RESUMO
Influenza B virus (IBV) infections can cause severe disease in children and the elderly. Commonly used antivirals have lower clinical effectiveness against IBV compared to influenza A viruses (IAV). Neuraminidase (NA), the second major surface protein on the influenza virus, is emerging as a target of broadly protective antibodies that recognize the NA active site of IAVs. However, similarly broadly protective antibodies against IBV NA have not been identified. Here, we isolated and characterized human monoclonal antibodies (mAbs) that target IBV NA from an IBV-infected patient. Two mAbs displayed broad and potent capacity to inhibit IBV NA enzymatic activity, neutralize the virus in vitro, and protect against lethal IBV infection in mice in prophylactic and therapeutic settings. These mAbs inserted long CDR-H3 loops into the NA active site, engaging residues highly conserved among IBV NAs. These mAbs provide a blueprint for the development of improved vaccines and therapeutics against IBVs.
Assuntos
Anticorpos Antivirais/imunologia , Domínio Catalítico/imunologia , Vírus da Influenza B/imunologia , Neuraminidase/imunologia , Proteínas Virais/imunologia , Animais , Anticorpos Monoclonais/imunologia , Linhagem Celular , Cães , Feminino , Células HEK293 , Humanos , Vírus da Influenza A/imunologia , Influenza Humana/imunologia , Leucócitos Mononucleares/imunologia , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Infecções por Orthomyxoviridae/imunologiaRESUMO
Coagulation factor V (fV) is the precursor of activated fV (fVa), an essential component of the prothrombinase complex required for the rapid activation of prothrombin in the penultimate step of the coagulation cascade. In addition, fV regulates the tissue factor pathway inhibitor α (TFPIα) and protein C pathways that inhibit the coagulation response. A recent cryogenic electron microscopy (cryo-EM) structure of fV has revealed the architecture of its A1-A2-B-A3-C1-C2 assembly but left the mechanism that keeps fV in its inactive state unresolved because of an intrinsic disorder in the B domain. A splice variant of fV, fV short, carries a large deletion of the B domain that produces constitutive fVa-like activity and unmasks epitopes for the binding of TFPIα. The cryo-EM structure of fV short was solved at 3.2 Å resolution and revealed the arrangement of the entire A1-A2-B-A3-C1-C2 assembly. The shorter B domain stretches across the entire width of the protein, making contacts with the A1, A2, and A3 domains but suspended over the C1 and C2 domains. In the portion distal to the splice site, several hydrophobic clusters and acidic residues provide a potential binding site for the basic C-terminal end of TFPIα. In fV, these epitopes may bind intramolecularly to the basic region of the B domain. The cryo-EM structure reported in this study advances our understanding of the mechanism that keeps fV in its inactive state, provides new targets for mutagenesis and facilitates future structural analysis of fV short in complex with TFPIα, protein S, and fXa.
Assuntos
Fator V , Fator Xa , Fator V/metabolismo , Microscopia Crioeletrônica , Fator Xa/metabolismo , Fator Va/química , Coagulação Sanguínea , EpitoposRESUMO
The hemostatic response to vascular injury entails a sequence of proteolytic events where several inactive zymogens of the trypsin family are converted to active proteases. The cascade starts with exposure of tissue factor from the damaged endothelium and culminates with conversion of prothrombin to thrombin in a reaction catalyzed by the prothrombinase complex composed of the enzyme factor Xa, cofactor Va, Ca2+, and phospholipids. This cofactor-dependent activation is paradigmatic of analogous reactions of the blood coagulation and complement cascades, which makes elucidation of its molecular mechanism of broad significance to the large class of trypsin-like zymogens to which prothrombin belongs. Because of its relevance as the most important reaction in the physiological response to vascular injury, as well as the main trigger of pathological thrombotic complications, the mechanism of prothrombin activation has been studied extensively. However, a molecular interpretation of this mechanism has become available only recently from important developments in structural biology. Here we review current knowledge on the prothrombin-prothrombinase interaction and outline future directions for the study of this key reaction of the coagulation cascade.
