RESUMEN
BACKGROUND: The X-linked bleeding disorder hemophilia B, caused by mutation(s) in the coagulation factor (F)IX gene, leads to partial or total loss of its function, requiring lifelong FIX replacement therapy. Although new recombinant FIX (rIX) therapeutics like albumin fusion proteins (rIX-FP) enable longer plasma half-life and thus less frequent administration, the complexity of intravenous (i.v.) injection remains. OBJECTIVES: The study aimed to characterize rIX-FP variants with anticipated enhanced specific activity, which would leverage rIX-FP's superior pharmacokinetic profile with beneficial characteristics for subcutaneous (s.c.) administration. METHODS: Two rIX-FP variants, R338L ("Padua variant") and R338L/E410K, were characterized in vitro. Pharmacokinetic profiles of FIX antigen and activity levels were evaluated in FIX-deficient mice after i.v. and s.c. administration of these variants (dosing based on antigen levels). The efficacy of the most promising variant was tested after i.v. and s.c. administration (dosing based on activity) in a tail clip bleeding model. A marketed wild-type (WT) rIX-FP product served as the comparator. RESULTS: Both rIX-FP variants showed a 4- to 5-fold increase in specific activity in vitro compared with rIX(WT)-FP, while FXIa-mediated activation was the fastest for rIX(WT)-FP and rIX(R338L)-FP. Compared with rIX(WT)-FP and rIX(R338L/E410K)-FP, rIX(R338L)-FP exhibited higher FIX activity exposure after i.v. and s.c. administration and demonstrated comparable efficacy with rIX(WT)-FP in reducing bleeding time and blood loss in FIX-deficient mice requiring â¼4 times lower protein amount. CONCLUSION: rIX(R338L)-FP was shown to be a promising candidate for s.c. administration, exhibiting increased specific activity combined with higher activity-based exposure and indicating efficacy at a lower protein dose.
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Factor XII (FXII) is a serine protease involved in multiple cascades, including the kallikrein-kinin system. It may play a role in diseases in which the downstream cascades are dysregulated, such as hereditary angioedema. Garadacimab (CSL312) is a first-in-class, fully human, monoclonal antibody targeting activated FXII (FXIIa). We describe how translational pharmacokinetic (PK) and pharmacodynamic (PD) modeling enabled dose selection for the phase I, first-in-human trial of garadacimab. The PK/PD data used for modeling were derived from preclinical PK/PD and safety studies. Garadacimab plasma concentrations rose with increasing dose, and clear dose-related PD effects were observed (e.g., a mechanism-based prolongation of activated partial thromboplastin time). The PK/PD profile from cynomolgus monkeys was used to generate minimal physiologically-based pharmacokinetic (mPBPK) models with target-mediated drug disposition (TMDD) for data prediction in cynomolgus monkeys. These models were later adapted for prediction of human data to establish dose selection. Based on the final mPBPK model with TMDD and assuming a weight of 70 kg for an adult human, a minimal inhibition (<10%) of FXIIa with a starting dose of 0.1 mg/kg garadacimab and a near maximal inhibition (>95%) at 10 mg/kg garadacimab were predicted. The phase I study is complete, and data on exposure profiles and inhibition of FXIIa-mediated kallikrein activity observed in the trial support and validate these simulations. This emphasizes the utility and relevance of translational modeling and simulation in drug development.
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Angioedemas Hereditarios , Factor XIIa , Animales , Anticuerpos Monoclonales/farmacocinética , Simulación por Computador , Humanos , Macaca fascicularisRESUMEN
BACKGROUND: 3F7 is a monoclonal antibody targeting the enzymatic pocket of activated factor XII (FXIIa), thereby inhibiting its catalytic activity. Given the emerging role of FXIIa in promoting thromboinflammation, along with its apparent redundancy for hemostasis, the selective inhibition of FXIIa represents a novel and highly attractive approach targeting pathogenic processes that cause thromboinflammation-driven cardiovascular diseases. METHODS: The effects of FXIIa inhibition were investigated using three distinct mouse models of cardiovascular disease-angiotensin II-induced abdominal aortic aneurysm (AAA), an ApoE-/- model of atherosclerosis, and a tandem stenosis model of atherosclerotic plaque instability. 3F7 or its isotype control, BM4, was administered to mice (10 mg/kg) on alternate days for 4 to 8 weeks, depending on the experimental model. Mice were examined for the development and size of AAAs, or the burden and instability of atherosclerosis and associated markers of inflammation. RESULTS: Inhibition of FXIIa resulted in a reduced incidence of larger AAAs, with less acute aortic ruptures and an associated fibro-protective phenotype. FXIIa inhibition also decreased stable atherosclerotic plaque burden and achieved plaque stabilization associated with increased deposition of fibrous structures, a >2-fold thicker fibrous cap, increased cap-to-core ratio, and reduction in localized and systemic inflammatory markers. CONCLUSION: Inhibition of FXIIa attenuates disease severity across three mouse models of thromboinflammation-driven cardiovascular diseases. Specifically, the FXIIa-inhibiting monoclonal antibody 3F7 reduces AAA severity, inhibits the development of atherosclerosis, and stabilizes vulnerable plaques. Ultimately, clinical trials in patients with cardiovascular diseases such as AAA and atherosclerosis are warranted to demonstrate the therapeutic potential of FXIIa inhibition.
