RESUMEN
Heparin cofactor II (HCII) is predominantly expressed in the liver and inhibits thrombin in blood plasma to influence the blood coagulation cascade. Its deficiency is associated with arterial thrombosis. Its cleavage by neutrophil elastase produces fragment that helps in neutrophil chemotaxis in the acute inflammatory response in human. In the present study, we have identified a novel alternatively spliced transcript of the HCII gene in human liver. This novel transcript includes an additional novel region in continuation with exon 3 called exon 3b. Exon 3b acts like an alternate last exon, and hence its inclusion in the transcript due to alternative splicing removes exon 4 and encodes for a different C-terminal region to give a novel protein, HCII-N. MD simulations of HCII-N and three-dimensional structure showed a unique 51 amino acid sequence at the C-terminal having unique RCL-like structure. The HCII-N protein purified from bacterial culture showed a protein migrating at lower molecular weight (MW 55 kDa) as compared to native HCII (MW 66 kDa). A fluorescence-based analysis revealed a more compact structure of HCII-N that was in a more hydrophilic environment. The HCII-N protein, however, showed no inhibitory activity against thrombin. Due to large conformational variation observed in comparison with native HCII, HCII-N may have alternate protease specificity or a non-inhibitory role. Western blot of HCII purified from large plasma volume showed the presence of a low MW 59 kDa band with no thrombin activity. This study provides the first evidence of alternatively spliced novel isoform of the HCII gene.
Asunto(s)
Cofactor II de Heparina/química , Cofactor II de Heparina/genética , Cofactor II de Heparina/metabolismo , Hígado/metabolismo , Empalme Alternativo , Factor Xa/metabolismo , Humanos , Modelos Moleculares , Simulación de Dinámica Molecular , Isoformas de Proteínas , Espectrometría de Fluorescencia , Trombina/metabolismo , Activador de Tejido Plasminógeno/antagonistas & inhibidores , Activador de Tejido Plasminógeno/metabolismoRESUMEN
Neuroserpin (NS) is predominantly expressed in brain and inhibits tissue-type plasminogen activator (tPA) with implications in brain development and memory. Nature of conformational change in pathological variants in strand 6B and helix B of NS that cause a relatively mild to severe epilepsy (and/or dementia) remains largely elusive. MD simulation with wild type (WT) NS, strand 6B and helix B variants indicated that substitution in this region affects the conformation of the strands 5B, 5A and reactive centre loop. Therefore, we designed variants of NS in strand 6B (I46D and F48S) and helix B (A54F, L55A and L55P) to investigate their role in tPA inhibition mechanism and propensity to aggregate. An interaction analysis showed disturbance of a hydrophobic patch centered at strands 5B, 6B and helix B in I46D and F48S but not in A54F, L55A, L55P and WT NS. Purified I46D, F48S and L55P variants showed decrease in fluorescence emission intensity but have similar α-helical content, however results of A54F and L55A were comparable to WT NS. Analysis of tPA inhibition showed marginal effect on A54F and L55A variant with tPA-NS complex formation. In contrast, I46D, F48S and L55P variants showed massive decrease in tPA inhibition, with no tPA-NS complex formation. Analysis of native PAGE under under polymerization condition showed prompt conversion of I46D, F48S and L55P to latent conformation but not A54F and L55A variants. Identification of these novel conformational changes will aid in the understanding of variable clinical phenotype of shutter region NS variants and other serpins.