Assuntos
Coagulação Sanguínea , Protrombina , Tromboplastina , Humanos , Protrombina/metabolismo , Protrombina/química , Tromboplastina/metabolismo , Tromboplastina/química , Coagulação Sanguínea/fisiologia , Animais , Ligação Proteica , Fator Xa/metabolismo , Fator VRESUMO
Patients with hereditary angioedema (HAE) experience episodes of bradykinin (BK)-induced swelling of skin and mucosal membranes. The most common cause is reduced plasma activity of C1 inhibitor, the main regulator of the proteases plasma kallikrein (PKa) and factor XIIa (FXIIa). Recently, patients with HAE were described with a Lys311 to glutamic acid substitution in plasminogen (Plg), the zymogen of the protease plasmin (Plm). Adding tissue plasminogen activator to plasma containing Plg-Glu311 vs plasma containing wild-type Plg (Plg-Lys311) results in greater BK generation. Similar results were obtained in plasma lacking prekallikrein or FXII (the zymogens of PKa and FXIIa) and in normal plasma treated with a PKa inhibitor, indicating Plg-Glu311 induces BK generation independently of PKa and FXIIa. Plm-Glu311 cleaves high and low molecular weight kininogens (HK and LK, respectively), releasing BK more efficiently than Plm-Lys311. Based on the plasma concentrations of HK and LK, the latter may be the source of most of the BK generated by Plm-Glu311. The lysine analog ε-aminocaproic acid blocks Plm-catalyzed BK generation. The Glu311 substitution introduces a lysine-binding site into the Plg kringle 3 domain, perhaps altering binding to kininogens. Plg residue 311 is glutamic acid in most mammals. Glu311 in patients with HAE, therefore, represents reversion to the ancestral condition. Substantial BK generation occurs during Plm-Glu311 cleavage of human HK, but not mouse HK. Furthermore, mouse Plm, which has Glu311, did not liberate BK from human kininogens more rapidly than human Plg-Lys311. This indicates Glu311 is pathogenic in the context of human Plm when human kininogens are the substrates.
Assuntos
Angioedemas Hereditários , Angioedemas Hereditários/genética , Angioedemas Hereditários/patologia , Animais , Bradicinina/metabolismo , Fator XIIa/metabolismo , Fibrinolisina , Ácido Glutâmico , Humanos , Cininogênios/metabolismo , Lisina , Mamíferos/metabolismo , Camundongos , Calicreína Plasmática , Plasminogênio/genética , Plasminogênio/metabolismo , Ativador de Plasminogênio TecidualRESUMO
Prekallikrein (PK) is the precursor of the trypsin-like plasma protease kallikrein (PKa), which cleaves kininogens to release bradykinin and converts the protease precursor factor XII (FXII) to the enzyme FXIIa. PK and FXII undergo reciprocal conversion to their active forms (PKa and FXIIa) by a process that is accelerated by a variety of biological and artificial surfaces. The surface-mediated process is referred to as contact activation. Previously, we showed that FXII expresses a low level of proteolytic activity (independently of FXIIa) that may initiate reciprocal activation with PK. The current study was undertaken to determine whether PK expresses similar activity. Recombinant PK that cannot be converted to PKa was prepared by replacing Arg371 with alanine at the activation cleavage site (PK-R371A, or single-chain PK). Despite being constrained to the single-chain precursor form, PK-R371A cleaves high-molecular-weight kininogen (HK) to release bradykinin with a catalytic efficiency â¼1500-fold lower than that of kallikrein cleavage of HK. In the presence of a surface, PK-R371A converts FXII to FXIIa with a specific activity â¼4 orders of magnitude lower than for PKa cleavage of FXII. These results support the notion that activity intrinsic to PK and FXII can initiate reciprocal activation of FXII and PK in solution or on a surface. The findings are consistent with the hypothesis that the putative zymogens of many trypsin-like proteases are actually active proteases, explaining their capacity to undergo processes such as autoactivation and to initiate enzyme cascades.