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Anticuerpos Monoclonales/administración & dosificación , Aneurisma de la Aorta Abdominal/prevención & control , Aterosclerosis/prevención & control , Factor XIIa/antagonistas & inhibidores , Placa Aterosclerótica/metabolismo , Animales , Aneurisma de la Aorta Abdominal/epidemiología , Apolipoproteínas E , Modelos Animales de Enfermedad , Inflamación , Masculino , RatonesRESUMEN
BACKGROUND: Effective inhibition of thrombosis without generating bleeding risks is a major challenge in medicine. Accumulating evidence suggests that this can be achieved by inhibition of coagulation factor XII (FXII), as either its knock-out or inhibition in animal models efficiently reduced thrombosis without affecting normal hemostasis. Based on these findings, highly specific inhibitors for human FXII(a) are under development. However, currently, in vivo studies on their efficacy and safety are impeded by the lack of an optimized animal model expressing the specific target, that is, human FXII. OBJECTIVE: The primary objective of this study is to develop and functionally characterize a humanized FXII mouse model. METHODS: A humanized FXII mouse model was generated by replacing the murine with the human F12 gene (genetic knock-in) and tested it in in vitro coagulation assays and in in vivo thrombosis models. RESULTS: These hF12KI mice were indistinguishable from wild-type mice in all tested assays of coagulation and platelet function in vitro and in vivo, except for reduced expression levels of hFXII compared to human plasma. Targeting FXII by the anti-human FXIIa antibody 3F7 increased activated partial thromboplastin time dose-dependently and protected hF12KI mice in an arterial thrombosis model without affecting bleeding times. CONCLUSION: These data establish the newly generated hF12KI mouse as a powerful and unique model system for in vivo studies on anti-FXII(a) biologics, supporting the development of efficient and safe human FXII(a) inhibitors.
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Factor XII , Trombosis , Animales , Coagulación Sanguínea , Modelos Animales de Enfermedad , Factor XII/genética , Hemostasis , Ratones , Trombosis/tratamiento farmacológico , Trombosis/genéticaRESUMEN
BACKGROUND: Hemophilia B is caused by coagulation factor IX (FIX) deficiency. Recombinant fusion protein linking coagulation FIX with recombinant albumin (rIX-FP; Idelvion® ) is used for replacement therapy with an extended half-life. A previous quantitative whole-body autoradiography (QWBA) study investigating the biodistribution of rIX-FP indicated equal biodistribution, but more prolonged tissue retention compared with a marketed recombinant FIX product. OBJECTIVES: To complete and confirm the QWBA study data by directly measuring rIX-FP protein and activity levels in tissues following intravenous (i.v.) administration to normal rats and FIX-deficient (hemophilia B) mice. METHODS: After i.v. administration of rIX-FP at a dose of 2000 IU/kg, animals were euthanized at specific time points up to 72 hours postdosing. Subsequently, plasma and various tissues, which were selected based on the previous QWBA results, were harvested and analyzed for FIX antigen levels using an ELISA (both species) or an immunohistochemistry method (mice only), as well as for FIX activity levels (mice only) using a chromogenic assay. RESULTS: In rats, rIX-FP distributed extravascularly into all tissues analyzed (ie, liver, kidney, skin and knee) with peak antigen levels reached between 1 and 7 hours postdosing. In hemophilia B mice, rIX-FP tissue distribution was comparable to rats. FIX antigen levels correlated well with FIX activity readouts. CONCLUSIONS: Our results confirm QWBA data showing that rIX-FP distributes into relevant target tissues. Importantly, it was demonstrated that rIX-FP available in tissues retains its functional activity and can thus facilitate its therapeutic activity at sites of potential injury.
