RESUMEN
Adeno-associated virus (AAV) based gene therapy has demonstrated effective disease control in hemophilia. However, pre-existing immunity from wild-type AAV exposure impacts gene therapy eligibility. The aim of this multicenter epidemiologic study was to determine the prevalence and persistence of preexisting immunity against AAV2, AAV5, and AAV8, in adult participants with hemophilia A or B. Blood samples were collected at baseline and annually for ≤3 years at trial sites in Austria, France, Germany, Italy, Spain, and the United States. At baseline, AAV8, AAV2, and AAV5 neutralizing antibodies (NAbs) were present in 46.9%, 53.1%, and 53.4% of participants, respectively; these values remained stable at Years 1 and 2. Co-prevalence of NAbs to at least two serotypes and all three serotypes was present at baseline for ~40% and 38.2% of participants, respectively. For each serotype, ~10% of participants who tested negative for NAbs at baseline were seropositive at Year 1. At baseline, 38.3% of participants had detectable cell mediated immunity by ELISpot, although no correlations were observed with the humoral response. In conclusion, participants with hemophilia may have significant preexisting immunity to AAV capsids. Insights from this study may assist in understanding capsid-based immunity trends in participants considering AAV vector-based gene therapy.
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Anticuerpos Neutralizantes , Anticuerpos Antivirales , Dependovirus , Terapia Genética , Hemofilia A , Humanos , Dependovirus/inmunología , Dependovirus/genética , Masculino , Hemofilia A/inmunología , Hemofilia A/terapia , Adulto , Estudios Longitudinales , Anticuerpos Antivirales/sangre , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunología , Terapia Genética/métodos , Inmunidad Adaptativa , Vectores Genéticos/genética , Vectores Genéticos/administración & dosificación , Vectores Genéticos/inmunología , Persona de Mediana Edad , Prevalencia , Adulto JovenRESUMEN
The blood-clotting protein fibrinogen has been implicated in host defense following Staphylococcus aureus infection, but precise mechanisms of host protection and pathogen clearance remain undefined. Peritonitis caused by staphylococci species is a complication for patients with cirrhosis, indwelling catheters, or undergoing peritoneal dialysis. Here, we sought to characterize possible mechanisms of fibrin(ogen)-mediated antimicrobial responses. Wild-type (WT) (Fib+) mice rapidly cleared S. aureus following intraperitoneal infection with elimination of â¼99% of an initial inoculum within 15 min. In contrast, fibrinogen-deficient (Fib-) mice failed to clear the microbe. The genotype-dependent disparity in early clearance resulted in a significant difference in host mortality whereby Fib+ mice uniformly survived whereas Fib- mice exhibited high mortality rates within 24 h. Fibrin(ogen)-mediated bacterial clearance was dependent on (pro)thrombin procoagulant function, supporting a suspected role for fibrin polymerization in this mechanism. Unexpectedly, the primary host initiator of coagulation, tissue factor, was found to be dispensable for this antimicrobial activity. Rather, the bacteria-derived prothrombin activator vWbp was identified as the source of the thrombin-generating potential underlying fibrin(ogen)-dependent bacterial clearance. Mice failed to eliminate S. aureus deficient in vWbp, but clearance of these same microbes in WT mice was restored if active thrombin was administered to the peritoneal cavity. These studies establish that the thrombin/fibrinogen axis is fundamental to host antimicrobial defense, offer a possible explanation for the clinical observation that coagulase-negative staphylococci are a highly prominent infectious agent in peritonitis, and suggest caution against anticoagulants in individuals susceptible to peritoneal infections.
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Fibrinógeno/metabolismo , Peritonitis/metabolismo , Protrombina/metabolismo , Animales , Antibacterianos/metabolismo , Antiinfecciosos/metabolismo , Coagulación Sanguínea , Coagulasa/metabolismo , Femenino , Fibrina/metabolismo , Fibrinógeno/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/metabolismo , Staphylococcus aureus/patogenicidad , TromboplastinaRESUMEN
Efficient migration of macrophages to sites of inflammation requires cell surface-bound plasmin(ogen). Here, we investigated the mechanisms underlying the deficits of plasmin(ogen)-mediated macrophage migration in 2 models: murine thioglycollate-induced peritonitis and in vitro macrophage migration. As previously reported, macrophage migration into the peritoneal cavity of mice in response to thioglycollate was significantly impaired in the absence of plasminogen. Fibrin(ogen) deposition was noted in the peritoneal cavity in response to thioglycollate, with a significant increase in fibrin(ogen) in the plasminogen-deficient mice. Interestingly, macrophage migration was restored in plasminogen-deficient mice by simultaneous imposition of fibrinogen deficiency. Consistent with this in vivo finding, chemotactic migration of cultured macrophages through a fibrin matrix did not occur in the absence of plasminogen. The macrophage requirement for plasmin-mediated fibrinolysis, both in vivo and in vitro, was negated by deletion of the major myeloid integrin αMß2-binding motif on the γ chain of fibrin(ogen). The study identifies a critical role of fibrinolysis in macrophage migration, presumably through the alleviation of migratory constraints imposed by the interaction of leukocytes with fibrin(ogen) through the integrin αMß2 receptor.