Assuntos
Coagulação Sanguínea , Bradicinina/metabolismo , Pré-Calicreína/metabolismo , Substituição de Aminoácidos , Animais , Fator XII/metabolismo , Células HEK293 , Humanos , Cininogênio de Alto Peso Molecular/metabolismo , Camundongos Endogâmicos C57BL , Pré-Calicreína/química , Pré-Calicreína/genética , Proteólise , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Biological roles for extracellular RNA (eRNA) have become apparent. For example, eRNA can induce contact activation in blood via activation of the plasma proteases factor XII (FXII) and factor XI (FXI). We sought to reveal the biological role of the secretory enzyme ribonuclease 1 (RNase 1) in an organismal context by generating and analyzing RNase 1 knockout (Rnase1-/-) mice. We found that these mice are viable, healthy, and fertile, though larger than Rnase1+/+ mice. Rnase1-/- plasma contains more RNA than does the plasma of Rnase1+/+ mice. Moreover, the plasma of Rnase1-/- mice clots more rapidly than does wild-type plasma. This phenotype appeared to be due to increased levels of the active form of FXII (FXIIa) in the plasma of Rnase1-/- mice compared to Rnase1+/+ mice, and is consistent with the known effects of eRNA on FXII activation. The apparent activity of FXI in the plasma of Rnase1-/- mice was 1000-fold higher when measured in an assay triggered by a low concentration of tissue factor than in assays based on recalcification, consistent with eRNA enhancing FXI activation by thrombin. These findings suggest that one of the physiological functions of RNase 1 is to degrade eRNA in blood plasma. Loss of this function facilitates FXII and FXI activation, which could have effects on inflammation and blood coagulation. We anticipate that Rnase1-/- mice will be a useful tool for evaluating other hypotheses about the functions of RNase 1 and of eRNA in vivo.
Assuntos
Neurotoxina Derivada de Eosinófilo/deficiência , Fator XII/metabolismo , RNA/química , Animais , Coagulação Sanguínea , Tamanho Corporal , Neurotoxina Derivada de Eosinófilo/genética , Fator XI/metabolismo , Feminino , Fertilidade , Técnicas de Inativação de Genes , Masculino , Camundongos , Modelos Animais , Fenótipo , RNA/sangue , Estabilidade de RNA , Regulação para CimaRESUMO
The plasma proteins factor XII (FXII) and prekallikrein (PK) undergo reciprocal activation to the proteases FXIIa and kallikrein by a process that is enhanced by surfaces (contact activation) and regulated by the serpin C1 inhibitor. Kallikrein cleaves high-molecular-weight kininogen (HK), releasing the vasoactive peptide bradykinin. Patients with hereditary angioedema (HAE) experience episodes of soft tissue swelling as a consequence of unregulated kallikrein activity or increased prekallikrein activation. Although most HAE cases are caused by reduced plasma C1-inhibitor activity, HAE has been linked to lysine/arginine substitutions for Thr309 in FXII (FXII-Lys/Arg309). Here, we show that FXII-Lys/Arg309 is susceptible to cleavage after residue 309 by coagulation proteases (thrombin and FXIa), resulting in generation of a truncated form of FXII (δFXII). The catalytic efficiency of δFXII activation by kallikrein is 15-fold greater than for full-length FXII. The enhanced rate of reciprocal activation of PK and δFXII in human plasma and in mice appears to overwhelm the normal inhibitory function of C1 inhibitor, leading to increased HK cleavage. In mice given human FXII-Lys/Arg309, induction of thrombin generation by infusion of tissue factor results in enhanced HK cleavage as a consequence of δFXII formation. The effects of δFXII in vitro and in vivo are reproduced when wild-type FXII is bound by an antibody to the FXII heavy chain (HC; 15H8). The results contribute to our understanding of the predisposition of patients carrying FXII-Lys/Arg309 to angioedema after trauma, and reveal a regulatory function for the FXII HC that normally limits PK activation in plasma.