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Hemofilia B , Roedores , Administración Intravenosa , Animales , Factor IX/metabolismo , Semivida , Hemofilia B/tratamiento farmacológico , Ratones , Ratas , Proteínas Recombinantes de Fusión/uso terapéutico , Roedores/metabolismo , Distribución TisularAsunto(s)
Anticuerpos Bloqueadores/farmacología , Bradiquinina/sangre , Factor XII/inmunología , Angioedema/tratamiento farmacológico , Animales , Anticuerpos Bloqueadores/uso terapéutico , Proteína Inhibidora del Complemento C1/genética , Humanos , Macaca fascicularis , Masculino , Ratones Endogámicos BALB C , Ratones Noqueados , Anafilaxis Cutánea Pasiva/efectos de los fármacosRESUMEN
OBJECTIVE: Ischemia-reperfusion (I/R) injury is a major clinical problem linked to vascular surgery. Currently, no drugs to prevent or to treat I/R injury are approved for clinical use. C1 inhibitor (C1 INH) is known to reduce activation of the plasma cascade systems that are involved in the pathophysiologic process of I/R injury. The aim of this study was therefore to investigate the effect of C1 INH on complement deposition and endothelial cell activation in a rat model of hind limb I/R injury. METHODS: Male Wistar rats (wild type, bred at the central animal facility, University of Bern), weighing 250 to 320 g, were used. The rats underwent 2-hour ischemia and 24-hour reperfusion by unilateral clamping of the femoral artery and additional use of a tourniquet. Five groups were divided according to intravenous treatment 5 minutes before ischemia: 50 IU/kg C1 INH (n = 5); 100 IU/kg C1 INH (n = 7); vehicle control (n = 5); nontreated control (n = 7); and normal, healthy control without intervention (n = 4). At the end, muscle edema, tissue viability, and histologic features were assessed. Deposition of immunoglobulin M, C1r, C4d, and fibrin and expression of plasminogen activator inhibitor 1, heparan sulfate (HS), E-selectin, and vascular cell adhesion molecule 1 were evaluated by fluorescence staining. In addition, high-mobility group box 1 protein was measured in plasma. RESULTS: Edema formation was reduced by C1 INH at two dosages, mirrored by improved histologic injury scores and preserved muscle viability. Deposition of immunoglobulin M, C4d, and fibrin was significantly decreased by 100 IU/kg C1 INH compared with nontreated controls. Pretreatment with 100 IU/kg C1 INH also significantly reduced HS shedding and expression of plasminogen activator inhibitor 1 as well as plasma levels of high-mobility group box 1 protein. CONCLUSIONS: Pretreatment with both 50 and 100 IU/kg C1 INH attenuated reperfusion injury of rat hind limbs. Pretreatment with 100 IU/kg also preserved the endothelial HS layer as well as the natural, profibrinolytic phenotype of the endothelium. Prevention of endothelial cell activation by C1 INH may therefore be a promising strategy to prevent I/R injury in the clinical setting of peripheral vascular diseases and elective surgery on extremities.
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Activación de Complemento/efectos de los fármacos , Proteína Inhibidora del Complemento C1/farmacología , Inactivadores del Complemento/farmacología , Células Endoteliales/efectos de los fármacos , Músculo Esquelético/irrigación sanguínea , Músculo Esquelético/efectos de los fármacos , Daño por Reperfusión/prevención & control , Animales , Complemento C1r/metabolismo , Complemento C4b/metabolismo , Modelos Animales de Enfermedad , Selectina E/metabolismo , Edema/inmunología , Edema/metabolismo , Edema/patología , Edema/prevención & control , Células Endoteliales/metabolismo , Células Endoteliales/patología , Fibrina/metabolismo , Proteína HMGB1/metabolismo , Heparitina Sulfato/metabolismo , Miembro Posterior , Inmunoglobulina M/metabolismo , Masculino , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Fragmentos de Péptidos/metabolismo , Inhibidor 1 de Activador Plasminogénico/metabolismo , Ratas Wistar , Daño por Reperfusión/inmunología , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Supervivencia Tisular/efectos de los fármacos , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
BACKGROUND: Traumatic brain injury (TBI) is a devastating neurological condition and a frequent cause of permanent disability. Posttraumatic inflammation and brain edema formation, two pathological key events contributing to secondary brain injury, are mediated by the contact-kinin system. Activation of this pathway in the plasma is triggered by activated factor XII. Hence, we set out to study in detail the influence of activated factor XII on the abovementioned pathophysiological features of TBI. METHODS: Using a cortical cryogenic lesion model in mice, we investigated the impact of genetic deficiency of factor XII and inhibition of activated factor XII with a single bolus injection of recombinant human albumin-fused Infestin-4 on the release of bradykinin, the brain lesion size, and contact-kinin system-dependent pathological events. We determined protein levels of bradykinin, intracellular adhesion molecule-1, CC-chemokine ligand 2, and interleukin-1ß by enzyme-linked immunosorbent assays and mRNA levels of genes related to inflammation by quantitative real-time PCR. Brain lesion size was determined by tetrazolium chloride staining. Furthermore, protein levels of the tight junction protein occludin, integrity of the blood-brain barrier, and brain water content were assessed by Western blot analysis, extravasated Evans Blue dye, and the wet weight-dry weight method, respectively. Infiltration of neutrophils and microglia/activated macrophages into the injured brain lesions was quantified by immunohistological stainings. RESULTS: We show that both genetic deficiency of factor XII and inhibition of activated factor XII in mice diminish brain injury-induced bradykinin release by the contact-kinin system and minimize brain lesion size, blood-brain barrier leakage, brain edema formation, and inflammation in our brain injury model. CONCLUSIONS: Stimulation of bradykinin release by activated factor XII probably plays a prominent role in expanding secondary brain damage by promoting brain edema formation and inflammation. Pharmacological blocking of activated factor XII could be a useful therapeutic principle in the treatment of TBI-associated pathologic processes by alleviating posttraumatic inflammation and brain edema formation.
