas y Péptidos Salivales/farmacología , Animales , Anticoagulantes/sangre , Anticoagulantes/química , Proteínas de Artrópodos/química , Proteínas de Artrópodos/genética , Coagulación Sanguínea/fisiología , Pruebas de Coagulación Sanguínea , Relación Dosis-Respuesta a Droga , Factor V/antagonistas & inhibidores , Inhibidores del Factor Xa , Conducta Alimentaria , Fibrinógeno/metabolismo , Humanos , Ixodes/química , Ixodes/genética , Ixodes/fisiología , Mutagénesis , Estructura Terciaria de Proteína , Conejos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Proteínas y Péptidos Salivales/química , Proteínas y Péptidos Salivales/genética , Resonancia por Plasmón de Superficie , Trombina/metabolismoRESUMEN
A proliferation-inducing ligand (APRIL), a member of the TNF ligand superfamily with an important role in humoral immunity, is also implicated in several cancers as a prosurvival factor. APRIL binds two different TNF receptors, B cell maturation antigen (BCMA) and transmembrane activator and cylclophilin ligand interactor (TACI), and also interacts independently with heparan sulfate proteoglycans. Because APRIL shares binding of the TNF receptors with B cell activation factor, separating the precise signaling pathways activated by either ligand in a given context has proven quite difficult. In this study, we have used the protein design algorithm FoldX to successfully generate a BCMA-specific variant of APRIL, APRIL-R206E, and two TACI-selective variants, D132F and D132Y. These APRIL variants show selective activity toward their receptors in several in vitro assays. Moreover, we have used these ligands to show that BCMA and TACI have a distinct role in APRIL-induced B cell stimulation. We conclude that these ligands are useful tools for studying APRIL biology in the context of individual receptor activation.
Asunto(s)
Proteínas Mutantes/química , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/química , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/genética , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Antígeno de Maduración de Linfocitos B , Linfocitos B/metabolismo , Linfocitos B/fisiología , Supervivencia Celular , Cristalografía por Rayos X , Células HEK293 , Humanos , Enlace de Hidrógeno , Inmunoglobulina A/biosíntesis , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Unión Proteica , Estructura Terciaria de Proteína , Transporte de Proteínas , Proteína Activadora Transmembrana y Interactiva del CAMLRESUMEN
BACKGROUND: The antiphospholipid syndrome (APS) is characterized by the persistent presence of anti-ß(2) -glycoprotein I (ß(2) -GPI) autoantibodies. ß(2) -GPI can exist in two conformations. In plasma it is a circular protein, whereas it adopts a fish-hook conformation after binding to phospholipids. Only the latter conformation is recognized by patient antibodies. ß(2) -GPI has been shown to interact with Streptococcus pyogenes. OBJECTIVE: To evaluate the potential of S. pyogenes-derived proteins to induce anti-ß(2) -GPI autoantibodies. METHODS AND RESULTS: Four S. pyogenes surface proteins (M1 protein, protein H, streptococcal collagen-like protein A [SclA], and streptococcal collagen-like protein B [SclB]) were found to interact with ß(2) -GPI. Only binding to protein H induces a conformational change in ß(2) -GPI, thereby exposing a cryptic epitope for APS-related autoantibodies. Mice were injected with the four proteins. Only mice injected with protein H developed antibodies against the patient antibody-related epitope in domain I of ß(2) -GPI. Patients with pharyngotonsillitis caused by S. pyogenes who developed anti-protein H antibodies also generated anti-ß(2) -GPI antibodies. CONCLUSIONS: Our study has demonstrated that a bacterial protein can induce a conformational change in ß(2) -GPI, resulting in the formation of antiß(2) -GPI autoantibodies. This constitutes a novel mechanism for the formation of anti-ß(2) -GPI autoantibodies.
Asunto(s)
Autoanticuerpos/biosíntesis , Proteínas Bacterianas/fisiología , Proteínas Portadoras/fisiología , Proteínas de la Membrana/fisiología , Streptococcus pyogenes/fisiología , beta 2 Glicoproteína I/inmunología , Animales , Síndrome Antifosfolípido/inmunología , Ensayo de Inmunoadsorción Enzimática , Humanos , Ratones , Ratones Endogámicos BALB C , Microscopía Electrónica de Transmisión , Resonancia por Plasmón de SuperficieRESUMEN
ß2-Glycoprotein I (ß2GPI) is a highly abundant plasma protein and the major antigen for autoantibodies in the antiphospholipid syndrome. Recently, we have described a novel function of ß2GPI as scavenger of lipopolysaccharide (LPS). With this in mind we investigated the conservation of ß2GPI in vertebrates and set out to identify the binding site of LPS within ß2GPI. The genome sequences of 42 species were surveyed. Surface plasmon resonance (SPR) was performed with peptides to characterise the binding site of ß2GPI for LPS. ß2GPI could be identified in most tested vertebrates with a high overall amino acid homology of 80% or more in mammals. SPR revealed that a synthesised peptide (LAFWKTDA) from domain V of ß2GPI was able to compete for binding of ß2GPI to LPS. The AFWKTDA sequence was completely conserved in all mammals. The peptide containing the LPS binding site attenuated the inhibition by ß2GPI in a cellular model of LPS-induced tissue factor expression. Other important sites, such as the binding site for anionic phospholipids and the antiphospholipid antibody binding epitope, were also preserved. ß2GPI is highly conserved across the animal kingdom, which suggests that the function of ß2GPI may be more important than anticipated.