Asunto(s)
Neuropéptidos/química , Serpinas/química , Epilepsias Mioclónicas/genética , Trastornos Heredodegenerativos del Sistema Nervioso/genética , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Simulación de Dinámica Molecular , Mutación , Neuropéptidos/genética , Neuropéptidos/aislamiento & purificación , Neuropéptidos/metabolismo , Fenotipo , Polimerizacion , Agregado de Proteínas , Conformación Proteica , Conformación Proteica en Hélice alfa , Serpinas/genética , Serpinas/aislamiento & purificación , Serpinas/metabolismo , Activador de Tejido Plasminógeno/antagonistas & inhibidores , NeuroserpinaRESUMEN
T cells play a key role in mounting an adaptive immune response. T cells are activated upon recognition of cognate Ag presented by an APC. Subsequently, T cells adhere to other activated T cells to form activation clusters, which lead to directed secretion of cytokines between communicating cells. T cell activation clusters have been implicated in regulating activation, proliferation, and memory formation in T cells. We previously reported the expression of the protease inhibitor neuroserpin by human T cells and showed that expression and intracellular localization is regulated following T cell activation. To gain a better understanding of neuroserpin in the proteolytic environment postactivation we assessed its role in human T cell clustering and proliferation. Neuroserpin knockdown increased T cell proliferation and cluster formation following T cell activation. This increased cluster formation was dependent on the proteases tissue plasminogen activator (tPA) and plasmin. Furthermore, neuroserpin knockdown or plasmin treatment of T cells increased the cleavage of annexin A2, a known plasmin target that regulates the actin cytoskeleton. Live cell imaging of activated T cells further indicated a role of the actin cytoskeleton in T cell clustering. The inhibition of actin regulators myosin ATPase and Rho-associated protein kinase signaling completely reversed the neuroserpin knockdown-induced effects. The results presented in this study reveal a novel role for neuroserpin and the proteolytic environment in the regulation of T cell activation biology.
Asunto(s)
Comunicación Celular , Proliferación Celular , Activación de Linfocitos , Neuropéptidos/farmacología , Inhibidores de Serina Proteinasa/farmacología , Serpinas/farmacología , Linfocitos T/citología , Activador de Tejido Plasminógeno/antagonistas & inhibidores , Citoesqueleto de Actina/metabolismo , Humanos , Neuropéptidos/antagonistas & inhibidores , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , NeuroserpinaRESUMEN
Hemorrhagic transformation (HT) is a devastating complication for patients with acute ischemic stroke (AIS) who are treated with tissue plasminogen activator (tPA). HT is associated with high morbidity and mortality, but no effective treatments are currently available to reduce the risk of HT. Therefore, methods to prevent HT are urgently needed. In this study, we used IM-12, an inhibitor of glycogen synthase kinase 3ß (GSK-3ß), to evaluate the role of the Wnt-ß-catenin signaling pathway in recombinant tPA (rtPA)-induced HT. Sprague-Dawley rats were subjected to a middle cerebral artery occlusion (MCAO) model of ischemic stroke, and then were either administered rtPA, rtPA combined with IM-12, or the vehicle at 4 h after stroke was induced. Our results indicate that rats subjected to HT had more severe neurological deficits, brain edema, and blood-brain barrier (BBB) breakdown, and had a greater infarction volume than the control group. Rats treated with IM-12 had improved outcomes compared with those of rats treated with rtPA alone. Moreover, IM-12 increased the protein expression of ß-catenin and downstream proteins while suppressing the expression of GSK-3ß. These results suggest that IM-12 reduces rtPA-induced HT and attenuates BBB disruption, possibly through activation of the Wnt-ß-catenin signaling pathway, and provides a potential therapeutic strategy for preventing tPA-induced HT after AIS.
Asunto(s)
Hemorragia/tratamiento farmacológico , Indoles/farmacología , Maleimidas/farmacología , Accidente Cerebrovascular/tratamiento farmacológico , Activador de Tejido Plasminógeno/antagonistas & inhibidores , Vía de Señalización Wnt/efectos de los fármacos , Animales , Hemorragia/metabolismo , Masculino , Ratas , Ratas Sprague-Dawley , Accidente Cerebrovascular/metabolismo , Activador de Tejido Plasminógeno/farmacologíaRESUMEN
Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, irreversible and the most common fatal interstitial lung disease, which is characterized by damaged alveolar structure, the massive proliferation of fibroblasts and deposition of extracellular matrix (ECM). While the pathogenesis of IPF remains unclear, it has been clearly established that the excessive proliferation of lung fibroblasts is the most direct cause of fibrogenesis. Numerous proliferating fibroblasts form fibrous foci and secrete a large amount of ECM to aggravate the process of pulmonary fibrosis. Tissue plasminogen activator (tPA) is a kind of serine protease, its main function is to activate zymogens into active enzymes involved in fibrinolysis. Our study found tPA functioned as a cytokine to promote the proliferation of lung fibroblasts through intracellular signaling events involving Erk1/2, p90RSK, GSK-3ß phosphorylation, and cyclinD1 induction. We also uncovered that tPA indirectly activated the Wnt/ß-catenin signaling pathway by regulating the GSK-3ß phosphorylation level. It's well-known that Wnt/ß-catenin signaling pathway plays an important role in the pathogenesis of pulmonary fibrosis, in which the accumulation of ß-catenin in the cytoplasm is an important signal of the activation of Wnt/ß-catenin signaling pathway. Our study unveiled that tPA can serve as a cytokine involved in Wnt/ß-catenin signaling pathway and be implicated in pulmonary fibrosis.