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Edema Encefálico/metabolismo , Edema Encefálico/prevención & control , Lesiones Traumáticas del Encéfalo/metabolismo , Lesiones Traumáticas del Encéfalo/prevención & control , Factor XIIa/antagonistas & inhibidores , Factor XIIa/metabolismo , Animales , Bradiquinina/metabolismo , Lesiones Encefálicas/metabolismo , Lesiones Encefálicas/prevención & control , Factor XIIa/genética , Inflamación/metabolismo , Inflamación/prevención & control , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
RATIONALE: Acute respiratory distress syndrome is characterized by alveolar epithelial cell injury, edema formation, and intraalveolar contact phase activation. OBJECTIVES: To explore whether C1 esterase inhibitor (C1INH), an endogenous inhibitor of the contact phase, may protect from lung injury in vivo and to decipher the possible underlying mechanisms mediating protection. METHODS: The ability of C1INH to control the inflammatory processes was studied in vitro and in vivo. MEASUREMENTS AND MAIN RESULTS: Here, we demonstrate that application of C1INH alleviates bleomycin-induced lung injury via direct interaction with extracellular histones. In vitro, C1INH was found to bind all histone types. Interaction with histones was independent of its protease inhibitory activity, as demonstrated by the use of reactive-center-cleaved C1INH, but dependent on its glycosylation status. C1INH sialylated-N- and -O-glycans were not only essential for its interaction with histones but also to protect against histone-induced cell death. In vivo, histone-C1INH complexes were detected in bronchoalveolar lavage fluid from patients with acute respiratory distress syndrome and multiple models of lung injury. Furthermore, reactive-center-cleaved C1INH attenuated pulmonary damage evoked by intravenous histone instillation. CONCLUSIONS: Collectively, C1INH administration provides a new therapeutic option for disorders associated with histone release.
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Proteína Inhibidora del Complemento C1/farmacología , Histonas/metabolismo , Lesión Pulmonar/prevención & control , Síndrome de Dificultad Respiratoria/fisiopatología , Animales , Líquido del Lavado Bronquioalveolar , Proteína Inhibidora del Complemento C1/metabolismo , Modelos Animales de Enfermedad , Humanos , Pulmón/metabolismo , Pulmón/fisiopatología , Lesión Pulmonar/fisiopatología , Ratones , Ratones Endogámicos C57BLRESUMEN
Atherosclerosis is considered a chronic inflammatory disease of the vessel wall. Coagulation pathways and immune responses contribute to disease development. The role of coagulation factor XII (FXII) in vascular inflammation, however, remains controversial. We here investigated the function of FXII in atherosclerosis using apolipoprotein E and FXII-deficient (F12-/-Apoe-/-) mice. Compared to F12+/+Apoe-/- controls, atherosclerotic lesion formation was reduced in F12-/-Apoe-/- mice. This was associated with a decrease in serum interleukin (IL)-1ß and IL-12 levels and reduced expression of pro-inflammatory cytokines in the aorta in atherosclerotic F12-/-Apoe-/- mice, as well as diminished Th1-cell differentiation in the aorta, blood, and lymphoid organs. No changes in circulating bradykinin, thrombin-antithrombin-complexes or plasminogen were observed. Mechanistically, activated FXII (FXIIa) was revealed to directly induce bone marrow-derived macrophages to secrete pro-inflammatory cytokines, including tumour necrosis factor-α, IL-1ß, IL-12, and IL-6. Exposure of bone marrow-derived antigen presenting cells to FXIIa similarly induced pro-inflammatory cytokines, and an enhanced capacity to trigger antigen-specific interferon γ-production in CD4+ T cells. Notably, bone-marrow derived macrophages were capable of directly activating FXII. Moreover, the induction of cytokine expression by FXIIa in macrophages occurred independently of FXII protease enzymatic activity and was decreased upon phospholipase C treatment, suggesting urokinase-type plasminogen activator receptor (uPAR) to confer FXIIa-induced cell signalling. These data reveal FXII to play an important role in atherosclerotic lesion formation by functioning as a strong inducer of pro-inflammatory cytokines in antigen-presenting cells. Targeting of FXII may thus be a promising approach for treating cardiovascular disease.
