RESUMO
Hemophilia is treated by IV replacement therapy with Factor VIII (FVIII) or Factor IX (FIX), either on demand to resolve bleeding, or as prophylaxis. Improved treatment may be provided by drugs designed for subcutaneous and less frequent administration with a reduced risk of inhibitor formation. Tissue factor pathway inhibitor (TFPI) down-regulates the initiation of coagulation by inhibition of Factor VIIa (FVIIa)/tissue factor/Factor Xa (FVIIa/TF/FXa). Blockage of TFPI inhibition may facilitate thrombin generation in a hemophilic setting. A high-affinity (K(D) = 25pM) mAb, mAb 2021, against TFPI was investigated. Binding of mAb 2021 to TFPI effectively prevented inhibition of FVIIa/TF/FXa and improved clot formation in hemophilia blood and plasma. The binding epitope on the Kunitz-type protease inhibitor domain 2 of TFPI was mapped by crystallography, and showed an extensive overlap with the FXa contact region highlighting a structural basis for its mechanism of action. In a rabbit hemophilia model, an intravenous or subcutaneous dose significantly reduced cuticle bleeding. mAb 2021 showed an effect comparable with that of rFVIIa. Cuticle bleeding in the model was reduced for at least 7 days by a single intravenous dose of mAb 2021. This study suggests that neutralization of TFPI by mAb 2021 may constitute a novel treatment option in hemophilia.
Assuntos
Anticorpos Bloqueadores/farmacologia , Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Fator Xa/metabolismo , Hemofilia A/tratamento farmacológico , Hemostasia/efeitos dos fármacos , Lipoproteínas/metabolismo , Modelos Moleculares , Animais , Anticorpos Bloqueadores/administração & dosagem , Anticorpos Bloqueadores/uso terapêutico , Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Neutralizantes/farmacologia , Tempo de Sangramento , Coagulação Sanguínea/efeitos dos fármacos , Reações Cruzadas/efeitos dos fármacos , Modelos Animais de Doenças , Epitopos/imunologia , Fator VIII/farmacologia , Fator Xa/imunologia , Feminino , Fibrina/metabolismo , Células HEK293 , Hemofilia A/sangue , Células Endoteliais da Veia Umbilical Humana , Humanos , Testes de Neutralização , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Coelhos , Especificidade da Espécie , Tromboplastina/farmacologiaRESUMO
Tralokinumab, a fully human mAb specifically targeting the IL-13 cytokine, has demonstrated clinical efficacy and safety in patients with moderate-to-severe atopic dermatitis. Tralokinumab binds IL-13 with high affinity, which prevents the interaction of IL-13 with IL-13Rα1 and subsequent signaling. Similarly, tralokinumab-bound IL-13 cannot bind to IL-13Rα2, a proposed decoy receptor that is reported to bind IL-13 with extraordinarily high affinity. It has however not been fully elucidated to what extent tralokinumab interferes with the endogenous regulation of IL-13 through IL-13Rα2. In this mechanistic study, we used biophysical, biochemical, and cellular assays to investigate the effect of tralokinumab on the interaction between IL-13 and IL-13Rα1 and IL-13Rα2, respectively, as well as the effects on IL-13Rα2-mediated IL-13 internalization. We demonstrate that IL-13Rα2 binds IL-13 with exceptionally high affinity and that tralokinumab is unable to displace IL-13 from IL-13Rα2. In contrast to this, tralokinumab is able to disrupt the IL-13/IL-13Rα1 and IL-13Rα1/IL-13/IL-4Rα complex. Furthermore, we demonstrate that whereas the IL-13/tralokinumab complex is unable to bind IL-13Rα2, any IL-13 that is not bound by tralokinumab (i.e., free IL-13) can be bound by IL-13Rα2 and subsequently internalized, regardless of the presence of tralokinumab. In summary, our study indicates that tralokinumab does not interfere with endogenous IL-13Rα2-mediated regulation of free IL-13.