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Quimiotaxis de Leucocito , Fibrinolisina/metabolismo , Fibrinólisis , Inflamación/etiología , Inflamación/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Animales , Biomarcadores , Quimiotaxis de Leucocito/inmunología , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Fibrinógeno/genética , Fibrinógeno/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Inmunofenotipificación , Inflamación/patología , Recuento de Leucocitos , Ratones , Ratones Noqueados , Plasminógeno/deficiencia , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Células RAW 264.7RESUMEN
Astrocytes protect neurons during cerebral injury through several postulated mechanisms. Recent therapeutic attention has focused on enhancing or augmenting the neuroprotective actions of astrocytes but in some instances astrocytes can assume a neurotoxic phenotype. The signaling mechanisms that drive astrocytes toward a protective versus toxic phenotype are not fully known but cell-cell signaling via proteases acting on cell-specific receptors underlies critical mechanistic steps in neurodevelopment and disease. The protease activated receptor (PAR), resides in multiple brain cell types, and most PARs are found on astrocytes. We asked whether neuron-generated thrombin constituted an important astrocyte activation signal because our previous studies have shown that neurons contain prothrombin gene and transcribed protein. We used neuron and astrocyte mono-cell cultures exposed to oxygen-glucose deprivation and a model of middle cerebral artery occlusion. We found that ischemic neurons secrete thrombin into culture media, which leads to astrocyte activation; such astrocyte activation can be reproduced with low doses of thrombin. Media from prothrombin-deficient neurons failed to activate astrocytes and adding thrombin to such media restored activation. Astrocytes lacking PAR1 did not respond to neuron-generated thrombin. Induced astrocyte activation was antagonized dose-dependently with thrombin inhibitors or PAR1 antagonists. Ischemia-induced astrocyte activation in vivo was inhibited after neuronal prothrombin knockout, resulting in larger strokes. Restoring prothrombin to neurons with a lentiviral gene vector restored astrocyte activation and reduced stroke damage. We conclude that neuron-generated thrombin, released during ischemia, acts via PAR1 and may cause astrocyte activation and paracrine neuroprotection.
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Astrocitos/metabolismo , Isquemia Encefálica/metabolismo , Neuronas/metabolismo , Accidente Cerebrovascular/etiología , Animales , Encéfalo/metabolismo , Supervivencia Celular/fisiología , Ratones , Neurogénesis/fisiología , Accidente Cerebrovascular/metabolismoRESUMEN
INTRODUCTION: Previous studies reported the efficacy and safety profile of extended half-life PEGylated recombinant factor VIII (FVIII) rurioctocog alfa pegol (TAK-660, SHP660, BAX 855) in preventing bleeding in haemophilia A patients. AIM: This study evaluated long-term safety and efficacy of rurioctocog alfa pegol for prophylaxis and treatment of bleeding in previously treated children and adults. METHODS: In this phase 3b, prospective, open-label, multicentre study (NCT01945593), eligible patients ≤ 75 years with severe haemophilia A (FVIII < 1%) received prophylactic rurioctocog alfa pegol in a fixed dose (FD, twice-weekly or less frequent) or pharmacokinetic (PK)-tailored dose regimen. Co-primary endpoints were incidence of confirmed FVIII inhibitory antibody development and spontaneous annualized bleed rate (ABR), analysed using a generalised linear model. Secondary endpoints included overall haemostatic efficacy, occurrence of adverse events and health-related quality of life (HRQoL). RESULTS: Overall, 216 patients were included; mean (SD) age at enrolment was 22.8 (15.7) years. No patients developed confirmed FVIII inhibitors. The point estimate (95% CI) of mean spontaneous ABR was 1.20 (0.92-1.56) among 186 patients receiving twice-weekly FD prophylaxis and 0.96 (0.54-1.71) among 25 patients receiving PK-tailored prophylaxis. Overall haemostatic efficacy was rated good or excellent in 88.6% of all bleeds. No new safety signals were observed. Patients reported improvements in HRQoL measures of pain, and physical and mental well-being. CONCLUSION: These results highlight the long-term safety and efficacy of rurioctocog alfa pegol prophylaxis in previously treated children and adults with severe haemophilia A, with a safety profile similar to previous studies and continuing ABR reduction.