Assuntos
Fator XII/química , Fator XIa/química , Angioedema Hereditário Tipo III/sangue , Angioedema Hereditário Tipo III/genética , Angioedemas Hereditários , Animais , Arginina/química , Coagulação Sanguínea , Bradicinina/sangue , Catálise , Proteína Inibidora do Complemento C1/química , Fator XIIa/química , Células HEK293 , Humanos , Cininogênios/sangue , Lisina/química , Camundongos , Camundongos Endogâmicos C57BL , Calicreína Plasmática/química , Pré-Calicreína/química , Ligação Proteica , Proteínas Recombinantes/química , Propriedades de Superfície , Trombina/genéticaRESUMO
Hemostasis is the normal process that produces a blood clot at a site of vascular injury. Mice are widely used to study hemostasis and abnormalities of blood coagulation because their hemostatic system is similar in most respects to that of humans, and their genomes can be easily manipulated to create models of inherited human coagulation disorders. Two of the most widely used techniques for assessing hemostasis in mice are the tail bleeding time (TBT) and saphenous vein bleeding (SVB) models. Here we discuss the use of these methods in the evaluation of hemostasis, and the advantages and limits of using mice as surrogates for studying hemostasis in humans.
Assuntos
Tempo de Sangramento/métodos , Coagulação Sanguínea , Modelos Animais de Doenças , Hemorragia/metabolismo , Animais , Hemostasia , Humanos , Lacerações/sangue , Lacerações/metabolismo , Fígado/lesões , Fígado/metabolismo , Camundongos , Veia Safena/lesões , Veia Safena/metabolismo , Cauda/lesões , Cauda/metabolismoRESUMO
INTRODUCTION: Tissue factor pathway inhibitor (TFPI) is an endogenous inhibitor of the extrinsic pathway that negatively regulates thrombin production during coagulation. Under haemophilic conditions, where the intrinsic coagulation pathway is impaired, inhibition of TFPI may improve clotting. AIM: We investigated the ex vivo effects of a human TFPI neutralizing antibody, marstacimab (previously PF-06741086), in coagulation assays including rotational thromboelastometry (ROTEM), thrombin generation assay (TGA) and the dilute prothrombin time (dPT) assay, performed in haemophilic whole blood and plasmas. We compared the effects of marstacimab to the effects of recombinant coagulation factors and investigated the reproducibility of marstacimab in restoring haemostasis by comparing its effect in whole blood collected from the same study participants on differing days. METHODS: Citrated whole blood and plasmas obtained from haemophilia participants were supplemented ex vivo with vehicle, marstacimab, recombinant FVIII (rFVIII) or recombinant factor IX (rFIX) and analysed in ROTEM, TGA and the dPT assay using low tissue factor concentrations to trigger coagulation. RESULTS: Marstacimab induced pro-coagulant responses in ROTEM parameters including reduction in clotting times and increases in angle. Similarly, participant plasmas supplemented with marstacimab exhibited improvements in TGA parameters, including reduced lag times, increased peak thrombin concentrations and reductions in dPT clotting time. Concentrations of marstacimab tested showed activity comparable to addition of rFVIII or rFIX and were reproducible. CONCLUSIONS: These studies show the ex vivo potency of marstacimab in restoring haemostasis in whole blood and plasmas from haemophilia participants and comparability to ex vivo reconstitution with recombination coagulation factors.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Coagulação Sanguínea/efeitos dos fármacos , Hemofilia A/tratamento farmacológico , Plasma/metabolismo , Tromboplastina/antagonistas & inibidores , Anticorpos Monoclonais Humanizados/farmacologia , Feminino , Hemofilia A/patologia , Humanos , MasculinoRESUMO
BACKGROUND: Mortality is high among patients with Middle East Respiratory Syndrome Coronavirus (MERS-CoV) infection. We aimed to determine hospital mortality and the factors associated with it in a cohort of MERS-CoV patients. METHODS: We reviewed hospital records of confirmed cases (detection of virus by polymerase chain reaction from respiratory tract samples) of MERS-CoV patients (n = 63) admitted to Buraidah Central Hospital in Al-Qassim, Saudi Arabia between 2014 and 2017. We abstracted data on demography, vital signs, associated conditions presented on admission, pre-existing chronic diseases, treatment, and vital status. Bi-variate comparisons and multiple logistic regressions were the choice of data analyses. RESULTS: The mean age was 60 years (SD = 18.2); most patients were male (74.6%) and Saudi citizens (81%). All but two patients were treated with Ribavirin plus Interferon. Hospital mortality was 25.4%. Patients who were admitted with septic shock and/or organ failure were significantly more likely to die than patients who were admitted with pneumonia and/or acute respiratory distress syndrome (OR = 47.9, 95% CI = 3.9, 585.5, p-value 0.002). Age, sex, and presence of chronic conditions were not significantly associated with mortality. CONCLUSION: Hospital mortality was 25%; septic shock/organ failure at admittance was a significant predictor of mortality.