RESUMO
Protease-activated receptor (PAR) signaling is closely linked to the cellular activation of the pro- and anticoagulant pathways. The endothelial protein C receptor (EPCR) is crucial for signaling by activated protein C through PAR1, but EPCR may have additional roles by interacting with the 4-carboxyglutamic acid domains of procoagulant coagulation factors VII (FVII) and X (FX). Here we show that soluble EPCR regulates the interaction of FX with human or mouse tissue factor (TF)-FVIIa complexes. Mutagenesis of the FVIIa 4-carboxyglutamic acid domain and dose titrations with FX showed that EPCR interacted primarily with FX to attenuate FX activation in lipid-free assay systems. In human cell models of TF signaling, antibody inhibition of EPCR selectively blocked PAR activation by the ternary TF-FVIIa-FXa complex but not by the non-coagulant TF-FVIIa binary complex. Heterologous expression of EPCR promoted PAR1 and PAR2 cleavage by FXa in the ternary complex but did not alter PAR2 cleavage by TF-FVIIa. In murine smooth muscle cells that constitutively express EPCR and TF, thrombin and FVIIa/FX but not FVIIa alone induced PAR1-dependent signaling. Although thrombin signaling was unchanged, cells with genetically reduced levels of EPCR no longer showed a signaling response to the ternary complex. These results demonstrate that EPCR interacts with the ternary TF coagulation initiation complex to enable PAR signaling and suggest that EPCR may play a role in regulating the biology of TF-expressing extravascular and vessel wall cells that are exposed to limited concentrations of FVIIa and FX provided by ectopic synthesis or vascular leakage.
Assuntos
Antígenos CD/metabolismo , Glicoproteínas/metabolismo , Miócitos de Músculo Liso/metabolismo , Receptor PAR-1/metabolismo , Receptor PAR-2/metabolismo , Receptores de Superfície Celular/metabolismo , Transdução de Sinais/fisiologia , Tromboplastina/metabolismo , Animais , Antígenos CD/genética , Células Cultivadas , Receptor de Proteína C Endotelial , Fator VIIa/genética , Fator VIIa/metabolismo , Fator X/genética , Fator X/metabolismo , Glicoproteínas/genética , Humanos , Camundongos , Camundongos Knockout , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Miócitos de Músculo Liso/citologia , Proteína C/genética , Proteína C/metabolismo , Estrutura Terciária de Proteína , Receptor PAR-1/genética , Receptor PAR-2/genética , Receptores de Superfície Celular/genética , Trombina/genética , Trombina/metabolismo , Tromboplastina/genéticaRESUMO
BACKGROUND: The regulation of factor X (FX) is critical to maintain the balance between blood coagulation and fluidity. OBJECTIVES: To functionally characterize the role of the FX autolysis loop in the regulation of the zymogen and active form of FX. METHODS: We introduced novel N-linked glycosylations on the surface-exposed loop spanning residues 143-150 (chymotrypsin numbering) of FX. The activity and inhibition of recombinant FX variants was quantified in pure component assays. The in vitro thrombin generation potential of the FX variants was evaluated in FX-depleted plasma. RESULTS: The factor VIIa (FVIIa)-mediated activation and prothrombin activation was reduced, presumably through steric hinderance. Prothrombin activation was, however, recovered in presence of cofactor factor Va (FVa) despite a reduced prothrombinase assembly. The introduced N-glycans exhibited position-specific effects on the interaction with two FXa inhibitors: tissue factor pathway inhibitor (TFPI) and antithrombin (ATIII). Ki for the inhibition by full-length TFPI of these FXa variants was increased by 7- to 1150-fold, whereas ATIII inhibition in the presence of the heparin-analog Fondaparinux was modestly increased by 2- to 15-fold compared with wild-type. When supplemented in zymogen form, the FX variants exhibited reduced thrombin generation activity relative to wild-type FX, whereas enhanced procoagulant activity was measured for activated FXa variants. CONCLUSION: The autolysis loop participates in all aspects of FX regulation. In plasma-based assays, a modest decrease in FX activation rate appeared to knock down the procoagulant response even when down regulation of FXa activity by inhibitors was reduced.