Asunto(s)
Fibroblastos/efectos de los fármacos , Fibrosis Pulmonar Idiopática/metabolismo , Activador de Tejido Plasminógeno/farmacología , Vía de Señalización Wnt/efectos de los fármacos , beta Catenina/metabolismo , Animales , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Ciclina D1/metabolismo , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patología , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Fibrosis Pulmonar Idiopática/inducido químicamente , Fibrosis Pulmonar Idiopática/genética , Fibrosis Pulmonar Idiopática/patología , Ratones , Ratones Endogámicos C57BL , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Activador de Tejido Plasminógeno/antagonistas & inhibidores , Activador de Tejido Plasminógeno/metabolismoRESUMEN
Urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) are two serine proteases that contribute to initiating fibrinolysis by activating plasminogen. uPA is also an important tumour-associated protease due to its role in extracellular matrix remodelling. Overexpression of uPA has been identified in several different cancers and uPA inhibition has been reported as a promising therapeutic strategy. Although several peptide-based uPA inhibitors have been developed, the extent to which uPA tolerates different tetrapeptide sequences that span the P1-P4 positions remains to be thoroughly explored. In this study, we screened a sequence-defined peptide aldehyde library against uPA and tPA. Preferred sequences from the library screen yielded potent inhibitors for uPA, led by Ac-GTAR-H (Ki =18â nm), but not for tPA. Additionally, synthetic peptide substrates corresponding to preferred inhibitor sequences were cleaved with high catalytic efficiency by uPA but not by tPA. These findings provide new insights into the binding specificity of uPA and tPA and the relative activity of tetrapeptide inhibitors and substrates against these enzymes.
Asunto(s)
Aldehídos/química , Inhibidores Enzimáticos/química , Péptidos/química , Activador de Tejido Plasminógeno/química , Activador de Plasminógeno de Tipo Uroquinasa/química , Aldehídos/síntesis química , Dominio Catalítico , Inhibidores Enzimáticos/síntesis química , Humanos , Biblioteca de Péptidos , Péptidos/síntesis química , Especificidad por Sustrato , Activador de Tejido Plasminógeno/antagonistas & inhibidores , Activador de Plasminógeno de Tipo Uroquinasa/antagonistas & inhibidoresRESUMEN
The invasion of glioblastoma is a complex process based on the interactions of tumor cells and the extracellular matrix. Tumors that are engineered using biomaterials are more physiologically relevant than a two-dimensional (2D) cell culture system. Matrix metalloproteinases and the plasminogen activator generated by tumor cells regulate a tumor's invasive behavior. In this study, microtumors were fabricated by encapsulating U87 glioma cells in Type I collagen and then glioma cell migration in the collagen hydrogels was investigated. Crosslinking of collagen with 8S-StarPEG increased the hydrogel viscosity and reduced the tumor cell migration speed in the hydrogels. The higher migration speed corresponded to the increased gene expression of MMP-2, MMP-9, urokinase plasminogen activator (uPA), and tissue plasminogen activator (tPA) in glioma cells grown in non-crosslinked collagen hydrogels. Inhibitors of these molecules hindered U87 and A172 cell migration in collagen hydrogels. Aprotinin and tranexamic acid did not inhibit U87 and A172 migration on the culture dish. This study demonstrated the differential effect of pharmacologic molecules on tumor cell motility in either a 2D or three-dimensional culture environment.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias Encefálicas/tratamiento farmacológico , Movimiento Celular/efectos de los fármacos , Colágeno/metabolismo , Glioblastoma/tratamiento farmacológico , Inhibidores de Proteasas/farmacología , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Hidrogeles , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Invasividad Neoplásica , Transducción de Señal , Estrés Mecánico , Factores de Tiempo , Activador de Tejido Plasminógeno/antagonistas & inhibidores , Activador de Tejido Plasminógeno/genética , Activador de Tejido Plasminógeno/metabolismo , ViscosidadRESUMEN