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Factor VIII/uso terapéutico , Hemofilia A/tratamiento farmacológico , Hemorragia/prevención & control , Hemostasis/efectos de los fármacos , Adolescente , Adulto , Niño , Factor VIII/administración & dosificación , Factor VIII/efectos adversos , Factor VIII/farmacocinética , Femenino , Hemofilia A/sangre , Hemofilia A/etnología , Humanos , Masculino , Medición de Resultados Informados por el Paciente , Estudios Prospectivos , Calidad de Vida , Proteínas Recombinantes , Seguridad , Índice de Severidad de la Enfermedad , Resultado del Tratamiento , Adulto JovenRESUMEN
This study analyzes and evaluates the use of cellulose nanocrystals (CNCs), stiff nanosized natural materials that have been modified to mimic heparin. These CNCs are simple polysaccharides with a similar backbone structure to heparin, which when modified reduces coagulation and potentially the long-term effects of solution-based anticoagulants. Thus, CNCs represent an ideal foundation for generating materials biocompatible with blood. In this study, we developed a biocompatible material that inhibits blood clotting through surface functionalization to mimic heparin. Surface chemistry of CNCs was modified from "plain" CNCs (70 mmol SO3-/kg) to 500 mmol COO-/kg (TEMPO-oxidized CNCs) and 330 mmol SO3-/kg CNCs (sulfonated CNCs). Platelet adherence and blood assays show that changes in functionalization reduce coagulation. By utilizing and modifying CNCs reactive functional groups, we create a material with unique and favorable mechanical properties while also reducing clotting.
Asunto(s)
Celulosa , Nanopartículas , Materiales Biocompatibles , Heparina , PolisacáridosRESUMEN
Multiple sclerosis (MS) is a neuroinflammatory, demyelinating disease of the CNS. Fibrinogen deposition at sites of blood-brain barrier breakdown is a prominent feature of neuroinflammatory disease and contributes to disease severity. Plasminogen, the primary fibrinolytic enzyme, also modifies inflammatory processes. We used a murine model of MS, experimental autoimmune encephalomyelitis (EAE), to evaluate the hypothesis that the loss of plasminogen would exacerbate neuroinflammatory disease. However, contrary to initial expectations, EAE-challenged plasminogen-deficient (Plg-) mice developed significantly delayed disease onset and reduced disease severity compared with wild-type (Plg+) mice. Similarly, pharmacologic inhibition of plasmin activation with tranexamic acid also delayed disease onset. The T-cell response to immunization was similar between genotypes, suggesting that the contribution of plasminogen was downstream of the T-cell response. Spinal cords from EAE-challenged Plg- mice demonstrated significantly decreased demyelination and microglial/macrophage accumulation compared with Plg+ mice. Although fibrinogen-deficient mice or mice with combined deficiencies of plasminogen and fibrinogen had decreased EAE severity, they did not exhibit the delay in EAE disease onset, as seen in mice with plasminogen deficiency alone. Together, these data suggest that plasminogen and plasmin-mediated fibrinolysis is a key modifier of the onset of neuroinflammatory demyelination.SIGNIFICANCE STATEMENT Multiple sclerosis is a severe, chronic, demyelinating disease. Understanding the pathobiology related to the autoreactive T-cell and microglial/macrophage demyelinating response is critical to effectively target therapeutics. We describe for the first time that deficiency of plasminogen, the key fibrinolytic enzyme, delays disease onset and protects from the development of the paralysis associated with a murine model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). Administration of a widely used, pharmacologic inhibitor of plasminogen activation, tranexamic acid, also delays the onset of neuroinflammation associated with EAE.
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Enfermedades Desmielinizantes/metabolismo , Enfermedades Desmielinizantes/prevención & control , Encefalomielitis Autoinmune Experimental/metabolismo , Encefalomielitis Autoinmune Experimental/prevención & control , Parálisis/metabolismo , Parálisis/prevención & control , Plasminógeno/deficiencia , Animales , Células Cultivadas , Enfermedades Desmielinizantes/patología , Encefalomielitis Autoinmune Experimental/patología , Femenino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Parálisis/patologíaRESUMEN
Thrombin-mediated proteolysis is central to hemostatic function but also plays a prominent role in multiple disease processes. The proteolytic conversion of fII to α-thrombin (fIIa) by the prothrombinase complex occurs through 2 parallel pathways: (1) the inactive intermediate, prethrombin; or (2) the proteolytically active intermediate, meizothrombin (fIIa(MZ)). FIIa(MZ) has distinct catalytic properties relative to fIIa, including diminished fibrinogen cleavage and increased protein C activation. Thus, fII activation may differentially influence hemostasis and disease depending on the pathway of activation. To determine the in vivo physiologic and pathologic consequences of restricting thrombin generation to fIIa(MZ), mutations were introduced into the endogenous fII gene, resulting in expression of prothrombin carrying 3 amino acid substitutions (R157A, R268A, and K281A) to limit activation events to yield only fIIa(MZ) Homozygous fII(MZ) mice are viable, express fII levels comparable with fII(WT) mice, and have reproductive success. Although in vitro studies revealed delayed generation of fIIa(MZ) enzyme activity, platelet aggregation by fII(MZ) is similar to fII(WT) Consistent with prior analyses of human fIIa(MZ), significant prolongation of clotting times was observed for fII(MZ) plasma. Adult fII(MZ) animals displayed significantly compromised hemostasis in tail bleeding assays, but did not demonstrate overt bleeding. More notably, fII(MZ) mice had 2 significant phenotypic advantages over fII(WT) animals: protection from occlusive thrombosis after arterial injury and markedly diminished metastatic potential in a setting of experimental tumor metastasis to the lung. Thus, these novel animals will provide a valuable tool to assess the role of both fIIa and fIIa(MZ) in vivo.