Assuntos
Fator X , Fator Va/química , Fator X/química , Fator Xa/metabolismo , Humanos , Protrombina/química , Tromboplastina/genética , Tromboplastina/metabolismoRESUMO
BACKGROUND & AIMS: The low density lipoprotein receptor-related protein-1 (LRP-1) is a large, multifunctional endocytic receptor from the LDL receptor family, highly expressed in liver parenchymal cells (PCs), neurons, activated astrocytes, and fibroblasts. The aim of the study was to investigate if liver sinusoidal endothelial cells (LSECs), highly specialized scavenger cells, express LRP-1. METHODS: To address this question, experiments were performed in vivo and in vitro to determine if receptor associated protein (RAP) and trypsin-activated α(2)-macroglobulin (α(2)M∗) were endocytosed in LSECs. RESULTS: Both ligands were cleared from the circulation mainly by the liver. Hepatocellular distribution of intravenously administered ligands, assessed after magnetic bead cell separation using LSEC- and KC-specific antibodies, showed that PCs contained 93% and 82% of liver-associated (125)I-RAP and (125)I-α(2)M∗, whereas 5% and 11% were associated with LSECs. Uptake of RAP and α(2)M∗ in the different liver cell population in vitro was specific and followed by degradation. The uptake of (125)I-RAP was not inhibited by ligands to known endocytosis receptors in LSECs, while uptake of (125)I-α(2)M∗ was significantly inhibited by RAP, suggesting the involvement of LRP-1. Immunofluorescence using LRP-1 antibody showed positive staining in LSECs. Ligand blot analyses using total cell proteins and (125)I-RAP followed by mass spectrometry further confirmed and identified LRP-1 in LSECs. CONCLUSIONS: LSECs express functional LRP-1. An important implication of our findings is that LSECs contribute to the rapid removal of blood borne ligands for LRP-1 and may thus play a role in lipid homeostasis.
Assuntos
Fígado/citologia , Fígado/metabolismo , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Animais , Endocitose , Células Endoteliais/metabolismo , Técnicas In Vitro , Cinética , Proteína Associada a Proteínas Relacionadas a Receptor de LDL/metabolismo , Ligantes , Circulação Hepática , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Masculino , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , alfa-Macroglobulinas/metabolismoRESUMO
Corticosteroid binding globulin (CBG) is the carrier for glucocorticoids in plasma. The protein is believed to keep the steroids inactive and to regulate the amount of free hormone acting on target tissues (free hormone hypothesis). Here, we generated a mouse model genetically deficient for CBG to test the contribution of the carrier to glucocorticoid action and adrenocortical stress response. The absence of CBG resulted in a lack of corticosterone binding activity in serum and in an approximately 10-fold increase in free corticosterone levels in CBG-null mice, consistent with its role in regulation of circulating free hormone levels. Surprisingly, cbg(-/-) animals did not exhibit features seen in organisms with enhanced glucocorticoid signaling. Rather, the mice exhibited increased activity of the pituitary axis of hormonal control, normal levels of gluconeogenetic enzymes, and fatigue, as well as an aggravated response to septic shock, indicating an inability to appropriately respond to the excess free corticosterone in the absence of CBG. Thus, our data suggest an active role for CBG in bioavailability, local delivery, and/or cellular signal transduction of glucocorticoids that extends beyond a function as a mere cargo transporter.
Assuntos
Glucocorticoides/metabolismo , Transcortina/deficiência , Transcortina/metabolismo , Hormônio Adrenocorticotrópico/sangue , Animais , Peso Corporal , Corticosterona/sangue , Éxons/genética , Feminino , Privação de Alimentos , Expressão Gênica , Marcação de Genes , Glucocorticoides/farmacologia , Inflamação/imunologia , Injeções Intraperitoneais , Rim/citologia , Lipopolissacarídeos/imunologia , Fígado/citologia , Masculino , Camundongos , Taxa de SobrevidaRESUMO
Decysin-1 (ADAMDEC1) is an orphan ADAM-like metalloprotease with unknown biological function and a short domain structure. ADAMDEC1 mRNA has previously been demonstrated primarily in macrophages and mature dendritic cells. Here, we generated monoclonal antibodies (mAbs) against the mature ADAMDEC1 protein, as well as mAbs specific for the ADAMDEC1 pro-form, enabling further investigations of the metalloprotease. The generated mAbs bind ADAMDEC1 with varying affinity and represent at least six different epitope bins. Binding of mAbs to one epitope bin in the C-terminal disintegrin-like domain efficiently reduces the proteolytic activity of ADAMDEC1. A unique mAb, also recognizing the disintegrin-like domain, stimulates the caseinolytic activity of ADAMDEC1 while having no significant effect on the proteolysis of carboxymethylated transferrin. Using two different mAbs binding the disintegrin-like domain, we developed a robust, quantitative sandwich ELISA and demonstrate secretion of mature ADAMDEC1 protein by primary human macrophages. Surprisingly, we also found ADAMDEC1 present in human plasma with an approximate concentration of 0.5 nM. The presence of ADAMDEC1 both in human plasma and in macrophage cell culture supernatant were biochemically validated using immunoprecipitation and Western blot analysis demonstrating that ADAMDEC1 is secreted in a mature form.