Some studies have indicated that ketamine has a rapid antidepressant effects, but the underlying molecular mechanism is still unclear. Researchers have found that mature brain-derived neurotrophic factor (mBDNF) and its precursor proBDNF are related to depression; they elicit opposite effects on cellular functions. It is clear that tissue plasminogen activator (tPA) is a key regulatory element in the conversion of proBDNF to mBDNF. The chronic unpredicted mild stress (CUMS) procedure is a classical and reliable method to establish the model of depression. This study found that sucrose preference and locomotor activity were both reduced in CUMS-treated rats while were increased in those who were injected with ketamine. The hippocampal proBDNF/mBDNF ratio was downregulated after ketamine treatment in those rats, together with an increased level of tPA in the hippocampus. However, tPA activity was unaltered after ketamine intraperitoneal injection. Intrahippocampal injection of active plasminogen activator inhibitor-1 (inhibitor of tPA) before ketamine treatment reversed the antidepressant effects and upregulated the proBDNF/mBDNF ratio. The results of this study suggest that the antidepressant action induced by ketamine may be related to tPA-mediated conversion of proBDNF to mBDNF in the hippocampus.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/metabolismo , Depresión/metabolismo , Hipocampo/metabolismo , Ketamina/administración & dosificación , Precursores de Proteínas/metabolismo , Activador de Tejido Plasminógeno/metabolismo , Anestésicos Disociativos/administración & dosificación , Animales , Antidepresivos/administración & dosificación , Depresión/tratamiento farmacológico , Hipocampo/efectos de los fármacos , Inyecciones Intraventriculares , Masculino , Proteínas de Plantas/administración & dosificación , Ratas , Ratas Sprague-Dawley , Estrés Psicológico/tratamiento farmacológico , Estrés Psicológico/metabolismo , Activador de Tejido Plasminógeno/antagonistas & inhibidoresAsunto(s)
Fibrinólisis/efectos de los fármacos , Polisacáridos/farmacología , Serpina E2/antagonistas & inhibidores , Trombosis/tratamiento farmacológico , Activador de Tejido Plasminógeno/antagonistas & inhibidores , Animales , Modelos Animales de Enfermedad , Ratones , Ratones Endogámicos BALB C , Serpina E2/metabolismo , Trombosis/sangre , Activador de Tejido Plasminógeno/metabolismo , Activador de Tejido Plasminógeno/farmacologíaRESUMEN
Delayed thrombolytic treatment with recombinant tissue plasminogen activator (tPA) may exacerbate blood-brain barrier breakdown after ischaemic stroke and lead to lethal haemorrhagic transformation. The immune system is a dynamic modulator of stroke response, and excessive immune cell accumulation in the cerebral vasculature is associated with compromised integrity of the blood-brain barrier. We previously reported that regulatory T cells, which function to suppress excessive immune responses, ameliorated blood-brain barrier damage after cerebral ischaemia. This study assessed the impact of regulatory T cells in the context of tPA-induced brain haemorrhage and investigated the underlying mechanisms of action. The number of circulating regulatory T cells in stroke patients was dramatically reduced soon after stroke onset (84 acute ischaemic stroke patients with or without intravenous tPA treatment, compared to 115 age and gender-matched healthy controls). Although stroke patients without tPA treatment gradually repopulated the numbers of circulating regulatory T cells within the first 7 days after stroke, post-ischaemic tPA treatment led to sustained suppression of regulatory T cells in the blood. We then used the murine suture and embolic middle cerebral artery occlusion models of stroke to investigate the therapeutic potential of adoptive regulatory T cell transfer against tPA-induced haemorrhagic transformation. Delayed administration of tPA (10 mg/kg) resulted in haemorrhagic transformation in the ischaemic territory 1 day after ischaemia. When regulatory T cells (2 × 106/mouse) were intravenously administered immediately after delayed tPA treatment in ischaemic mice, haemorrhagic transformation was significantly decreased, and this was associated with improved sensorimotor functions. Blood-brain barrier disruption and tight junction damages were observed in the presence of delayed tPA after stroke, but were mitigated by regulatory T cell transfer. Mechanistic studies demonstrated that regulatory T cells completely abolished the tPA-induced elevation of MMP9 and CCL2 after stroke. Using MMP9 and CCL2 knockout mice, we discovered that both molecules partially contributed to the protective actions of regulatory T cells. In an in vitro endothelial cell-based model of the blood-brain barrier, we confirmed that regulatory T cells inhibited tPA-induced endothelial expression of CCL2 and preserved blood-brain barrier integrity after an ischaemic challenge. Lentivirus-mediated CCL2 knockdown in endothelial cells completely abolished the blood-brain barrier protective effect of regulatory T cells in vitro. Altogether, our studies suggest that regulatory T cell adoptive transfer may alleviate thrombolytic treatment-induced haemorrhage in stroke victims. Furthermore, regulatory T cell-afforded protection in the tPA-treated stroke model is mediated by two inhibitory mechanisms involving CCL2 and MMP9. Thus, regulatory T cell adoptive transfer may be useful as a cell-based therapy to improve the efficacy and safety of thrombolytic treatment for ischaemic stroke.