Asunto(s)
Evolución Molecular , Lipopolisacáridos/metabolismo , Monocitos/metabolismo , Fragmentos de Péptidos/metabolismo , beta 2 Glicoproteína I/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Unión Competitiva , Células Cultivadas , Secuencia Conservada/genética , Regulación hacia Abajo/efectos de los fármacos , Humanos , Datos de Secuencia Molecular , Monocitos/efectos de los fármacos , Monocitos/patología , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/farmacología , Alineación de Secuencia , Resonancia por Plasmón de Superficie , Tromboplastina/genética , Tromboplastina/metabolismo , beta 2 Glicoproteína I/genética , beta 2 Glicoproteína I/farmacologíaRESUMEN
Streptococcus pyogenes is the causative agent in a wide range of diseases in humans. Thrombin-activatable fibrinolysis inhibitor (TAFI) binds to collagen-like proteins SclA and SclB at the surface of S. pyogenes. Activation of TAFI at this surface redirects inflammation from a transient to chronic state by modulation of the kallikrein/kinin system. We investigated TAFI binding characteristics to SclA/SclB. Thirty-four overlapping TAFI peptides of ~20 amino acids were generated. Two of these peptides (P18: residues G205-S221, and P19: R214-D232) specifically bound to SclA/SclB with high affinity, and competed in a dose-dependent manner with TAFI binding to SclA/SclB. In another series of experiments, the binding properties of activated TAFI (TAFIa) to SclA/SclB were studied with a quadruple TAFI mutant (TAFI-IIYQ) that after activation is a 70-fold more stable enzyme than wild-type TAFIa. TAFI and TAFI-IIYQ bound to the bacterial proteins with similar affinities. The rate of dissociation was different between the proenzyme (both TAFI and TAFI-IIYQ) and the stable enzyme TAFIa-IIYQ. TAFIa-IIYQ bound to SclA/SclB, but dissociated faster than TAFI-IIYQ. In conclusion, the bacterial proteins SclA and SclB bind to a TAFI fragment encompassing residues G205-D232. Binding of TAFI to the bacteria may allow activation of TAFI, whereafter the enzyme easily dissociates.
Asunto(s)
Trastornos de la Coagulación Sanguínea/sangre , Carboxipeptidasa B2/química , Fragmentos de Péptidos/química , Infecciones Estreptocócicas/sangre , Streptococcus pyogenes/patogenicidad , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Trastornos de la Coagulación Sanguínea/etiología , Trastornos de la Coagulación Sanguínea/microbiología , Carboxipeptidasa B2/genética , Carboxipeptidasa B2/metabolismo , Activación Enzimática , Exotoxinas/química , Exotoxinas/metabolismo , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Mutación/genética , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Unión Proteica , Estabilidad Proteica , Infecciones Estreptocócicas/complicaciones , Infecciones Estreptocócicas/microbiología , Streptococcus pyogenes/metabolismo , Trombina/metabolismoRESUMEN
Within the framework of REACH, an assessment regarding local dermal effects and skin sensitisation should be performed for substances. Quantitative hazard information for these effects is often not available. Furthermore, it is difficult to relate the way in which animals are exposed in dermal toxicity studies directly to dermal exposure in practice. In the absence of quantitative information, a qualitative assessment for dermal effects is the most reasonable option. The qualitative approach as proposed in the REACH guidance recommends only general risk management measures (RMM) for three categories with a low, moderate and high identified hazard, without specifying which RMM are needed for a specific exposure scenario. We propose to differentiate frequency of exposure based on differences in activities and to compare measured and estimated local skin exposure levels with rules of thumb for evaluation of control of risks per hazard category. For workers, specific RMM regimes are assigned to each combination of hazard category and process category (PROC). For consumers, a strategy in which RMM are arranged from product-integrated measures to the use of personal protective equipment (PPE) is presented. Our approach may be transferred into automated assessment tools like Chesar and CEFIC GES.