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Precursores Enzimáticos/metabolismo , Hemostasis , Protrombina/metabolismo , Trombina/metabolismo , Alelos , Animales , Coagulación Sanguínea , Retracción del Coagulo , Venenos de Crotálidos , Embrión de Mamíferos/metabolismo , Fibrosis , Metaloendopeptidasas , Ratones Endogámicos C57BL , Miocardio/patología , Metástasis de la Neoplasia , Agregación Plaquetaria , Análisis de Supervivencia , Trombosis/metabolismo , Trombosis/patologíaRESUMEN
Sickle cell disease (SCD) results in vascular occlusions, chronic hemolytic anemia, and cumulative organ damage. A conspicuous feature of SCD is chronic inflammation and coagulation system activation. Thrombin (factor IIa [FIIa]) is both a central protease in hemostasis and a key modifier of inflammatory processes. To explore the hypothesis that reduced prothrombin (factor II [FII]) levels in SCD will limit vaso-occlusion, vasculopathy, and inflammation, we used 2 strategies to suppress FII in SCD mice. Weekly administration of FII antisense oligonucleotide "gapmer" to Berkeley SCD mice to selectively reduce circulating FII levels to â¼10% of normal for 15 weeks significantly diminished early mortality. More comprehensive, long-term comparative studies were done using mice with genetic diminution of circulating FII. Here, cohorts of FII(lox/-) mice (constitutively carrying â¼10% normal FII) and FII(WT) mice were tracked in parallel for a year following the imposition of SCD via hematopoietic stem cell transplantation. This genetically imposed suppression of FII levels resulted in an impressive reduction in inflammation (reduction in leukocytosis, thrombocytosis, and circulating interleukin-6 levels), reduced endothelial cell dysfunction (reduced endothelial activation and circulating soluble vascular cell adhesion molecule), and a significant improvement in SCD-associated end-organ damage (nephropathy, pulmonary hypertension, pulmonary inflammation, liver function, inflammatory infiltration, and microinfarctions). Notably, all of these benefits were achieved with a relatively modest 1.25-fold increase in prothrombin times, and in the absence of hemorrhagic complications. Taken together, these data establish that prothrombin is a powerful modifier of SCD-induced end-organ damage, and present a novel therapeutic target to ameliorate SCD pathologies.
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Anemia de Células Falciformes/complicaciones , Terapia Genética , Hipertensión Pulmonar/prevención & control , Inflamación/prevención & control , Protrombina/fisiología , Enfermedades Vasculares/prevención & control , Anemia de Células Falciformes/mortalidad , Anemia de Células Falciformes/fisiopatología , Animales , Coagulación Sanguínea , Células Cultivadas , Hipertensión Pulmonar/etiología , Técnicas para Inmunoenzimas , Inflamación/etiología , Imagen por Resonancia Magnética , Masculino , Ratones , Ratones Noqueados , Oligorribonucleótidos Antisentido/farmacología , Protrombina/antagonistas & inhibidores , Tasa de Supervivencia , Trombina/metabolismo , Enfermedades Vasculares/etiologíaRESUMEN
Rheumatoid arthritis is a chronic inflammatory disease characterized by synovial hyperplasia, inflammatory cell infiltration, irreversible cartilage and bone destruction, and exuberant coagulation system activity within joint tissue. Here, we demonstrate that the coagulation transglutaminase, factor XIII (fXIII), drives arthritis pathogenesis by promoting local inflammatory and tissue degradative and remodeling events. All pathological features of collagen-induced arthritis (CIA) were significantly reduced in fXIII-deficient mice. However, the most striking difference in outcome was the preservation of cartilage and bone in fXIIIA(-/-) mice concurrent with reduced osteoclast numbers and activity. The local expression of osteoclast effectors receptor activator of nuclear factor-κB ligand (RANKL) and tartrate resistant acid phosphatase were significantly diminished in CIA-challenged and even unchallenged fXIIIA(-/-) mice relative to wild-type animals, but were similar in wild-type and fibrinogen-deficient mice. Impaired osteoclast formation in fXIIIA(-/-) mice was not due to an inherent deficiency of monocyte precursors, but it was linked to reduced RANKL-driven osteoclast formation. Furthermore, treatment of mice with the pan-transglutaminase inhibitor cystamine resulted in significantly diminished CIA pathology and local markers of osteoclastogenesis. Thus, eliminating fXIIIA limits inflammatory arthritis and protects from cartilage and bone destruction in part through mechanisms linked to reduced RANKL-mediated osteoclastogenesis. In summary, therapeutic strategies targeting fXIII activity may prove beneficial in limiting arthropathies and other degenerative bone diseases.