Asunto(s)
Hemorragias Intracraneales/terapia , Linfocitos T Reguladores/trasplante , Activador de Tejido Plasminógeno/efectos adversos , Animales , Barrera Hematoencefálica , Isquemia Encefálica/complicaciones , Estudios de Casos y Controles , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Técnicas de Silenciamiento del Gen , Infarto de la Arteria Cerebral Media , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones Noqueados , Accidente Cerebrovascular/complicaciones , Activador de Tejido Plasminógeno/antagonistas & inhibidoresRESUMEN
Tissue plasminogen activator (tPA) is the only recommended pharmacological treatment for acute ischemic stroke. However, tPA can induce intracerebral hemorrhage by blood-brain barrier breakdown through an increase in matrix metalloproteinases (MMPs). Previously, we showed that isoflurane postconditioning reduced intracranial hemorrhage following tPA treatment after cerebral ischemia. Here, we investigated the mechanism by which isoflurane postconditioning reduces tPA-induced MMP-2 and MMP-9 activation following hypoxia/reoxygenation (H/R) in brain endothelial cells. Mouse brain endothelial cells (bEnd.3) were exposed to 6 h of oxygen-glucose deprivation and 3 h of reoxygenation with tPA. Cells were treated with isoflurane for 1 h of the reoxygenation condition and the effect of isoflurane postconditioning on MMP-2 and MMP-9 activation was assessed. Involvement of low-density lipoprotein receptor-related protein (LRP), which is a receptor for tPA, and the extracellular signal-regulated kinase (ERK) and NF-κB pathway in isoflurane postconditioning was assessed using LRP inhibitor (receptor-associated protein, RAP) and ERK-1/2 inhibitor (PD98059). Isoflurane postconditioning decreased tPA-induced MMP-2 and MMP-9 activation under H/R. tPA treatment under H/R increased expression of LRP and the active form of NF-κB. Isoflurane postconditioning suppressed LRP expression, increased ERK-1/2 activation, and suppressed MMP-2 and MMP-9 activation, comparable to the effect of RAP. Activation of ERK-1/2, inhibition of NF-κB activation, and suppression of MMP-2 and MMP-9 activation by isoflurane postconditioning were abolished with PD98059 treatment. These finding indicate that isoflurane postconditioning inhibits tPA-induced MMP-2 and MMP-9 activation following H/R via the LRP/ERK/NF-κB pathway in bEnd.3.