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Sustancias Peligrosas/toxicidad , Exposición Profesional/efectos adversos , Medición de Riesgo/legislación & jurisprudencia , Piel/efectos de los fármacos , Animales , Unión Europea , Humanos , Inmunización , Medición de Riesgo/métodos , Gestión de Riesgos , Absorción CutáneaRESUMEN
The mechanism by which the intrinsic pathway of coagulation contributes to physiological hemostasis is enigmatic. Thrombin activates factor XI, a key zymogen in this pathway, which leads to increased thrombin generation. As thrombin-dependent activation of factor XI in vitro is relatively inefficient, we hypothesized that a physiological cofactor supports this reaction in a plasma environment. We therefore investigated whether the cofactors of coagulation, activated factor V, activated factor VIII, high-molecular weight kininogen, or protein S, influenced activation of factor XI by thrombin. Only activated factor V stimulated activation of factor XI by thrombin in a purified system. Binding studies demonstrated that factor XI specifically interacts with both factor V and factor Va through multiple binding sites. We further investigated this cofactor function of activated factor V in plasma. Depletion of factor V, or the addition of activated protein C, decreased the activation of the intrinsic pathway by thrombin in plasma. However, activated protein C did not exert this effect in the plasma of a homozygous carrier of the prothrombotic factor V Leiden mutation. In conclusion, we propose a role for (activated) factor V as a cofactor in the activation of factor XI by thrombin. These findings offer insights into the coagulation system in both health and disease.
Asunto(s)
Coagulación Sanguínea/fisiología , Factor Va/metabolismo , Factor XI/metabolismo , Hemostasis/fisiología , Trombina/metabolismo , Análisis de Varianza , Ensayo de Inmunoadsorción Enzimática , Humanos , Plasma/metabolismo , Unión Proteica , Resonancia por Plasmón de SuperficieRESUMEN
The antiphospholipid syndrome is defined by the presence of antiphospholipid antibodies in blood of patients with thrombosis or fetal loss. There is ample evidence that beta(2)-glycoprotein I (beta(2)GPI) is the major antigen for antiphospholipid antibodies. The autoantibodies recognize beta(2)GPI when bound to anionic surfaces and not in solution. We showed that beta(2)GPI can exist in at least 2 different conformations: a circular plasma conformation and an "activated" open conformation. We also showed that the closed, circular conformation is maintained by interaction between the first and fifth domain of beta(2)GPI. By changing pH and salt concentration, we were able to convert the conformation of beta(2)GPI from the closed to the open conformation and back. In the activated open conformation, a cryptic epitope in the first domain becomes exposed that enables patient antibodies to bind and form an antibody-beta(2)GPI complex. We also demonstrate that the open conformation of beta(2)GPI prolonged the activated partial thromboplastin time when added to normal plasma, whereas the activated partial thromboplastin time is further prolonged by addition of anti-beta(2)GPI antibodies. The conformational change of beta(2)GPI, and the influence of the autoantibodies may have important consequences for our understanding of the antiphospholipid syndrome.
Asunto(s)
Anticuerpos Antifosfolípidos/inmunología , Síndrome Antifosfolípido/metabolismo , beta 2 Glicoproteína I/química , Anticuerpos Antifosfolípidos/aislamiento & purificación , Anticoagulantes/farmacología , Síndrome Antifosfolípido/patología , Cardiolipinas/metabolismo , Humanos , Tiempo de Tromboplastina Parcial , Conformación Proteica , Pliegue de Proteína , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Resonancia por Plasmón de Superficie , beta 2 Glicoproteína I/genéticaRESUMEN
A proliferation-inducing ligand (APRIL) (also known as TALL-2 and TRDL-1) is a member of the tumor necrosis factor (TNF) superfamily that has tumorigenic properties but is also important for the induction of humoral immune responses. APRIL binds two TNF receptors: transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) and B-cell maturation antigen (BCMA) as well as heparan sulfate proteoglycans (HSPGs). The aim of this study was to clarify the role of the HSPG interaction in canonical APRIL signaling, because it has been proposed to act as a docking site and also to play a role in direct signaling. In this study, we generated point mutants of soluble APRIL that lack either the capacity to bind HSPGs or TACI and BCMA and then tested the function of these mutants in mouse B-cell assays. In contrast to previous reports, we found that APRIL alone is sufficient to costimulate B-cell proliferation and drive IgA production and does not require artificial antibody cross-linking. We found no evidence that APRIL requires signaling through HSPGs but, notably, were able to show that binding of APRIL to HSPGs is crucial for mediating natural APRIL cross-linking to allow for optimal activation of murine B cells.