Assuntos
Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/mortalidade , Mortalidade Hospitalar , Adulto , Idoso , Antivirais/uso terapêutico , Estudos de Coortes , Infecções por Coronavirus/complicações , Feminino , Humanos , Pacientes Internados/estatística & dados numéricos , Interferon-alfa/uso terapêutico , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Pneumonia/tratamento farmacológico , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/etiologia , Pneumonia Viral/mortalidade , Reação em Cadeia da Polimerase , Ribavirina/uso terapêutico , Arábia Saudita/epidemiologia , Resultado do TratamentoRESUMO
INTRODUCTION: The thrombin generation assay (TGA) can be used to monitor factor replacement therapy in patients with haemophilia. The TGA assay is typically performed using tissue factor as the reaction activator; however, activating with FIXa or FXIa can enhance assay sensitivity when FVIII < 1%. AIMS: To evaluate the sensitivity of the TGA when FIXa (5 nmol/L) and FXIa (0.22 nmol/L) are used to activate the assay in platelet-poor plasma and to compare these data to the one-stage and chromogenic assays. METHODS: Plasma from 10 severe FVIII-deficient subjects was supplemented with FVIII (0%, 0.1%, 0.4%, 1.2%, 4%, 11% and 33%), using either Novo Eight® , Advate® , Eloctate® , turoctocog alfa pegol or a control standard. The one-stage and chromogenic assays quantified the FVIII levels. The TGA assay was activated using either FIXa or FXIa. RESULTS: Both FIXa- and FXIa-activated TGA were sensitive across FVIII concentrations, with intra-assay coefficient of variation (CV) < 10%. The FXIa-activated assay had 25% CV at the lowest level of FVIII compared to 10% CV with FIXa activation. There were strong correlations between the FIXa- and FXIa-activated TGA tests (R2 = 0.9912) and between the one-stage and chromogenic assays (R2 = 0.9469). However, there were poor relationships between the TGA tests and one-stage and chromogenic assays. CONCLUSIONS: Both FIXa- and FXIa activation results in similar TGA profiles across a FVIII range of 0.1%-33%; however, FIXa activation was more robust at the lowest levels of FVIII compared with FXIa activation.
Assuntos
Fator VIII/metabolismo , Fator XIa/metabolismo , Hemofilia A/sangue , Trombina/metabolismo , HumanosRESUMO
Hemodialysis (HD) and peritoneal dialysis (PD) are the primary means of managing end stage renal disease (ESRD). However, these treatment modalities are associated with the onset of coagulation abnormalities. Effective management of coagulation risk among these patients requires the identification of surrogate markers that provide an early indication of the coagulation abnormalities. The role of sphingolipids in the manifestation and prediction of coagulation abnormalities among dialysis patients have never been investigated. Herein, we report the first instance of an in depth investigation into the sphingolipid changes among ESRD patients undergoing HD and PD. The results reveal distinct differences in terms of perturbations to specific sphingolipid biosynthetic pathways that are highly dependent on the treatment modality. Our studies also demonstrated strong correlation between specific sphingolipids and coagulation parameters, such as HexCer(d18:1/26:0) and maximal amplitude (MA), SM(d18:1/24:1) and tissue factor pathway inhibitor, and sphingosine 1-phosphate d18:1 and FX (Spearman ρ of 0.93, 0.89, and -0.89, respectively). Furthermore, our study revealed the potential for using HexCer(d18:1/22:0), HexCer(d18:1/24:0), and HexCer(d18:1/26:0) (r2 = 0.71, 0.82, and 0.63, respectively) and coagulation parameter MA (r2 = 0.7) for successful diagnosis of differential coagulopathies among ESRD patients undergoing HD, providing an opportunity toward personalized disease management.