Assuntos
Proteínas ADAM/antagonistas & inibidores , Anticorpos Monoclonais/farmacologia , Ensaio de Imunoadsorção Enzimática/métodos , Epitopos/imunologia , Inibidores de Proteases/farmacologia , Proteólise/efeitos dos fármacos , Proteínas ADAM/sangue , Proteínas ADAM/imunologia , Animais , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Sítios de Ligação de Anticorpos , Ácidos Carboxílicos/metabolismo , Células Cultivadas , Humanos , Cinética , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Metilação , Camundongos , Inibidores de Proteases/imunologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Especificidade por Substrato , Transferrina/análogos & derivados , Transferrina/metabolismoRESUMO
The intracellular domain (ICD) of the low-density lipoprotein receptor-related protein (LRP) functionally interacts with adaptor proteins both as an integral part of the receptor polypeptide and after proteolytic release. Identification of such adaptors has been difficult because the ICD is self-activating in conventional transcription factor-based yeast two-hybrid screens. We adopted an alternative screen for the ICD that depends on the activation of the Ras-signaling pathway and uncovered the transcription factor MafB as novel ICD interacting protein. MafB is a regulator of hindbrain segmentation and interacts with the ICD through a leucine zipper domain. The ICD co-localizes with MafB to the nucleus and negatively regulates its transcriptional activity, suggesting a possible role for LRP in brain development.
Assuntos
Proteínas Aviárias , Encéfalo/embriologia , Proteínas de Ligação a DNA , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Fatores de Transcrição , Sequência de Aminoácidos , Animais , Encéfalo/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , DNA Complementar/metabolismo , Genes Reporter , Glutationa Transferase/metabolismo , Humanos , Imuno-Histoquímica , Ligantes , Fator de Transcrição MafB , Camundongos , Camundongos Knockout , Microscopia de Fluorescência , Modelos Genéticos , Dados de Sequência Molecular , Proteínas Oncogênicas/fisiologia , Ligação Proteica , Estrutura Terciária de Proteína , Ressonância de Plasmônio de Superfície , Fatores de Tempo , Transativadores/fisiologia , Transcrição Gênica , Transfecção , Técnicas do Sistema de Duplo-HíbridoRESUMO
Megalin is a member of the LDL receptor gene family that plays an important role in forebrain development and in cellular vitamin D metabolism through endocytic uptake of vitamin D metabolites. Similar to other receptors in this gene family, megalin is believed to functionally interact with intracellular proteins through adaptors that bind to the receptor tail and regulate its endocytic and signal transducing activities. Using yeast two-hybrid screens, we identified a novel scaffold protein with tetratrico peptide repeats, the megalin-binding protein (MegBP) that associates with the receptor. The binding site of MegBP was mapped to an N-terminal region on the receptor tail harboring a proline-rich peptide element. MegBP binding did not block the endocytic activity of the receptor; however, overexpression resulted in cellular lethality. In further screens, we identified proteins that bound to MegBP and thus might be recruited to the megalin tail. MegBP-interacting partners included several transcriptional regulators such as the SKI-interacting protein (SKIP), a co-activator of the vitamin D receptor. These finding suggest a model whereby megalin directly participates in transcriptional regulation through controlled sequestration or release of transcription factors via MegBP.