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Células Presentadoras de Antígenos/metabolismo , Enfermedades de la Aorta/metabolismo , Aterosclerosis/metabolismo , Citocinas/metabolismo , Deficiencia del Factor XII/metabolismo , Factor XII/metabolismo , Mediadores de Inflamación/metabolismo , Macrófagos/metabolismo , Animales , Células Presentadoras de Antígenos/inmunología , Enfermedades de la Aorta/sangre , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/inmunología , Aterosclerosis/sangre , Aterosclerosis/genética , Aterosclerosis/inmunología , Proliferación Celular , Citocinas/inmunología , Modelos Animales de Enfermedad , Factor XII/genética , Deficiencia del Factor XII/sangre , Deficiencia del Factor XII/genética , Deficiencia del Factor XII/inmunología , Factor XIIa/genética , Factor XIIa/metabolismo , Predisposición Genética a la Enfermedad , Mediadores de Inflamación/inmunología , Activación de Linfocitos , Macrófagos/inmunología , Ratones Endogámicos C57BL , Ratones Noqueados para ApoE , Fenotipo , Placa Aterosclerótica , Receptores del Activador de Plasminógeno Tipo Uroquinasa/metabolismo , Células TH1/inmunología , Células TH1/metabolismo , Factores de TiempoRESUMEN
In relation to the recent trials of Intravenous Immunoglobulin (IVIG) in Alzheimer's Disease (AD) it was demonstrated that different IgG preparations contain varying amounts of natural anti-amyloid ß (Aß) antibodies as measured by ELISA. We therefore investigated the relevance of ELISA data for measuring low-affinity antibodies, such as anti-Aß. We analysed the binding of different commercial Immunoglobulin G (IgG) preparations to Aß, actin and tetanus toxoid in different binding assays to further investigate the possible cause for observed differences in binding to Aß and actin between different IgG preparations. We show that the differences of commercial IgG preparations in binding to Aß and actin in ELISA assays are artefactual and only evident in in vitro binding assays. In functional assays and in vivo animal studies the different IVIG preparations exhibited very similar potency. ELISA data alone are not appropriate to analyse and rank the binding capacity of low-affinity antibodies to Aß or other endogenous self-antigens contained in IgG preparations. Additional analytical methods should be adopted to complement ELISA data.
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Antígenos/inmunología , Autoantígenos/inmunología , Inmunoglobulina G/inmunología , Inmunoglobulinas Intravenosas/inmunología , Actinas/inmunología , Actinas/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/inmunología , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/inmunología , Péptidos beta-Amiloides/metabolismo , Animales , Afinidad de Anticuerpos/efectos de los fármacos , Afinidad de Anticuerpos/inmunología , Antígenos/metabolismo , Autoantígenos/metabolismo , Ensayo de Inmunoadsorción Enzimática , Femenino , Herpesvirus Humano 3/inmunología , Herpesvirus Humano 3/metabolismo , Humanos , Inmunoglobulina G/metabolismo , Inmunoglobulinas Intravenosas/farmacología , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Unión Proteica/efectos de los fármacos , Unión Proteica/inmunología , Ratas , Toxoide Tetánico/inmunología , Toxoide Tetánico/metabolismoRESUMEN
Aberrant immune responses represent the underlying cause of central nervous system (CNS) autoimmunity, including multiple sclerosis (MS). Recent evidence implicated the crosstalk between coagulation and immunity in CNS autoimmunity. Here we identify coagulation factor XII (FXII), the initiator of the intrinsic coagulation cascade and the kallikrein-kinin system, as a specific immune cell modulator. High levels of FXII activity are present in the plasma of MS patients during relapse. Deficiency or pharmacologic blockade of FXII renders mice less susceptible to experimental autoimmune encephalomyelitis (a model of MS) and is accompanied by reduced numbers of interleukin-17A-producing T cells. Immune activation by FXII is mediated by dendritic cells in a CD87-dependent manner and involves alterations in intracellular cyclic AMP formation. Our study demonstrates that a member of the plasmatic coagulation cascade is a key mediator of autoimmunity. FXII inhibition may provide a strategy to combat MS and other immune-related disorders.
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Inmunidad Adaptativa , Células Dendríticas/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Factor XII/inmunología , Esclerosis Múltiple/inmunología , Adulto , Anciano , Animales , Diferenciación Celular , Factor XII/metabolismo , Femenino , Humanos , Interleucina-17/metabolismo , Calicreínas/metabolismo , Cininas/metabolismo , Masculino , Ratones Endogámicos C57BL , Persona de Mediana Edad , Esclerosis Múltiple/sangre , Receptores del Activador de Plasminógeno Tipo Uroquinasa/metabolismo , Linfocitos T/metabolismo , Adulto JovenRESUMEN
OBJECTIVE: Traumatic brain injury is a major global public health problem for which specific therapeutic interventions are lacking. There is, therefore, a pressing need to identify innovative pathomechanism-based effective therapies for this condition. Thrombus formation in the cerebral microcirculation has been proposed to contribute to secondary brain damage by causing pericontusional ischemia, but previous studies have failed to harness this finding for therapeutic use. The aim of this study was to obtain preclinical evidence supporting the hypothesis that targeting factor XII prevents thrombus formation and has a beneficial effect on outcome after traumatic brain injury. METHODS: We investigated the impact of genetic deficiency of factor XII and acute inhibition of activated factor XII with a single bolus injection of recombinant human albumin-fused infestin-4 (rHA-Infestin-4) on trauma-induced microvascular thrombus formation and the subsequent outcome in 2 mouse models of traumatic brain injury. RESULTS: Our study showed that both genetic deficiency of factor XII and an inhibition of activated factor XII in mice minimize trauma-induced microvascular thrombus formation and improve outcome, as reflected by better motor function, reduced brain lesion volume, and diminished neurodegeneration. Administration of human factor XII in factor XII-deficient mice fully restored injury-induced microvascular thrombus formation and brain damage. INTERPRETATION: The robust protective effect of rHA-Infestin-4 points to a novel treatment option that can decrease ischemic injury after traumatic brain injury without increasing bleeding tendencies. Ann Neurol 2016;79:970-982.