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Artritis Experimental/patología , Enfermedades Óseas/fisiopatología , Factor XIII/fisiología , Inflamación/fisiopatología , Osteoclastos/patología , Animales , Artritis Experimental/inducido químicamente , Artritis Experimental/inmunología , Western Blotting , Enfermedades Óseas/complicaciones , Diferenciación Celular , Células Cultivadas , Colágeno/toxicidad , Femenino , Inflamación/complicaciones , Masculino , Ratones , Ratones Endogámicos DBA , Ratones Noqueados , Osteoclastos/metabolismo , Ligando RANK/genética , Ligando RANK/metabolismo , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Fibrin(ogen) is central to hemostasis and thrombosis and also contributes to multiple physiologic and pathologic processes beyond coagulation. However, the precise contribution of soluble fibrinogen vs insoluble fibrin matrices to vascular integrity, tissue repair, inflammation, and disease has been undefined and unapproachable. To establish the means to distinguish fibrinogen- and fibrin-dependent processes in vivo, Fib(AEK) mice were generated that carry normal levels of circulating fibrinogen but lack the capacity for fibrin polymer formation due to a germ-line mutation in the Aα chain thrombin cleavage site. Homozygous Fib(AEK) mice developed to term and exhibited postnatal survival superior to that of fibrinogen-deficient mice. Unlike fibrinogen-deficient mice, platelet-rich plasma from Fib(AEK) mice supported normal platelet aggregation in vitro, highlighting that fibrinogen(AEK) retains the functional capacity to support interactions with platelets. Thrombin failed to release fibrinopeptide-A from fibrinogen(AEK) and failed to induce polymer formation with Fib(AEK) plasma or purified fibrinogen(AEK) in 37°C mixtures regardless of incubation time. Fib(AEK) mice displayed both an absence of fibrin polymer formation following liver injury, as assessed by electron microscopy, and a failure to generate stable occlusive thrombi following FeCl3 injury of carotid arteries. Fib(AEK) mice exhibited a profound impediment in Staphylococcus aureus clearance following intraperitoneal infection similar to fibrinogen-deficient mice, yet Fib(AEK) mice displayed a significant infection dose-dependent survival advantage over fibrinogen-deficient mice following peritonitis challenge. Collectively, these findings establish for the first time that fibrin polymer is the molecular form critical for antimicrobial mechanisms while simultaneously highlighting biologically meaningful contributions and functions of the soluble molecule.
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Fibrina/metabolismo , Fibrinógeno/fisiología , Interacciones Huésped-Patógeno , Mutación/genética , Peritonitis/etiología , Infecciones Estafilocócicas/inmunología , Staphylococcus aureus/inmunología , Animales , Pruebas de Coagulación Sanguínea , Células Cultivadas , Citometría de Flujo , Perfilación de la Expresión Génica , Hemostáticos , Ratones , Ratones Noqueados , Mutagénesis Sitio-Dirigida , Peritonitis/patología , Agregación Plaquetaria , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/patologíaRESUMEN
Although multiple sclerosis (MS) has been associated with the coagulation system, the temporal and spatial regulation of coagulation activity in neuroinflammatory lesions is unknown. Using a novel molecular probe, we characterized the activity pattern of thrombin, the central protease of the coagulation cascade, in experimental autoimmune encephalomyelitis. Thrombin activity preceded onset of neurological signs, increased at disease peak, and correlated with fibrin deposition, microglial activation, demyelination, axonal damage, and clinical severity. Mice with a genetic deficit in prothrombin confirmed the specificity of the thrombin probe. Thrombin activity might be exploited for developing sensitive probes for preclinical detection and monitoring of neuroinflammation and MS progression.
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Encefalomielitis Autoinmune Experimental/metabolismo , Encefalomielitis Autoinmune Experimental/patología , Trombina/metabolismo , Animales , Axones/patología , Factores de Coagulación Sanguínea/química , Conexina 30 , Conexinas/genética , Enfermedades Desmielinizantes/etiología , Enfermedades Desmielinizantes/patología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Encefalomielitis Autoinmune Experimental/inducido químicamente , Fibrina/metabolismo , Proteínas Fluorescentes Verdes/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteína Básica de Mielina/metabolismo , Glicoproteína Mielina-Oligodendrócito/toxicidad , Fragmentos de Péptidos/toxicidad , Poli I-C/toxicidad , Trombina/químicaRESUMEN
Polyurea-based synthetic glycopolymers containing sulfated glucose, mannose, glucosamine, or lactose as pendant groups have been synthesized by step-growth polymerization of hexamethylene diisocyanate and corresponding secondary diamines. The obtained polymers were characterized by gel permeation chromatography, nuclear magnetic resonance spectroscopy, and Fourier transform infrared spectroscopy. The nonsulfated polymers showed similar results to the commercially available biomaterial polyurethane TECOFLEX in a platelet adhesion assay. The average degree of sulfation after reaction with SO3 was calculated from elemental analysis and found to be between three and four -OSO3 groups per saccharide. The blood-compatibility of the synthetic polymers was measured using activated partial thromboplastin time, prothrombin time, thrombin time, anti-IIa, and anti-Xa assays. Activated partial thromboplastin time, prothrombin time, and thrombin time results indicated that the mannose and lactose based polymers had the highest anticoagulant activities among all the sulfated polymers. The mechanism of action of the polymers appears to be mediated via an anti-IIa pathway rather than an anti-Xa pathway.
