RESUMO
Inhibitors of complement and coagulation are present in the saliva of a variety of blood-feeding arthropods that transmit parasitic and viral pathogens. Here, we describe the structure and mechanism of action of the sand fly salivary protein lufaxin, which inhibits the formation of the central alternative C3 convertase (C3bBb) and inhibits coagulation factor Xa (fXa). Surface plasmon resonance experiments show that lufaxin stabilizes the binding of serine protease factor B (FB) to C3b but does not detectably bind either C3b or FB alone. The crystal structure of the inhibitor reveals a novel all ß-sheet fold containing 2 domains. A structure of the lufaxin-C3bB complex obtained via cryo-electron microscopy (EM) shows that lufaxin binds via its N-terminal domain at an interface containing elements of both C3b and FB. By occupying this spot, the inhibitor locks FB into a closed conformation in which proteolytic activation of FB by FD cannot occur. C3bB-bound lufaxin binds fXa at a separate site in its C-terminal domain. In the cryo-EM structure of a C3bB-lufaxin-fXa complex, the inhibitor binds to both targets simultaneously, and lufaxin inhibits fXa through substrate-like binding of a C-terminal peptide at the active site as well as other interactions in this region. Lufaxin inhibits complement activation in ex vivo models of atypical hemolytic uremic syndrome (aHUS) and paroxysmal nocturnal hemoglobinuria (PNH) as well as thrombin generation in plasma, providing a rationale for the development of a bispecific inhibitor to treat complement-related diseases in which thrombosis is a prominent manifestation.
Assuntos
Coagulação Sanguínea , Fator B do Complemento , Microscopia Crioeletrônica , Fator B do Complemento/química , Fator B do Complemento/metabolismo , Ativação do Complemento , Serina Endopeptidases , Complemento C3b/químicaRESUMO
Adenoviruses are used extensively as gene transfer agents, both experimentally and clinically. However, targeting of liver cells by adenoviruses compromises their potential efficacy. In cell culture, the adenovirus serotype 5 fiber protein engages the coxsackievirus and adenovirus receptor (CAR) to bind cells. Paradoxically, following intravascular delivery, CAR is not used for liver transduction, implicating alternate pathways. Recently, we demonstrated that coagulation factor (F)X directly binds adenovirus leading to liver infection. Here, we show that FX binds to the Ad5 hexon, not fiber, via an interaction between the FX Gla domain and hypervariable regions of the hexon surface. Binding occurs in multiple human adenovirus serotypes. Liver infection by the FX-Ad5 complex is mediated through a heparin-binding exosite in the FX serine protease domain. This study reveals an unanticipated function for hexon in mediating liver gene transfer in vivo.
Assuntos
Adenovírus Humanos/fisiologia , Proteínas do Capsídeo/metabolismo , Fator X/metabolismo , Fígado/virologia , Transdução Genética , Internalização do Vírus , Adenovírus Humanos/química , Adenovírus Humanos/classificação , Animais , Proteínas do Capsídeo/química , Proteínas de Transporte/metabolismo , Microscopia Crioeletrônica , Fator X/química , Hepatócitos/virologia , Humanos , Imageamento Tridimensional , Camundongos , Camundongos Transgênicos , Modelos Moleculares , Filogenia , Ligação Proteica/efeitos dos fármacos , Domínios e Motivos de Interação entre Proteínas , Ressonância de Plasmônio de Superfície , Varfarina/farmacologiaRESUMO
Neutrophil extracellular traps (NETs) evolved as a unique effector mechanism contributing to resistance against infection that can also promote tissue damage in inflammatory conditions. Malaria infection can trigger NET release, but the mechanisms and consequences of NET formation in this context remain poorly characterized. Here we show that patients suffering from severe malaria had increased amounts of circulating DNA and increased neutrophil elastase (NE) levels in plasma. We used cultured erythrocytes and isolated human neutrophils to show that Plasmodium-infected red blood cells release macrophage migration inhibitory factor (MIF), which in turn caused NET formation by neutrophils in a mechanism dependent on the C-X-C chemokine receptor type 4 (CXCR4). NET production was dependent on histone citrullination by peptidyl arginine deiminase-4 (PAD4) and independent of reactive oxygen species (ROS), myeloperoxidase (MPO) or NE. In vitro, NETs functioned to restrain parasite dissemination in a mechanism dependent on MPO and NE activities. Finally, C57/B6 mice infected with P. berghei ANKA, a well-established model of cerebral malaria, presented high amounts of circulating DNA, while treatment with DNAse increased parasitemia and accelerated mortality, indicating a role for NETs in resistance against Plasmodium infection.