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Lesiones Traumáticas del Encéfalo/tratamiento farmacológico , Factor XII/uso terapéutico , Factor XIIa/antagonistas & inhibidores , Proteínas de Insectos/uso terapéutico , Trombosis Intracraneal/tratamiento farmacológico , Proteínas Recombinantes de Fusión/uso terapéutico , Albúmina Sérica/uso terapéutico , Adulto , Anciano , Animales , Lesiones Traumáticas del Encéfalo/fisiopatología , Estudios de Casos y Controles , Modelos Animales de Enfermedad , Factor XII/genética , Femenino , Humanos , Imagen por Resonancia Magnética , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Neuroimagen , Agregación Plaquetaria/fisiología , Albúmina Sérica Humana , Adulto JovenRESUMEN
Haemostasis including blood coagulation is initiated upon vessel wall injury and indispensable to limit excessive blood loss. However, unregulated pathological coagulation may lead to vessel occlusion, causing thrombotic disorders, most notably myocardial infarction and stroke. Furthermore, blood exposure to foreign surfaces activates the intrinsic pathway of coagulation. Hence, various clinical scenarios, such as extracorporeal membrane oxygenation, require robust anticoagulation consequently leading to an increased bleeding risk. This study aimed to further assess the antithrombotic efficacy of the activated factor XII (FXIIa) inhibitor, rHA-Infestin-4, in several thrombosis models. In mice, rHA-Infestin-4 decreased occlusion rates in the mechanically-induced arterial (Folt's) and the FeCl3 -induced venous thrombosis model. rHA-Infestin-4 also protected from FeCl3 -induced arterial thrombosis and from stasis-prompted venous thrombosis in rabbits. Furthermore, rHA-Infestin-4 prevented occlusion in the arterio-venous shunt model in mice and rabbits where thrombosis was induced via a foreign surface. In contrast to heparin, the haemostatic capacity in rabbits was unaffected by rHA-Infestin-4. Using rodent and non-rodent species, our data demonstrate that the FXIIa inhibitor rHA-Infestin-4 decreased arterial, venous and foreign surface-induced thrombosis without affecting physiological haemostasis. Hence, we provide further evidence that targeting FXIIa represents a potent yet safe antithrombotic treatment approach, especially in foreign surface-triggered thrombosis.
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Factor XIIa/antagonistas & inhibidores , Proteínas de Insectos/farmacología , Proteínas Recombinantes de Fusión/farmacología , Albúmina Sérica/farmacología , Trombosis/tratamiento farmacológico , Animales , Arteriopatías Oclusivas/tratamiento farmacológico , Arteriopatías Oclusivas/etiología , Modelos Animales de Enfermedad , Fibrinolíticos/farmacología , Hemostasis/efectos de los fármacos , Proteínas de Insectos/uso terapéutico , Cinética , Ratones , Conejos , Proteínas Recombinantes de Fusión/uso terapéutico , Albúmina Sérica/uso terapéutico , Albúmina Sérica Humana , Trombosis/etiología , Resultado del Tratamiento , Trombosis de la Vena/tratamiento farmacológico , Trombosis de la Vena/etiologíaRESUMEN
BACKGROUND AND PURPOSE: Ischemic stroke provokes severe brain damage and remains a predominant disease in industrialized countries. The coagulation factor XII (FXII)-driven contact activation system plays a central, but not yet fully defined pathogenic role in stroke development. Here, we investigated the efficacy of the FXIIa inhibitor rHA-Infestin-4 in a rat model of ischemic stroke using both a prophylactic and a therapeutic approach. METHODS: For prophylactic treatment, animals were treated intravenously with 100 mg/kg rHA-Infestin-4 or an equal volume of saline 15 min prior to transient middle cerebral artery occlusion (tMCAO) of 90 min. For therapeutic treatment, 100 mg/kg rHA-Infestin-4, or an equal volume of saline, was administered directly after the start of reperfusion. At 24 h after tMCAO, rats were tested for neurological deficits and blood was drawn for coagulation assays. Finally, brains were removed and analyzed for infarct area and edema formation. RESULTS: Within prophylactic rHA-Infestin-4 treatment, infarct areas and brain edema formation were reduced accompanied by better neurological scores and survival compared to controls. Following therapeutic treatment, neurological outcome and survival were still improved although overall effects were less pronounced compared to prophylaxis. CONCLUSIONS: With regard to the central role of the FXII-driven contact activation system in ischemic stroke, inhibition of FXIIa may represent a new and promising treatment approach to prevent cerebral ischemia/reperfusion injury.