Assuntos
Transtornos da Coagulação Sanguínea/metabolismo , Falência Renal Crônica/sangue , Diálise Peritoneal/efeitos adversos , Diálise Renal/efeitos adversos , Insuficiência Renal Crônica/sangue , Adulto , Biomarcadores/sangue , Coagulação Sanguínea , Transtornos da Coagulação Sanguínea/etiologia , Transtornos da Coagulação Sanguínea/patologia , Cromatografia Líquida de Alta Pressão , Feminino , Humanos , Falência Renal Crônica/complicações , Falência Renal Crônica/patologia , Falência Renal Crônica/terapia , Metabolismo dos Lipídeos , Lisofosfolipídeos/metabolismo , Masculino , Pessoa de Meia-Idade , Insuficiência Renal Crônica/complicações , Insuficiência Renal Crônica/patologia , Insuficiência Renal Crônica/terapia , Esfingolipídeos/metabolismo , Esfingomielinas/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismoRESUMO
Vitamin C (VitC) or ascorbic acid (AscA), a cofactor for collagen synthesis and a primary antioxidant, is rapidly consumed post-wounding. Parenteral VitC administration suppresses pro-inflammatory responses while promoting anti-inflammatory and pro-resolution effects in human/murine sepsis. We hypothesised that VitC could promote wound healing by altering the inflammatory, proliferative and remodelling phases of wound healing. Mice unable to synthesise VitC (Gulo(-/-) ) were used in this study. VitC was provided in the water (sufficient), withheld from another group (deficient) and supplemented by daily intra-peritoneal infusion (200 mg/kg, deficient + AscA) in a third group. Full thickness excisional wounds (6 mm) were created and tissue collected on days 7 and 14 for histology, quantitative polymerase chain reaction (qPCR) and Western blotting. Human neonatal dermal fibroblasts (HnDFs) were used to assess effects of In conclusion, VitC favorably on proliferation. Histological analysis showed improved wound matrix deposition and organisation in sufficient and deficient +AscA mice. Wounds from VitC sufficient and deficient + AscA mice had reduced expression of pro-inflammatory mediators and higher expression of wound healing mediators. Supplementation of HnDF with AscA induced the expression of self-renewal genes and promoted fibroblast proliferation. VitC favourably impacts the spatiotemporal expression of transcripts associated with early resolution of inflammation and tissue remodelling.
Assuntos
Cicatrização , Animais , Antioxidantes , Ácido Ascórbico , Fibroblastos , Humanos , Inflamação , CamundongosRESUMO
INTRODUCTION: Macrophage reprogramming is vital for resolution of acute inflammation. Parenteral vitamin C (VitC) attenuates proinflammatory states in murine and human sepsis. However information about the mechanism by which VitC regulates resolution of inflammation is limited. METHODS: To examine whether physiological levels of VitC modulate resolution of inflammation, we used transgenic mice lacking L-gulono-γ-lactone oxidase. VitC sufficient/deficient mice were subjected to a thioglycollate-elicited peritonitis model of sterile inflammation. Some VitC deficient mice received daily parenteral VitC (200 mg/kg) for 3 or 5 days following thioglycollate infusion. Peritoneal macrophages harvested on day 3 or day 5 were examined for intracellular VitC levels, pro- and anti-inflammatory protein and lipid mediators, mitochondrial function, and response to lipopolysaccharide (LPS). The THP-1 cell line was used to determine the modulatory activities of VitC in activated human macrophages. RESULTS: VitC deficiency significantly delayed resolution of inflammation and generated an exaggerated proinflammatory response to in vitro LPS stimulation. VitC sufficiency and in vivo VitC supplementation restored macrophage phenotype and function in VitC deficient mice. VitC loading of THP-1 macrophages attenuated LPS-induced proinflammatory responses. CONCLUSION: VitC sufficiency favorably modulates macrophage function. In vivo or in vitro VitC supplementation restores macrophage phenotype and function leading to timely resolution of inflammation.