Assuntos
Eritrócitos/imunologia , Armadilhas Extracelulares/imunologia , Fatores Inibidores da Migração de Macrófagos/metabolismo , Malária/imunologia , Neutrófilos/imunologia , Plasmodium/imunologia , Receptores CXCR4/metabolismo , Animais , Eritrócitos/metabolismo , Eritrócitos/parasitologia , Armadilhas Extracelulares/metabolismo , Armadilhas Extracelulares/parasitologia , Humanos , Malária/metabolismo , Malária/parasitologia , Malária/patologia , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/metabolismo , Neutrófilos/parasitologia , Parasitemia/imunologia , Parasitemia/metabolismo , Parasitemia/parasitologia , Parasitemia/patologiaRESUMO
Ixolaris is a potent tick salivary anticoagulant that binds coagulation factor Xa (FXa) and zymogen FX, with formation of a quaternary tissue factor (TF)/FVIIa/ FX(a)/Ixolaris inhibitory complex. Ixolaris blocks TF-induced coagulation and PAR2 signaling and prevents thrombosis, tumor growth, and immune activation. We present a high-resolution structure and dynamics of Ixolaris and describe the structural basis for recognition of FX. Ixolaris consists of 2 Kunitz domains (K1 and K2) in which K2 is strikingly dynamic and encompasses several residues involved in FX binding. This indicates that the backbone plasticity of K2 is critical for Ixolaris biological activity. Notably, a nuclear magnetic resonance-derived model reveals a mechanism for an electrostatically guided, high-affinity interaction between Ixolaris and FX heparin-binding (pro)exosite, resulting in an allosteric switch in the catalytic site. This is the first report revealing the structure-function relationship of an anticoagulant targeting a zymogen serving as a scaffold for TF inhibition.
Assuntos
Inibidores do Fator Xa/química , Inibidores do Fator Xa/farmacologia , Fator Xa/metabolismo , Proteínas e Peptídeos Salivares/química , Proteínas e Peptídeos Salivares/farmacologia , Animais , Fator Xa/química , Humanos , Simulação de Acoplamento Molecular , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Domínios Proteicos , Carrapatos/químicaRESUMO
Recently it was shown that the hematophagous salivary gland protein agaphelin exhibits multiple antithrombotic effects without promoting the risk of bleeding. Agaphelin inhibits neutrophil elastase and thereby reduces cathepsin G-induced platelet aggregation. However, it is still unclear, whether pharmacological treatment with agaphelin in brain ischemia is protective and, regarding its bleeding risk, safe. To elucidate this issue, male C57BL/6 mice were subjected to 60 min of transient middle cerebral artery occlusion (tMCAO) and treated with 0.25 mg/kg agaphelin intravenously immediately after tMCAO. On day 1 and 7, infarct volume and functional neurological outcome were assessed by behavioural tests, histochemistry and magnetic resonance imaging. Thrombus formation, intracerebral bleeding risk, blood-brain barrier damage and the local inflammatory response were determined on day 1. This study shows for the first time a protective effect of agaphelin characterized by smaller infarct volume, reduced neurological deficits and reduced animal mortality. This protective effect was associated with reduced local thrombus formation, increased blood-brain barrier integrity and reduced brain inflammatory response. It is essential to mention that the protective effect of agaphelin was not linked to an increased risk of intracerebral bleeding. The promotion of brain tissue survival and inhibition of thromboinflammation identifies agaphelin as a promising treatment option in ischemic stroke, which considering the lack of bleeding risk should potentially be safe.
Assuntos
Isquemia Encefálica , Proteínas de Insetos/farmacologia , AVC Isquêmico , Elastase Pancreática/antagonistas & inibidores , Proteínas e Peptídeos Salivares/farmacologia , Trombose , Animais , Barreira Hematoencefálica , Modelos Animais de Doenças , Infarto da Artéria Cerebral Média/complicações , Infarto da Artéria Cerebral Média/tratamento farmacológico , Inflamação/tratamento farmacológico , AVC Isquêmico/tratamento farmacológico , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: Invasion of mosquito salivary glands (SGs) by Plasmodium falciparum sporozoites is an essential step in the malaria life cycle. How infection modulates gene expression, and affects hematophagy remains unclear. PRINCIPAL FINDINGS: Using Affimetrix chip microarray, we found that at least 43 genes are differentially expressed in the glands of Plasmodium falciparum-infected Anopheles gambiae mosquitoes. Among the upregulated genes, one codes for Agaphelin, a 58-amino acid protein containing a single Kazal domain with a Leu in the P1 position. Agaphelin displays high homology to orthologs present in Aedes sp and Culex sp salivary glands, indicating an evolutionarily expanded family. Kinetics and surface plasmon resonance experiments determined that chemically synthesized Agaphelin behaves as a slow and tight inhibitor of neutrophil elastase (K(D) â¼ 10 nM), but does not affect other enzymes, nor promotes vasodilation, or exhibit antimicrobial activity. TAXIscan chamber assay revealed that Agaphelin inhibits neutrophil chemotaxis toward fMLP, affecting several parameter associated with cell migration. In addition, Agaphelin reduces paw edema formation and accumulation of tissue myeloperoxidase triggered by injection of carrageenan in mice. Agaphelin also blocks elastase/cathepsin-mediated platelet aggregation, abrogates elastase-mediated cleavage of tissue factor pathway inhibitor, and attenuates neutrophil-induced coagulation. Notably, Agaphelin inhibits neutrophil extracellular traps (NETs) formation and prevents FeCl3-induced arterial thrombosis, without impairing hemostasis. CONCLUSIONS: Blockade of neutrophil elastase emerges as a novel antihemostatic mechanism in hematophagy; it also supports the notion that neutrophils and the innate immune response are targets for antithrombotic therapy. In addition, Agaphelin is the first antihemostatic whose expression is induced by Plasmodium sp infection. These results suggest that an important interplay takes place in parasite-vector-host interactions.