Asunto(s)
Factor XIIa/antagonistas & inhibidores , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Proteínas de Insectos/farmacología , Proteínas Recombinantes de Fusión/farmacología , Daño por Reperfusión/prevención & control , Inhibidores de Serina Proteinasa/farmacología , Albúmina Sérica/farmacología , Animales , Encéfalo/irrigación sanguínea , Encéfalo/efectos de los fármacos , Encéfalo/patología , Células CHO , Cricetulus , Evaluación Preclínica de Medicamentos , Factor XIIa/metabolismo , Proteínas de Insectos/uso terapéutico , Masculino , Ratas , Proteínas Recombinantes de Fusión/uso terapéutico , Prueba de Desempeño de Rotación con Aceleración Constante , Inhibidores de Serina Proteinasa/uso terapéutico , Albúmina Sérica/uso terapéutico , Albúmina Sérica HumanaRESUMEN
Coated platelets, formed by collagen and thrombin activation, have been characterized in different ways: i) by the formation of a protein coat of α-granular proteins; ii) by exposure of procoagulant phosphatidylserine; or iii) by high fibrinogen binding. Yet, their functional role has remained unclear. Here we used a novel transglutaminase probe, Rhod-A14, to identify a subpopulation of platelets with a cross-linked protein coat, and compared this with other platelet subpopulations using a panel of functional assays. Platelet stimulation with convulxin/thrombin resulted in initial integrin α(IIb)ß3 activation, the appearance of a platelet population with high fibrinogen binding, (independently of active integrins, but dependent on the presence of thrombin) followed by phosphatidylserine exposure and binding of coagulation factors Va and Xa. A subpopulation of phosphatidylserine-exposing platelets bound Rhod-A14 both in suspension and in thrombi generated on a collagen surface. In suspension, high fibrinogen and Rhod-A14 binding were antagonized by combined inhibition of transglutaminase activity and integrin α(IIb)ß3 Markedly, in thrombi from mice deficient in transglutaminase factor XIII, platelet-driven fibrin formation and Rhod-A14 binding were abolished by blockage of integrin α(IIb)ß3. Vice versa, star-like fibrin formation from platelets of a patient with deficiency in α(IIb)ß3(Glanzmann thrombasthenia) was abolished upon blockage of transglutaminase activity. We conclude that coated platelets, with initial α(IIb)ß3 activation and high fibrinogen binding, form a subpopulation of phosphatidylserine-exposing platelets, and function in platelet-dependent star-like fibrin fiber formation via transglutaminase factor XIII and integrin α(IIb)ß3.
Asunto(s)
Plaquetas/metabolismo , Factor XIII/metabolismo , Fibrina/metabolismo , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Trombastenia/sangre , Animales , Coagulación Sanguínea , Plaquetas/efectos de los fármacos , Plaquetas/patología , Venenos de Crotálidos/farmacología , Factor Va/química , Factor Va/metabolismo , Factor XIII/química , Factor Xa/química , Factor Xa/metabolismo , Fibrina/química , Fibrinógeno/química , Fibrinógeno/metabolismo , Humanos , Lectinas Tipo C , Ratones , Ratones Noqueados , Sondas Moleculares/química , Fosfatidilserinas/química , Fosfatidilserinas/metabolismo , Activación Plaquetaria/efectos de los fármacos , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/química , Cultivo Primario de Células , Unión Proteica , Trombastenia/patología , Trombina/farmacologíaRESUMEN
Hereditary angioedema type III (HAEIII) is a rare inherited swelling disorder that is associated with point mutations in the gene encoding the plasma protease factor XII (FXII). Here, we demonstrate that HAEIII-associated mutant FXII, derived either from HAEIII patients or recombinantly produced, is defective in mucin-type Thr309-linked glycosylation. Loss of glycosylation led to increased contact-mediated autoactivation of zymogen FXII, resulting in excessive activation of the bradykinin-forming kallikrein-kinin pathway. In contrast, both FXII-driven coagulation and the ability of C1-esterase inhibitor to bind and inhibit activated FXII were not affected by the mutation. Intravital laser-scanning microscopy revealed that, compared with control animals, both F12-/- mice reconstituted with recombinant mutant forms of FXII and humanized HAEIII mouse models with inducible liver-specific expression of Thr309Lys-mutated FXII exhibited increased contact-driven microvascular leakage. An FXII-neutralizing antibody abolished bradykinin generation in HAEIII patient plasma and blunted edema in HAEIII mice. Together, the results of this study characterize the mechanism of HAEIII and establish FXII inhibition as a potential therapeutic strategy to interfere with excessive vascular leakage in HAEIII and potentially alleviate edema due to other causes.