Assuntos
Ácido Ascórbico/metabolismo , Ácido Ascórbico/uso terapêutico , Inflamação/tratamento farmacológico , Animais , Western Blotting , Linhagem Celular , Humanos , Inflamação/induzido quimicamente , Inflamação/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Microscopia de Fluorescência , Peritonite/induzido quimicamente , Peritonite/tratamento farmacológico , Peritonite/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Tioglicolatos/toxicidadeRESUMO
BACKGROUND: Coagulation factor (F)V features an A1-A2-B-A3-C1-C2 domain organization and functions as the inactive precursor of FVa, a component of the prothrombinase complex required for rapid thrombin generation in the penultimate step of the coagulation cascade. An intramolecular interaction within the large B domain (residues 710-1545) involves the basic region (BR, residues 963-1008) and acidic region (AR, residues 1493-1537) and locks FV in its inactive state. However, structural information on this important regulatory interaction or on the separate architecture of the AR and BR remains elusive due to conformational disorder of the B domain. OBJECTIVES: To reveal the structure of the BR-AR interaction or of its separate components. METHODS: The structure of FV is solved by cryogenic electron microscopy. RESULTS: A new 3.05 Å resolution cryogenic electron microscopy structure of FV confirms the overall organization of the A and C domains but resolves the segment 1507 to 1545 within a largely disordered B domain. The segment contains most of the AR and is organized as recently reported in FV short, a spliced variant of FV with a significantly shorter and less disordered B domain. CONCLUSION: The similar architecture of the AR in FV and FV short provides structural context for physiologically important interactions of this region with the BR in FV and with the basic C-terminal end of tissue factor pathway inhibitor α in FV short.
Assuntos
Coagulação Sanguínea , Fator V , Humanos , Fator V/metabolismo , Domínios Proteicos , Microscopia EletrônicaRESUMO
BACKGROUND: In plasma, high molecular weight kininogen (HK) is either free or bound to prekallikrein (PK) or factor (F) XI (FXI). During contact activation, HK is thought to anchor PK and FXI to surfaces, facilitating their conversion to the proteases plasma kallikrein and FXIa. Mice lacking HK have normal hemostasis but are resistant to injury-induced arterial thrombosis. OBJECTIVES: To identify amino acids on the HK-D6 domain involved in PK and FXI binding and study the importance of the HK-PK and HK-FXI interactions to coagulation. METHODS: Twenty-four HK variants with alanine replacements spanning residues 542-613 were tested in PK/FXI binding and activated partial thromboplastin time clotting assays. Surface-induced FXI and PK activation in plasma were studied in the presence or absence of HK. Kng1-/- mice lacking HK were supplemented with human or murine HK and tested in an arterial thrombosis model. RESULTS: Overlapping binding sites for PK and FXI were identified in the HK-D6 domain. HK variants with defects only in FXI binding corrected the activated partial thromboplastin time of HK-deficient plasma poorly compared to a variant defective only in PK-binding. In plasma, HK deficiency appeared to have a greater deleterious effect on FXI activation than PK activation. Human HK corrected the defect in arterial thrombus formation in HK-deficient mice poorly due to a specific defect in binding to mouse FXI. CONCLUSION: Clinical observations indicate FXI is required for hemostasis, while HK is not. Yet, the HK-FXI interaction is required for contact activation-induced clotting in vitro and in vivo suggesting an important role in thrombosis and perhaps other FXI-related activities.
Assuntos
Cininogênio de Alto Peso Molecular , Trombose , Animais , Humanos , Camundongos , Cininogênio de Alto Peso Molecular/metabolismo , Fator XI/metabolismo , Pré-Calicreína/metabolismo , Coagulação SanguíneaRESUMO
Germinal centers (GC) are microanatomical lymphoid structures where affinity-matured memory B cells and long-lived bone marrow plasma cells are primarily generated. It is unclear how the maturation of B cells within the GC impacts the breadth and durability of B cell responses to influenza vaccination in humans. We used fine needle aspiration of draining lymph nodes to longitudinally track antigen-specific GC B cell responses to seasonal influenza vaccination. Antigen-specific GC B cells persisted for at least 13 wk after vaccination in two out of seven individuals. Monoclonal antibodies (mAbs) derived from persisting GC B cell clones exhibit enhanced binding affinity and breadth to influenza hemagglutinin (HA) antigens compared with related GC clonotypes isolated earlier in the response. Structural studies of early and late GC-derived mAbs from one clonal lineage in complex with H1 and H5 HAs revealed an altered binding footprint. Our study shows that inducing sustained GC reactions after influenza vaccination in humans supports the maturation of responding B cells.