Assuntos
Anopheles/parasitologia , Hemostasia/fisiologia , Interações Hospedeiro-Parasita , Proteínas de Insetos/metabolismo , Neutrófilos/imunologia , Plasmodium falciparum/patogenicidade , Proteínas e Peptídeos Salivares/metabolismo , Trombose/prevenção & controle , Sequência de Aminoácidos , Animais , Anopheles/metabolismo , Dicroísmo Circular , Edema/etiologia , Edema/metabolismo , Edema/prevenção & controle , Feminino , Proteínas de Insetos/química , Proteínas de Insetos/genética , Insetos Vetores , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Glândulas Salivares/metabolismo , Glândulas Salivares/parasitologia , Proteínas e Peptídeos Salivares/química , Proteínas e Peptídeos Salivares/genética , Homologia de Sequência de Aminoácidos , Ressonância de Plasmônio de SuperfícieRESUMO
The identity of vampire bat saliva anticoagulant remained elusive for almost a century. Sequencing the salivary gland genes from the vampire bat Desmodus rotundus identified Desmolaris as a novel 21.5-kDa naturally deleted (Kunitz 1-domainless) form of tissue factor pathway inhibitor. Recombinant Desmolaris was expressed in HEK293 cells and characterized as a slow, tight, and noncompetitive inhibitor of factor (F) XIa by a mechanism modulated by heparin. Desmolaris also inhibits FXa with lower affinity, independently of protein S. In addition, Desmolaris binds kallikrein and reduces bradykinin generation in plasma activated with kaolin. Truncated and mutated forms of Desmolaris determined that Arg32 in the Kunitz-1 domain is critical for protease inhibition. Moreover, Kunitz-2 and the carboxyl-terminus domains mediate interaction of Desmolaris with heparin and are required for optimal inhibition of FXIa and FXa. Notably, Desmolaris (100 µg/kg) inhibited FeCl3-induced carotid artery thrombus without impairing hemostasis. These results imply that FXIa is the primary in vivo target for Desmolaris at antithrombotic concentrations. Desmolaris also reduces the polyphosphate-induced increase in vascular permeability and collagen- and epinephrine-mediated thromboembolism in mice. Desmolaris emerges as a novel anticoagulant targeting FXIa under conditions in which the coagulation activation, particularly the contact pathway, plays a major pathological role.
Assuntos
Anticoagulantes/química , Anticoagulantes/farmacologia , Quirópteros , Inibidores do Fator Xa , Proteínas e Peptídeos Salivares/química , Proteínas e Peptídeos Salivares/farmacologia , Trombose/tratamento farmacológico , Animais , Bradicinina/química , Bradicinina/genética , Bradicinina/metabolismo , Cloretos/efeitos adversos , Cloretos/farmacologia , Modelos Animais de Doenças , Fator Xa/química , Fator Xa/genética , Fator Xa/metabolismo , Compostos Férricos/efeitos adversos , Compostos Férricos/farmacologia , Células HEK293 , Humanos , Inflamação/tratamento farmacológico , Inflamação/genética , Inflamação/metabolismo , Calicreínas/química , Calicreínas/genética , Calicreínas/metabolismo , Camundongos , Noxas/efeitos adversos , Noxas/farmacologia , Estrutura Terciária de Proteína , Proteínas e Peptídeos Salivares/genética , Trombose/induzido quimicamente , Trombose/genética , Trombose/metabolismoRESUMO
Saliva of blood-sucking arthropods contains a complex cocktail of pharmacologically active compounds that assists feeding by counteracting their hosts' hemostatic and inflammatory reactions. Panstrongylus megistus (Burmeister) is an important vector of Chagas disease in South America, but despite its importance there is only one salivary protein sequence publicly deposited in GenBank. In the present work, we used Illumina technology to disclose and publicly deposit 3,703 coding sequences obtained from the assembly of >70 million reads. These sequences should assist proteomic experiments aimed at identifying pharmacologically active proteins and immunological markers of vector exposure. A supplemental file of the transcriptome and deducted protein sequences can be obtained from http://exon.niaid.nih.gov/transcriptome/P_megistus/Pmeg-web.xlsx.