Asunto(s)
Coagulación Sanguínea , Factor XII/metabolismo , Angioedema Hereditario Tipo III/metabolismo , Mutación Missense , Adulto , Sustitución de Aminoácidos , Animales , Anticuerpos Neutralizantes/farmacología , Bradiquinina/genética , Bradiquinina/metabolismo , Modelos Animales de Enfermedad , Factor XII/genética , Femenino , Glicosilación/efectos de los fármacos , Angioedema Hereditario Tipo III/tratamiento farmacológico , Angioedema Hereditario Tipo III/genética , Angioedema Hereditario Tipo III/patología , Humanos , Ratones , Ratones NoqueadosRESUMEN
Traumatic brain injury (TBI) induces a strong inflammatory response which includes blood-brain barrier damage, edema formation and infiltration of different immune cell subsets. More recently, microvascular thrombosis has been identified as another pathophysiological feature of TBI. The contact-kinin system represents an interface between inflammatory and thrombotic circuits and is activated in different neurological diseases. C1-Inhibitor counteracts activation of the contact-kinin system at multiple levels. We investigated the therapeutic potential of C1-Inhibitor in a model of TBI. Male and female C57BL/6 mice were subjected to cortical cryolesion and treated with C1-Inhibitor after 1 h. Lesion volumes were assessed between day 1 and day 5 and blood-brain barrier damage, thrombus formation as well as the local inflammatory response were determined post TBI. Treatment of male mice with 15.0 IU C1-Inhibitor, but not 7.5 IU, 1 h after cryolesion reduced lesion volumes by ~75% on day 1. This protective effect was preserved in female mice and at later stages of trauma. Mechanistically, C1-Inhibitor stabilized the blood-brain barrier and decreased the invasion of immune cells into the brain parenchyma. Moreover, C1-Inhibitor had strong antithrombotic effects. C1-Inhibitor represents a multifaceted anti-inflammatory and antithrombotic compound that prevents traumatic neurodegeneration in clinically meaningful settings.
RESUMEN
Fibrinogen, a soluble 340kDa plasma glycoprotein, is critical in achieving and maintaining hemostasis. Reduced fibrinogen levels are associated with an increased risk of bleeding and recent research has investigated the efficacy of fibrinogen concentrate for controlling perioperative bleeding. European guidelines on the management of perioperative bleeding recommend the use of fibrinogen concentrate if significant bleeding is accompanied by plasma fibrinogen levels less than 1.5-2.0g/l. Plasma-derived human fibrinogen concentrate has been available for therapeutic use since 1956. The overall aim of the comprehensive series of non-clinical investigations presented was to evaluate i) the pharmacodynamic and pharmacokinetic characteristics and ii) the safety and tolerability profile of human fibrinogen concentrate Haemocomplettan P® (RiaSTAP®). Pharmacodynamic characteristics were assessed in rabbits, pharmacokinetic parameters were determined in rabbits and rats and a safety pharmacology study was performed in beagle dogs. Additional toxicology tests included: single-dose toxicity tests in mice and rats; local tolerance tests in rabbits; and neoantigenicity tests in rabbits and guinea pigs following the introduction of pasteurization in the manufacturing process. Human fibrinogen concentrate was shown to be pharmacodynamically active in rabbits and dogs and well tolerated, with no adverse events and no influence on circulation, respiration or hematological parameters in rabbits, mice, rats and dogs. In these non-clinical investigations, human fibrinogen concentrate showed a good safety profile. This data adds to the safety information available to date, strengthening the current body of knowledge regarding this hemostatic agent.
Asunto(s)
Fibrinógeno/metabolismo , Fibrinógeno/toxicidad , Animales , Peso Corporal/efectos de los fármacos , Peso Corporal/fisiología , Perros , Relación Dosis-Respuesta a Droga , Femenino , Fibrinógeno/farmacología , Cobayas , Hemostasis/efectos de los fármacos , Hemostasis/fisiología , Humanos , Masculino , Ratones , Conejos , Ratas , Ratas Wistar , Especificidad de la EspecieRESUMEN
Human plasma-derived C1-esterase inhibitor (C1-INH) is an efficacious and safe treatment for hereditary angioedema. However, thrombotic events in subjects treated with C1-INH at recommended or off-label, high doses have been reported. In this study, we addressed the potential prothrombotic risk of C1-INH treatment in high doses using a non-clinical rabbit model. Following intravenous infusion of C1-INH to rabbits at doses up to 800 IU/kg, the exposure and the pharmacodynamic efficacy of C1-INH in rabbits were confirmed by activity measurements of C1-esterase, and coagulation factors XIa and XIIa, respectively. Potential prothrombotic effects were assessed following induction of venous and arterial thrombosis using in vivo models of venous and arterial stasis, complemented by various in vitro assays of coagulation markers. Administration of C1-INH at doses up to 800 IU/kg did not potentiate thrombus formation during venous stasis. In contrast, inhibition of arterial occlusion was observed upon C1-INH administration when compared with isotonic saline treatment, indicating antithrombotic rather than prothrombotic activity of high dose C1-INH treatment in vivo. This was further confirmed in vitro by decreased thrombin generation, increased activated partial thromboplastin time, clotting time and clot formation time, and inhibition of platelet aggregation. No relevant changes in fibrinolysis or in the levels of thrombin-antithrombin complexes, and prothrombin fragment 1+2 were observed upon high dose C1-INH treatment. The data suggest that treatment of healthy rabbits with high doses of C1-INH could potentially inhibit coagulation and thrombus formation rather than induce a prothrombotic risk.