Assuntos
Linfócitos B , Centro Germinativo , Vacinas contra Influenza , Vacinação , Centro Germinativo/imunologia , Humanos , Vacinas contra Influenza/imunologia , Linfócitos B/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Influenza Humana/imunologia , Influenza Humana/prevenção & controle , Anticorpos Antivirais/imunologia , Anticorpos Monoclonais/imunologia , Adulto , Feminino , Masculino , Pessoa de Meia-IdadeRESUMO
Evaluating prospective anticoagulant therapies in animal thrombosis and bleeding models are standard pre-clinical approaches. Mice are frequently used for initial evaluations because a variety of models have been developed in this well-characterized species, and mice are relatively inexpensive to maintain. Because mice seem to be resistant to forming "spontaneous" thrombosis, vessel injury is used to induce intravascular clot formation. For the purpose of testing heparin-based drugs, we adapted a well-established model in which thrombus formation in the carotid artery is induced by exposing the vessel to ferric chloride. For studying anticoagulant effects on venous thrombosis, we use a model in which the inferior vena cava is ligated and the size of the resulting clots are measured. The most common adverse effect of anticoagulation therapy is bleeding. We describe a simple tail bleeding time that has been used for many years to study the effects of anticoagulants on hemostasis. We also describe a more reproducible, but more technically challenging, saphenous vein bleeding model that is also used for this purpose.
Assuntos
Anticoagulantes/química , Trombose , Animais , Anticoagulantes/farmacologia , Modelos Animais de Doenças , Hemorragia , Heparitina Sulfato , Camundongos , Estudos Prospectivos , Trombose/tratamento farmacológicoRESUMO
A State of the Art lecture titled "Cryo-EM structures of coagulation factors" was presented at the ISTH Congress in 2022. Cryogenic electron microscopy (cryo-EM) is a revolutionary technique capable of solving the structure of high molecular weight proteins and their complexes, unlike nuclear magnetic resonance (NMR), and under conditions not biased by crystal contacts, unlike X-ray crystallography. These features are particularly relevant to the analysis of coagulation factors that are too big for NMR and often recalcitrant to X-ray investigation. Using cryo-EM, we have solved the structures of coagulation factors V and Va, prothrombinase on nanodiscs, and the prothrombin-prothrombinase complex. These structures have advanced basic knowledge in the field of thrombosis and hemostasis, especially on the function of factor V and the molecular mechanism for prothrombin activation, and set the stage for exciting new lines of investigation. Finally, we summarize relevant new data on this topic presented during the 2022 ISTH Congress.
RESUMO
Factor XII (FXII) is the zymogen of a plasma protease (FXIIa) that contributes to bradykinin generation by converting prekallikrein to the protease plasma kallikrein (PKa). FXII conversion to FXIIa by autocatalysis or PKa-mediated cleavage is enhanced when the protein binds to negatively charged surfaces such as polymeric orthophosphate. FXII is composed of noncatalytic (heavy chain) and catalytic (light chain) regions. The heavy chain promotes FXII surface-binding and surface-dependent activation but restricts activation when FXII is not surface bound. From the N terminus, the heavy chain contains fibronectin type 2 (FN2), epidermal growth factor-1 (EGF1), fibronectin type 1 (FN1), EGF2, and kringle (KNG) domains and a proline-rich region. It shares this organization with its homolog, pro-hepatocyte growth factor activator (Pro-HGFA). To study the importance of heavy chain domains in FXII function, we prepared FXII with replacements of each domain with corresponding Pro-HGFA domains and tested them in activation and activity assays. EGF1 is required for surface-dependent FXII autoactivation and surface-dependent prekallikrein activation by FXIIa. KNG and FN2 are important for limiting FXII activation in the absence of a surface by a process that may require interactions between a lysine/arginine binding site on KNG and basic residues elsewhere on FXII. This interaction is disrupted by the lysine analog ε-aminocaproic acid. A model is proposed in which an ε-aminocaproic acid-sensitive interaction between the KNG and FN2 domains maintains FXII in a conformation that restricts activation. Upon binding to a surface through EGF1, the KNG/FN2-dependent mechanism is inactivated, exposing the FXII activation cleavage site.