Assuntos
Proteínas de Insetos/genética , Panstrongylus/genética , Proteínas e Peptídeos Salivares/genética , Sialoglicoproteínas/genética , Animais , Proteínas de Insetos/metabolismo , Dados de Sequência Molecular , Ninfa/genética , Ninfa/crescimento & desenvolvimento , Ninfa/metabolismo , Panstrongylus/crescimento & desenvolvimento , Panstrongylus/metabolismo , Filogenia , Proteômica , Saliva/química , Proteínas e Peptídeos Salivares/metabolismo , Sialoglicoproteínas/metabolismoRESUMO
The malaria parasite, Plasmodium falciparum, and the human immune system have coevolved to ensure that the parasite is not eliminated and reinfection is not resisted. This relationship is likely mediated through a myriad of host-parasite interactions, although surprisingly few such interactions have been identified. Here we show that the 33-kDa fragment of P. falciparum merozoite surface protein 1 (MSP1(33)), an abundant protein that is shed during red blood cell invasion, binds to the proinflammatory protein, S100P. MSP1(33) blocks S100P-induced NFκB activation in monocytes and chemotaxis in neutrophils. Remarkably, S100P binds to both dimorphic alleles of MSP1, estimated to have diverged >27 Mya, suggesting an ancient, conserved relationship between these parasite and host proteins that may serve to attenuate potentially damaging inflammatory responses.
Assuntos
Proteínas de Ligação ao Cálcio/antagonistas & inibidores , Proteína 1 de Superfície de Merozoito/fisiologia , Proteínas de Neoplasias/antagonistas & inibidores , Plasmodium falciparum/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Ligação ao Cálcio/química , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Humanos , Microscopia Confocal , Dados de Sequência Molecular , Proteínas de Neoplasias/química , Homologia de Sequência de Aminoácidos , Ressonância de Plasmônio de SuperfícieRESUMO
The function of the antigen-5/CAP family of proteins found in the salivary gland of bloodsucking animals has remained elusive for decades. Antigen-5 members from the hematophagous insects Dipetalogaster maxima (DMAV) and Triatoma infestans (TIAV) were expressed and discovered to attenuate platelet aggregation, ATP secretion, and thromboxane A2 generation by low doses of collagen (<1 µg/ml) but no other agonists. DMAV did not interact with collagen, glycoprotein VI, or integrin α2ß1. This inhibitory profile resembles the effects of antioxidants Cu,Zn-superoxide dismutase (Cu,Zn-SOD) in platelet function. Accordingly, DMAV was found to inhibit cytochrome c reduction by O2[Symbol: see text] generated by the xanthine/xanthine oxidase, implying that it exhibits antioxidant activity. Moreover, our results demonstrate that DMAV blunts the luminescence signal of O2[Symbol: see text] generated by phorbol 12-myristate 13-acetate-stimulated neutrophils. Mechanistically, inductively coupled plasma mass spectrometry and fluorescence spectroscopy revealed that DMAV, like Cu,Zn-SOD, interacts with Cu(2+), which provides redox potential for catalytic removal of O2[Symbol: see text]. Notably, surface plasmon resonance experiments (BIAcore) determined that DMAV binds sulfated glycosaminoglycans (e.g. heparin, KD ~100 nmol/liter), as reported for extracellular SOD. Finally, fractions of the salivary gland of D. maxima with native DMAV contain Cu(2+) and display metal-dependent antioxidant properties. Antigen-5/CAP emerges as novel family of Cu(2+)-dependent antioxidant enzymes that inhibit neutrophil oxidative burst and negatively modulate platelet aggregation by a unique salivary mechanism.
Assuntos
Cobre/metabolismo , Sequestradores de Radicais Livres/metabolismo , Neutrófilos/metabolismo , Agregação Plaquetária , Explosão Respiratória , Triatoma/enzimologia , Sequência de Aminoácidos , Animais , Antioxidantes/metabolismo , Bovinos , Colágeno/metabolismo , Glicosaminoglicanos/metabolismo , Cavalos , Humanos , Peróxido de Hidrogênio/análise , Dados de Sequência Molecular , Oxigênio/metabolismo , Filogenia , Adesividade Plaquetária , Glândulas Salivares/enzimologia , Alinhamento de Sequência , Tubarões , Enxofre/química , Ressonância de Plasmônio de Superfície , SuínosRESUMO
OBJECTIVE: Polyphosphate and heparin are anionic polymers released by activated mast cells and platelets that are known to stimulate the contact pathway of coagulation. These polymers promote both the autoactivation of factor XII and the assembly of complexes containing factor XI, prekallikrein, and high-molecular-weight kininogen. We are searching for salivary proteins from blood-feeding insects that counteract the effect of procoagulant and proinflammatory factors in the host, including elements of the contact pathway. APPROACH AND RESULTS: Here, we evaluate the ability of the sand fly salivary proteins, PdSP15a and PdSP15b, to inhibit the contact pathway by disrupting binding of its components to anionic polymers. We attempt to demonstrate binding of the proteins to polyphosphate, heparin, and dextran sulfate. We also evaluate the effect of this binding on contact pathway reactions. We also set out to determine the x-ray crystal structure of PdSP15b and examine the determinants of relevant molecular interactions. Both proteins bind polyphosphate, heparin, and dextran sulfate with high affinity. Through this mechanism they inhibit the autoactivation of factor XII and factor XI, the reciprocal activation of factor XII and prekallikrein, the activation of factor XI by thrombin and factor XIIa, the cleavage of high-molecular-weight kininogen in plasma, and plasma extravasation induced by polyphosphate. The crystal structure of PdSP15b contains an amphipathic helix studded with basic side chains that forms the likely interaction surface. CONCLUSIONS: The results of these studies indicate that the binding of anionic polymers by salivary proteins is used by blood feeders as an antihemostatic/anti-inflammatory mechanism.