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Anticoagulantes/síntesis química , Anticoagulantes/farmacología , Carbohidratos/química , Polímeros/síntesis química , Polímeros/farmacología , Sulfatos/química , Animales , Materiales Biocompatibles/síntesis química , Materiales Biocompatibles/farmacología , Coagulación Sanguínea/efectos de los fármacos , Cromatografía en Gel , Ratones , Tiempo de Tromboplastina Parcial , Adhesividad Plaquetaria/efectos de los fármacos , Poliuretanos/síntesis química , Poliuretanos/farmacología , Tiempo de Protrombina , Espectroscopía Infrarroja por Transformada de Fourier , Tiempo de TrombinaRESUMEN
Transgender and gender-expansive (TG) people-those who identify with a gender other than their assigned sex at birth-frequently experience gender dysphoria, which is associated with negative health outcomes. One key strategy for improving gender dysphoria is the use of gender-affirming hormone therapy (GAHT): estrogen for feminization and testosterone for masculinization. Estrogen use in cisgender women is associated with well-established changes in hemostatic parameters, including increases in prothrombotic factors and decreases in inhibitors of coagulation. Cisgender women using estrogen have an increased risk of thrombosis. Studies of thrombosis risk associated with estrogen GAHT in TG people are less robust, with some studies limited by the use of hormones and hormone management strategies that are no longer recommended. However, TG women using estrogen appear to be at increased risk of both arterial and venous thrombosis, which may increase with longer time on estrogen. Testosterone use in both cisgender and transgender men is associated with increases in hemoglobin and hematocrit, which can lead to erythrocytosis and thus increased risk of thrombosis. The results of studies evaluating thrombosis risk in the setting of testosterone use are mixed. This review presents an overview of alterations in hemostatic parameters and thrombosis risk associated with use of exogenous estrogen and testosterone. Understanding what is known and unknown about thrombosis risk associated with use of these hormones is essential for hematologists who may be asked to evaluate TG people and provide guidance on management of those who may be at increased risk of thrombosis.
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Estrógenos , Testosterona , Trombosis , Personas Transgénero , Humanos , Femenino , Factores de Riesgo , Testosterona/efectos adversos , Testosterona/sangre , Estrógenos/efectos adversos , Masculino , Trombosis/etiología , Trombosis/prevención & control , Trombosis/sangre , Medición de Riesgo , Disforia de Género/tratamiento farmacológico , Disforia de Género/sangre , Coagulación Sanguínea/efectos de los fármacos , Transexualidad/tratamiento farmacológico , Hemostasis/efectos de los fármacos , Terapia de Reemplazo de Hormonas/efectos adversos , Factores Sexuales , Identidad de Género , Procedimientos de Reasignación de Sexo/efectos adversosRESUMEN
BACKGROUND: Von Willebrand Disease (VWD) is the most common inherited bleeding disorder. VWD is characterized by an abnormal quantity or quality of von Willebrand Factor (VWF). Anemia is often found at presentation for a bleeding disorder evaluation due to chronic blood loss. OBJECTIVES/HYPOTHESIS: We hypothesized that anemia is associated with elevations in both VWF and factor VIII (FVIII) over baseline. We also hypothesized that obesity would be associated with increased levels of VWF. METHODS: We conducted a single-center review of the electronic health record for patients that had proximal von Willebrand profiles and Hb data. RESULTS: We identified 4552 unique subjects with VWF studies and a CBC within 24 h. We found that decreasing hemoglobin inversely correlated with VWF antigen, VWF ristocetin cofactor activity, and FVIII activity. We also found that obesity and Black race were independently associated with increased VWF antigen, activity, and FVIII activity. Hb, race, and body mass index (BMI) continued to be determinants of VWF and FVIII levels in multivariable analysis. CONCLUSION: Our study demonstrates that anemia, race, and BMI were found to be associated with elevation of VWF antigen, VWF activity, and FVIII levels. As many individuals with anemia present for evaluation for a bleeding disorder, these variables need to be considered. KEY POINTS: - Anemia was found to be associated with elevation of VWF antigen, VWF activity and FVIII levels. - Testing von Willebrand factor at times of anemia may mask a diagnosis of von Willebrand Disease.