Assuntos
Anticoagulantes/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Sulfato de Dextrana/metabolismo , Heparina/metabolismo , Proteínas de Insetos/farmacologia , Polifosfatos/metabolismo , Psychodidae/química , Saliva/química , Animais , Anticoagulantes/química , Anticoagulantes/isolamento & purificação , Anticoagulantes/metabolismo , Testes de Coagulação Sanguínea , Permeabilidade Capilar/efeitos dos fármacos , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Fator XIIa/antagonistas & inibidores , Fator XIIa/metabolismo , Fator XIa/antagonistas & inibidores , Fator XIa/metabolismo , Humanos , Proteínas de Insetos/química , Proteínas de Insetos/isolamento & purificação , Proteínas de Insetos/metabolismo , Cininogênio de Alto Peso Molecular/antagonistas & inibidores , Cininogênio de Alto Peso Molecular/metabolismo , Camundongos , Modelos Moleculares , Pré-Calicreína/antagonistas & inibidores , Pré-Calicreína/metabolismo , Conformação Proteica , Relação Estrutura-Atividade , Trombina/metabolismo , Fatores de TempoRESUMO
The antihemostatic/antiangiogenic protein tablysin-15 is a member of the CAP (cysteine-rich secretory, antigen 5, and pathogenesis-related 1 protein) superfamily and has been shown to bind the integrins α(IIb)ß(3) and α(V)ß(3) by means of an Arg-Gly-Asp (RGD) tripeptide sequence. Here we describe the x-ray crystal structure of tablysin-15 and show that the RGD motif is located in a novel structural context. The motif itself is contained in a type II ß-turn structure that is similar in its conformation to the RGD sequence of the cyclic pentapeptide cilengitide when bound to integrin α(V)ß(3). The CAP domain also contains a hydrophobic channel that appears to bind a fatty acid molecule in the crystal structure after purification from Escherichia coli. After delipidation of the protein, tablysin-15 was found to bind proinflammatory cysteinyl leukotrienes with submicromolar affinities. The structure of the leukotriene E(4)-tablysin-15 complex shows that the ligand binds with the nonfunctionalized end of the fatty acid chain buried in the hydrophobic pocket, whereas the carboxylate end of the ligand binds forms hydrogen bond/salt bridge interactions with polar side chains at the channel entrance. Therefore, tablysin-15 functions as an inhibitor of integrin function and as an anti-inflammatory scavenger of eicosanoids.
Assuntos
Desintegrinas/metabolismo , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Leucotrienos/metabolismo , Proteínas e Peptídeos Salivares/química , Proteínas e Peptídeos Salivares/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Ácidos Graxos/metabolismo , Humanos , Proteínas de Insetos/farmacologia , Integrinas/antagonistas & inibidores , Modelos Moleculares , Dados de Sequência Molecular , Oligopeptídeos , Agregação Plaquetária/efeitos dos fármacos , Estrutura Terciária de Proteína , Proteínas e Peptídeos Salivares/farmacologiaRESUMO
Aegyptin is a mosquito salivary gland protein and potent inhibitor of platelet aggregation. Aegyptin binds to the von Willebrand factor-binding site on collagen and prevents its interaction with platelets. Because collagen also induces plasma clotting by activation of factor XII, we evaluated the effects of aegyptin on collagen-induced coagulation activation and how it interferes with thrombosis in three different in vivo models. Our results demonstrate that aegyptin abolishes collagen-induced clot formation and thrombin generation in platelet-free plasma. Aegyptin has no antithrombotic activity in the arteriovenous shunt model (collagen-independent) but it prevents laser-induced collagen-mediated thrombus formation in rats. Furthermore, aegyptin protects mice from collagen and epinephrine-induced thromboembolism. Therefore, aegyptin has a dual antithrombotic mechanism: inhibition of platelet-collagen interaction and collagen's pro-coagulant activity. This is the first description of a collagen-binding protein that also inhibits collagen-mediated coagulant activity.
Assuntos
Coagulação Sanguínea/efeitos dos fármacos , Colágeno/farmacologia , Proteínas de Insetos/farmacologia , Embolia Pulmonar/prevenção & controle , Proteínas e Peptídeos Salivares/farmacologia , Animais , Culicidae/metabolismo , Relação Dose-Resposta a Droga , Epinefrina/farmacologia , Feminino , Células HEK293 , Humanos , Proteínas de Insetos/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Ratos , Ratos Wistar , Proteínas Recombinantes/farmacologia , Proteínas e Peptídeos Salivares/genética , Trombina/metabolismoRESUMO
Platelet aggregation and acute inflammation are key processes in vertebrate defense to a skin injury. Recent studies uncovered the mediation of 2 serine proteases, cathepsin G and chymase, in both mechanisms. Working with a mouse model of acute inflammation, we revealed that an exogenous salivary protein of Ixodes ricinus, the vector of Lyme disease pathogens in Europe, extensively inhibits edema formation and influx of neutrophils in the inflamed tissue. We named this tick salivary gland secreted effector as I ricinus serpin-2 (IRS-2), and we show that it primarily inhibits cathepsin G and chymase, while in higher molar excess, it affects thrombin activity as well. The inhibitory specificity was explained using the crystal structure, determined at a resolution of 1.8 Å. Moreover, we disclosed the ability of IRS-2 to inhibit cathepsin G-induced and thrombin-induced platelet aggregation. For the first time, an ectoparasite protein is shown to exhibit such pharmacological effects and target specificity. The stringent specificity and biological activities of IRS-2 combined with the knowledge of its structure can be the basis for the development of future pharmaceutical applications.
Assuntos
Catepsina G/imunologia , Quimases/imunologia , Inflamação/imunologia , Proteínas de Insetos/imunologia , Ixodes/genética , Serpinas/imunologia , Sequência de Aminoácidos , Animais , Catepsina G/metabolismo , Quimases/metabolismo , Cristalização , Modelos Animais de Doenças , Feminino , Expressão Gênica , Humanos , Inflamação/metabolismo , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Ixodes/imunologia , Ixodes/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Agregação Plaquetária/genética , Agregação Plaquetária/imunologia , Estrutura Quaternária de Proteína , Proteínas e Peptídeos Salivares/genética , Proteínas e Peptídeos Salivares/imunologia , Proteínas e Peptídeos Salivares/metabolismo , Análise de Sequência de Proteína , Serpinas/genética , Serpinas/metabolismoRESUMO
The highly expressed D7 protein family of mosquito saliva has previously been shown to act as an anti-inflammatory mediator by binding host biogenic amines and cysteinyl leukotrienes (CysLTs). In this study we demonstrate that AnSt-D7L1, a two-domain member of this group from Anopheles stephensi, retains the CysLT binding function seen in the homolog AeD7 from Aedes aegypti but has lost the ability to bind biogenic amines. Unlike any previously characterized members of the D7 family, AnSt-D7L1 has acquired the important function of binding thromboxane A(2) (TXA(2)) and its analogs with high affinity. When administered to tissue preparations, AnSt-D7L1 abrogated Leukotriene C(4) (LTC(4))-induced contraction of guinea pig ileum and contraction of rat aorta by the TXA(2) analog U46619. The protein also inhibited platelet aggregation induced by both collagen and U46619 when administered to stirred platelets. The crystal structure of AnSt-D7L1 contains two OBP-like domains and has a structure similar to AeD7. In AnSt-D7L1, the binding pocket of the C-terminal domain has been rearranged relative to AeD7, making the protein unable to bind biogenic amines. Structures of the ligand complexes show that CysLTs and TXA(2) analogs both bind in the same hydrophobic pocket of the N-terminal domain. The TXA(2) analog U46619 is stabilized by hydrogen bonding interactions of the ω-5 hydroxyl group with the phenolic hydroxyl group of Tyr 52. LTC(4) and occupies a very similar position to LTE(4) in the previously determined structure of its complex with AeD7. As yet, it is not known what, if any, new function has been acquired by the rearranged C-terminal domain. This article presents, to our knowledge, the first structural characterization of a protein from mosquito saliva that inhibits collagen mediated platelet activation.