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Índice de Masa Corporal , Factor VIII , Hemoglobinas , Factor de von Willebrand , Humanos , Factor de von Willebrand/análisis , Factor de von Willebrand/metabolismo , Factor VIII/análisis , Factor VIII/metabolismo , Masculino , Femenino , Persona de Mediana Edad , Adulto , Hemoglobinas/análisis , Enfermedades de von Willebrand/sangre , Anemia/sangre , Anciano , Obesidad/sangre , Obesidad/complicacionesRESUMEN
Thrombomodulin (TM) is a predominantly endothelial transmembrane glycoprotein that modulates hemostatic function through a domain that controls thrombin-mediated proteolysis and an N-terminal lectin-like domain that controls inflammatory processes. To test the hypothesis that TM is a determinant of malignancy and dissect the importance of these functional domains in cancer biology, metastatic potential was evaluated in TM(Pro) mice expressing a mutant form of TM with reduced thrombin affinity and TM(LeD) mice lacking the N-terminal lectin-like domain. Studies of TM(Pro) mice revealed that TM is a powerful determinant of hematogenous metastasis. TM(Pro) mice exhibited a strongly prometastatic phenotype relative to control mice that was found to result from increased survival of tumor cells newly localized to the lung rather than any alteration in tumor growth. The impact of the TM(Pro) mutation on metastasis was dependent on both tumor cell-associated tissue factor and thrombin procoagulant function. In contrast, expression of a mutant form of TM lacking the lectin-like domain had no significant impact on metastasis. These studies directly demonstrate for the first time that TM-mediated regulation of tumor cell-driven procoagulant function strongly influences metastatic potential and suggest that endothelial cell-associated modulators of hemostasis may represent novel therapeutic targets in limiting tumor dissemination.
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Lectinas/metabolismo , Neoplasias Hepáticas/secundario , Neoplasias Pulmonares/secundario , Mutación/genética , Trombina/metabolismo , Trombomodulina/fisiología , Animales , Carcinoma Pulmonar de Lewis/metabolismo , Carcinoma Pulmonar de Lewis/patología , Femenino , Hirudinas/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Pulmonares/metabolismo , Metástasis Linfática , Masculino , Melanoma Experimental/metabolismo , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Células Neoplásicas Circulantes , Oligonucleótidos Antisentido/farmacología , Recuento de Plaquetas , Protrombina/antagonistas & inhibidores , Protrombina/genética , Proteínas Recombinantes/metabolismo , Sarcoma Experimental/metabolismo , Sarcoma Experimental/patologíaRESUMEN
Thrombin is a positive mediator of thrombus formation through the proteolytic activation of protease-activated receptors (PARs), fibrinogen, factor XI (fXI), and other substrates, and a negative regulator through activation of protein C, a natural anticoagulant with anti-inflammatory/cytoprotective properties. Protease-engineering studies have established that 2 active-site substitutions, W215A and E217A (fII(WE)), result in dramatically reduced catalytic efficiency with procoagulant substrates while largely preserving thrombomodulin (TM)-dependent protein C activation. To explore the hypothesis that a prothrombin variant favoring antithrombotic pathways would be compatible with development but limit inflammatory processes in vivo, we generated mice carrying the fII(WE) mutations within the endogenous prothrombin gene. Unlike fII-null embryos, fII(WE/WE) mice uniformly developed to term. Nevertheless, these mice ultimately succumbed to spontaneous bleeding events shortly after birth. Heterozygous fII(WT/WE) mice were viable and fertile despite a shift toward an antithrombotic phenotype exemplified by prolonged tail-bleeding times and times-to-occlusion after FeCl3 vessel injury. More interestingly, prothrombin(WE) expression significantly ameliorated the development of inflammatory joint disease in mice challenged with collagen-induced arthritis (CIA). The administration of active recombinant thrombin(WE) also suppressed the development of CIA in wild-type mice. These studies provide a proof-of-principle that pro/thrombin variants engineered with altered substrate specificity may offer therapeutic opportunities for limiting inflammatory disease processes.