Assuntos
Anopheles/metabolismo , Insetos Vetores/metabolismo , Leucotrienos/metabolismo , Malária/transmissão , Saliva/metabolismo , Tromboxano A2/metabolismo , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/metabolismo , Animais , Aorta/efeitos dos fármacos , Calorimetria , Cobaias , Humanos , Íleo/efeitos dos fármacos , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Proteínas de Insetos/farmacologia , Leucotrieno C4/farmacologia , Contração Muscular/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Ligação Proteica , Estrutura Secundária de Proteína , Ratos , Saliva/química , Tromboxano A2/análogos & derivadosRESUMO
OBJECTIVE: Blood-sucking arthropods' salivary glands contain a remarkable diversity of antihemostatics. The aim of the present study was to identify the unique salivary anticoagulant of the sand fly Lutzomyia longipalpis, which remained elusive for decades. METHODS AND RESULTS: Several L. longipalpis salivary proteins were expressed in human embryonic kidney 293 cells and screened for inhibition of blood coagulation. A novel 32.4-kDa molecule, named Lufaxin, was identified as a slow, tight, noncompetitive, and reversible inhibitor of factor Xa (FXa). Notably, Lufaxin's primary sequence does not share similarity to any physiological or salivary inhibitors of coagulation reported to date. Lufaxin is specific for FXa and does not interact with FX, Dansyl-Glu-Gly-Arg-FXa, or 15 other enzymes. In addition, Lufaxin blocks prothrombinase and increases both prothrombin time and activated partial thromboplastin time. Surface plasmon resonance experiments revealed that FXa binds Lufaxin with an equilibrium constant ≈3 nM, and isothermal titration calorimetry determined a stoichiometry of 1:1. Lufaxin also prevents protease-activated receptor 2 activation by FXa in the MDA-MB-231 cell line and abrogates edema formation triggered by injection of FXa in the paw of mice. Moreover, Lufaxin prevents FeCl(3)-induced carotid artery thrombus formation and prolongs activated partial thromboplastin time ex vivo, implying that it works as an anticoagulant in vivo. Finally, salivary gland of sand flies was found to inhibit FXa and to interact with the enzyme. CONCLUSIONS: Lufaxin belongs to a novel family of slow-tight FXa inhibitors, which display antithrombotic and anti-inflammatory activities. It is a useful tool to understand FXa structural features and its role in prohemostatic and proinflammatory events.
Assuntos
Anti-Inflamatórios/farmacologia , Inibidores do Fator Xa , Fibrinolíticos/farmacologia , Inflamação/prevenção & controle , Proteínas de Insetos/farmacologia , Psychodidae/química , Receptor PAR-2/antagonistas & inibidores , Glândulas Salivares/química , Trombose/prevenção & controle , Sequência de Aminoácidos , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/isolamento & purificação , Coagulação Sanguínea/efeitos dos fármacos , Calorimetria , Linhagem Celular Tumoral , Cloretos , Clonagem Molecular , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Fator Xa/metabolismo , Feminino , Compostos Férricos , Fibrinolíticos/química , Fibrinolíticos/isolamento & purificação , Células HEK293 , Humanos , Inflamação/sangue , Inflamação/metabolismo , Proteínas de Insetos/química , Proteínas de Insetos/isolamento & purificação , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Peso Molecular , Tempo de Tromboplastina Parcial , Ligação Proteica , Tempo de Protrombina , Ratos , Receptor PAR-2/metabolismo , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/metabolismo , Ressonância de Plasmônio de Superfície , Tromboplastina/antagonistas & inibidores , Tromboplastina/metabolismo , Trombose/sangue , Trombose/induzido quimicamente , Trombose/metabolismo , Fatores de TempoRESUMO
OBJECTIVE: The coagulation-inflammation cycle has been implicated as a critical component in malaria pathogenesis. Defibrotide (DF), a mixture of DNA aptamers, displays anticoagulant, anti-inflammatory, and endothelial cell (EC)-protective activities and has been successfully used to treat comatose children with veno-occlusive disease. DF was investigated here as a drug to treat cerebral malaria. METHODS AND RESULTS: DF blocks tissue factor expression by ECs incubated with parasitized red blood cells and attenuates prothrombinase activity, platelet aggregation, and complement activation. In contrast, it does not affect nitric oxide bioavailability. We also demonstrated that Plasmodium falciparum glycosylphosphatidylinositol (Pf-GPI) induces tissue factor expression in ECs and cytokine production by dendritic cells. Notably, dendritic cells, known to modulate coagulation and inflammation systemically, were identified as a novel target for DF. Accordingly, DF inhibits Toll-like receptor ligand-dependent dendritic cells activation by a mechanism that is blocked by adenosine receptor antagonist (8-p-sulfophenyltheophylline) but not reproduced by synthetic poly-A, -C, -T, and -G. These results imply that aptameric sequences and adenosine receptor mediate dendritic cells responses to the drug. DF also prevents rosetting formation, red blood cells invasion by P. falciparum and abolishes oocysts development in Anopheles gambiae. In a murine model of cerebral malaria, DF affected parasitemia, decreased IFN-γ levels, and ameliorated clinical score (day 5) with a trend for increased survival. CONCLUSION: Therapeutic use of DF in malaria is proposed.