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Artritis Experimental/metabolismo , Mutación Missense , Protrombina/metabolismo , Sustitución de Aminoácidos , Animales , Artritis Experimental/genética , Hemorragia/genética , Hemorragia/metabolismo , Humanos , Ratones , Ratones Mutantes , Proteína C/genética , Proteína C/metabolismo , Protrombina/genéticaRESUMEN
Mice carrying a conditional prothrombin knockout allele (fII(lox)) were established to develop an experimental setting for exploring the importance of thrombin in the maintenance of vascular integrity, the inflammatory response, and disease processes in adult animals. In the absence of Cre-mediated recombination, homozygous fII(lox/lox) mice or compound heterozygous mice carrying one fII(lox) allele and one constitutive-null allele were viable. Young adults exhibited neither spontaneous bleeding events nor diminished reproductive success. However, the induction of Cre recombinase in fII(lox) mice using the poly I:C-inducible Mx1-Cre system resulted in the rapid and near-complete recombination of the fII(lox) allele within the liver, the loss of circulating prothrombin, and profound derangements in coagulation function. Consistent with the notion that thrombin regulates coagulation and inflammatory pathways, an additional early consequence of reducing prothrombin was impaired antimicrobial function in mice challenged with Staphylococcus aureus peritonitis. However, life expectancy in unchallenged adults genetically depleted of prothrombin was very short ( approximately 5-7 days). The loss of viability was associated with the development of severe hemorrhagic events within multiple tissues, particularly in the heart and brain. Unlike the constitutive loss of either clotting or platelet function alone, the conditional loss of prothrombin is uniformly not compatible with maintenance of hemostasis or long-term survival.
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Encéfalo/patología , Hemorragia/genética , Miocardio/patología , Protrombina/metabolismo , Animales , Coagulación Sanguínea/genética , Northern Blotting , Inflamación/genética , Integrasas/genética , Ratones , Ratones Noqueados , Peritonitis/genética , Protrombina/genéticaRESUMEN
BACKGROUND AND OBJECTIVES: Many transgender youth experience gender dysphoria, a risk factor for suicide. Gender-affirming hormone therapy (GAHT) ameliorates this risk but may increase the risk for thrombosis, as seen from studies in adults. The aim with this study was to examine thrombosis and thrombosis risk factors among an exclusively adolescent and young adult transgender population. METHODS: This retrospective chart review was conducted at a pediatric hospital-associated transgender health clinic. The primary outcome was incidence of arterial or venous thrombosis during GAHT. Secondary measures included the prevalence of thrombosis risk factors. RESULTS: Among 611 participants, 28.8% were transgender women and 68.1% were transgender men. Median age was 17 years at GAHT initiation. Median follow-up time was 554 and 577 days for estrogen and testosterone users, respectively. Individuals starting GAHT had estradiol and testosterone levels titrated to physiologic normal. Multiple thrombotic risk factors were noted among the cohort, including obesity, tobacco use, and personal and family history of thrombosis. Seventeen youth with risk factors for thrombosis were referred for hematologic evaluation. Five individuals were treated with anticoagulation during GAHT: 2 with a previous thrombosis and 3 for thromboprophylaxis. No participant developed thrombosis while on GAHT. CONCLUSIONS: In this study, we examined thrombosis and thrombosis risk factors in an exclusively adolescent and young adult population of transgender people receiving GAHT. These data suggest that GAHT in youth, titrated within physiologic range, does not carry a significant risk of thrombosis in the short-term, even with the presence of preexisting thrombosis risk factors.
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Trombosis/epidemiología , Personas Transgénero , Adolescente , Anticoagulantes/uso terapéutico , Estudios de Cohortes , Femenino , Predisposición Genética a la Enfermedad , Hormonas Esteroides Gonadales/administración & dosificación , Humanos , Masculino , Obesidad/epidemiología , Estudios Retrospectivos , Factores de Riesgo , Trombosis/tratamiento farmacológico , Uso de Tabaco/epidemiología , Adulto JovenRESUMEN
Sickle cell anemia (SCA) is caused by a point mutation in the ß-globin gene that leads to devastating downstream consequences including chronic hemolytic anemia, episodic vascular occlusion, and cumulative organ damage resulting in death. SCA patients show coagulation activation and inflammation even in the absence of vascular occlusion. The coagulation factor fibrinogen is not only central to hemostasis but also plays important roles in pathologic inflammatory processes, in part by engaging neutrophils/macrophages through the αMß2 integrin receptor. To determine whether fibrin(ogen)-mediated inflammation is a driver of SCA-associated pathologies, hematopoietic stem cells from Berkeley sickle mice were transplanted into homozygous Fibγ390-396A mice that express normal levels of a mutant form of fibrin(ogen) that does not engage αMß2 Fibγ390-396A mice with SCA displayed an impressive reduction of reactive oxygen species (ROS) in white blood cells (WBCs), decreased circulating inflammatory cytokines/chemokines, and significantly improved SCA-associated glomerular pathology highlighted by reduced glomerulosclerosis, inflammatory cell infiltration, ischemic lesions, mesangial thickening, mesangial hypercellularity, and glomerular enlargement. In addition, Fibγ390-396A mice with SCA had improved glomerular protective responses and podocyte/mesangial transcriptional signatures that resulted in reduced albuminuria. Interestingly, the fibrinogen γ390-396A mutation had a negligible effect on cardiac, lung, and liver functions and pathologies in the context of SCA over a year-long observation period. Taken together, our data support that fibrinogen significantly contributes to WBC-driven inflammation and ROS production, which is a key driver of SCA-associated glomerulopathy, and may represent a novel therapeutic target against irreversible kidney damage in SCA.