Assuntos
Anti-Inflamatórios/farmacologia , Anticoagulantes/farmacologia , Antimaláricos/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Malária Cerebral/tratamento farmacológico , Plasmodium berghei/efeitos dos fármacos , Plasmodium falciparum/efeitos dos fármacos , Polidesoxirribonucleotídeos/farmacologia , Animais , Células Cultivadas , Ativação do Complemento/efeitos dos fármacos , Citocinas/sangue , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Células Dendríticas/parasitologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Células Endoteliais/imunologia , Células Endoteliais/metabolismo , Células Endoteliais/parasitologia , Feminino , Glicosilfosfatidilinositóis/metabolismo , Hemoglobinas/metabolismo , Humanos , Mediadores da Inflamação/sangue , Malária Cerebral/sangue , Malária Cerebral/imunologia , Malária Cerebral/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Óxido Nítrico/metabolismo , Plasmodium berghei/patogenicidade , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/metabolismo , Plasmodium falciparum/patogenicidade , Agregação Plaquetária/efeitos dos fármacos , Receptores Purinérgicos P1/efeitos dos fármacos , Receptores Purinérgicos P1/metabolismo , Índice de Gravidade de Doença , Tromboplastina/metabolismo , Fatores de TempoRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection results in coagulation activation although it is usually not associated with consumption coagulopathy. D-dimers are also commonly elevated despite systemic hypofibrinolysis. To understand these unusual features of coronavirus disease 2019 (COVID-19) coagulopathy, 64 adult patients with SARS-CoV-2 infection (36 moderate and 28 severe) and 16 controls were studied. We evaluated the repertoire of plasma protease inhibitors (Serpins, Kunitz, Kazal, Cystatin-like) targeting the fibrinolytic system: Plasminogen Activator Inhibitor-1 (PAI-1), Tissue Plasminogen Activator/Plasminogen Activator Inhibitor-1 complex (t-PA/PAI-1), α-2-Antiplasmin, Plasmin-α2-Antiplasmin Complex, Thrombin-activatable Fibrinolysis Inhibitor (TAFI)/TAFIa, Protease Nexin-1 (PN-1), and Neuroserpin (the main t-PA inhibitor of the central nervous system). Inhibitors of the common (Antithrombin, Thrombin-antithrombin complex, Protein Z [PZ]/PZ inhibitor, Heparin Cofactor II, and α2-Macroglobulin), Protein C ([PC], Protein C inhibitor, and Protein S), contact (Kallistatin, Protease Nexin-2/Amyloid Beta Precursor Protein, and α-1-Antitrypsin), and complement (C1-Inhibitor) pathways, in addition to Factor XIII, Histidine-rich glycoprotein (HRG) and Vaspin were also investigated by enzyme-linked immunosorbent assay. The association of these markers with disease severity was evaluated by logistic regression. Pulmonary expression of PAI-1 and Neuroserpin in the lungs from eight post-mortem cases was assessed by immunohistochemistry. Results show that six patients (10%) developed thrombotic events, and mortality was 11%. There was no significant reduction in plasma anticoagulants, in keeping with a compensated state. However, an increase in fibrinolysis inhibitors (PAI-1, Neuroserpin, PN-1, PAP, and t-PA/PAI-1) was consistently observed, while HRG was reduced. Furthermore, these markers were associated with moderate and/or severe disease. Notably, immunostains demonstrated overexpression of PAI-1 in epithelial cells, macrophages, and endothelial cells of fatal COVID-19, while Neuroserpin was found in intraalveolar macrophages only. These results imply that the lungs in SARS-CoV-2 infection provide anti-fibrinolytic activity resulting in a shift toward a local and systemic hypofibrinolytic state predisposing to (immuno)thrombosis, often in a background of compensated disseminated intravascular coagulation.
RESUMO
Dendritic cells (DCs) are powerful initiators of innate and adaptive immune responses. Ticks are blood-sucking ectoparasite arthropods that suppress host immunity by secreting immunomodulatory molecules in their saliva. Here, compounds present in Rhipicephalus sanguineus tick saliva with immunomodulatory effects on DC differentiation, cytokine production, and costimulatory molecule expression were identified. R. sanguineus tick saliva inhibited IL-12p40 and TNF-α while potentiating IL-10 cytokine production by bone marrow-derived DCs stimulated by Toll-like receptor-2, -4, and -9 agonists. To identify the molecules responsible for these effects, we fractionated the saliva through microcon filtration and reversed-phase HPLC and tested each fraction for DC maturation. Fractions with proven effects were analyzed by micro-HPLC tandem mass spectrometry or competition ELISA. Thus, we identified for the first time in tick saliva the purine nucleoside adenosine (concentration of â¼110 pmol/µl) as a potent anti-inflammatory salivary inhibitor of DC cytokine production. We also found prostaglandin E(2) (PGE(2) â¼100 nM) with comparable effects in modulating cytokine production by DCs. Both Ado and PGE(2) inhibited cytokine production by inducing cAMP-PKA signaling in DCs. Additionally, both Ado and PGE(2) were able to inhibit expression of CD40 in mature DCs. Finally, flow cytometry analysis revealed that PGE(2), but not Ado, is the differentiation inhibitor of bone marrow-derived DCs. The presence of non-protein molecules adenosine and PGE(2) in tick saliva indicates an important evolutionary mechanism used by ticks to subvert host immune cells and allow them to successfully complete their blood meal and life cycle.