Asunto(s)
Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Poscondicionamiento Isquémico/métodos , Isoflurano/farmacología , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Metaloproteinasas de la Matriz/metabolismo , Activador de Tejido Plasminógeno/farmacología , Anestésicos por Inhalación/farmacología , Animales , Hipoxia de la Célula/efectos de los fármacos , Hipoxia de la Célula/fisiología , Línea Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Relación Dosis-Respuesta a Droga , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Ratones , Activador de Tejido Plasminógeno/antagonistas & inhibidoresRESUMEN
Neuroserpin (NS) is a serpin inhibitor of tissue plasminogen activator (tPA) in the brain. The polymerisation of NS pathologic mutants is responsible for a genetic dementia known as familial encephalopathy with neuroserpin inclusion bodies (FENIB). So far, a pharmacological treatment of FENIB, i.e. an inhibitor of NS polymerisation, remains an unmet challenge. Here, we present a biophysical characterisation of the effects caused by embelin (EMB a small natural compound) on NS conformers and NS polymerisation. EMB destabilises all known NS conformers, specifically binding to NS molecules with a 1:1 NS:EMB molar ratio without unfolding the NS fold. In particular, NS polymers disaggregate in the presence of EMB, and their formation is prevented. The NS/EMB complex does not inhibit tPA proteolytic activity. Both effects are pharmacologically relevant: firstly by inhibiting the NS polymerisation associated to FENIB, and secondly by potentially antagonizing metastatic processes facilitated by NS activity in the brain.
Asunto(s)
Benzoquinonas/metabolismo , Neuropéptidos/metabolismo , Multimerización de Proteína , Serpinas/metabolismo , Benzoquinonas/química , Dicroismo Circular , Humanos , Cinética , Ligandos , Espectrometría de Masas/métodos , Neuropéptidos/química , Unión Proteica , Conformación Proteica , Estabilidad Proteica , Serpinas/química , Activador de Tejido Plasminógeno/antagonistas & inhibidores , NeuroserpinaRESUMEN
INTERVENTIONS: Helium has been shown to provide neuroprotection in mechanical model of acute ischemic stroke by inducing hypothermia, a condition shown by itself to reduce the thrombolytic and proteolytic properties of tissue plasminogen activator. However, whether or not helium interacts with the thrombolytic drug tissue plasminogen activator, the only approved therapy of acute ischemic stroke still remains unknown. This point is not trivial since previous data have shown the critical importance of the time at which the neuroprotective noble gases xenon and argon should be administered, during or after ischemia, in order not to block tissue plasminogen activator-induced thrombolysis and to obtain neuroprotection and inhibition of tissue plasminogen activator-induced brain hemorrhages. MEASUREMENTS AND MAIN RESULTS: We show that helium of 25-75 vol% inhibits in a concentration-dependent fashion the catalytic and thrombolytic activity of tissue plasminogen activator in vitro and ex vivo. In vivo, in rats subjected to thromboembolic brain ischemia, we found that intraischemic helium at 75 vol% inhibits tissue plasminogen activator-induced thrombolysis and subsequent reduction of ischemic brain damage and that postischemic helium at 75 vol% reduces ischemic brain damage and brain hemorrhages. CONCLUSIONS: In a clinical perspective for the treatment of acute ischemic stroke, these data suggest that helium 1) should not be administered before or together with tissue plasminogen activator therapy due to the risk of inhibiting the benefit of tissue plasminogen activator-induced thrombolysis; and 2) could be an efficient neuroprotective agent if given after tissue plasminogen activator-induced reperfusion.
Asunto(s)
Antifibrinolíticos/administración & dosificación , Helio/administración & dosificación , Accidente Cerebrovascular/tratamiento farmacológico , Tromboembolia/tratamiento farmacológico , Activador de Tejido Plasminógeno/antagonistas & inhibidores , Activador de Tejido Plasminógeno/uso terapéutico , Animales , Antifibrinolíticos/farmacología , Isquemia Encefálica/tratamiento farmacológico , Isquemia Encefálica/etiología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Quimioterapia Combinada , Helio/farmacología , Hemorragias Intracraneales/tratamiento farmacológico , Masculino , Ratas , Accidente Cerebrovascular/etiología , Tromboembolia/complicacionesRESUMEN
In addition to degrading fibrinogen as a source of consumptive coagulopathy, rattlesnake venom has also been demonstrated to enhance fibrinolysis and degrade alpha-2-antiplasmin. The goals of this investigation was to characterize the kinetic fibrinolytic profile of Crotalus atrox venom in the absence and presence of tissue-type plasminogen activator (tPA), and to also ascertain if iron and carbon monoxide (CO, a positive modulator of alpha-2-antiplasmin) could attenuate venom-enhanced fibrinolysis. Utilizing thrombelastographic methods, the coagulation and fibrinolytic kinetic profiles of human plasma exposed to C. atrox venom (0-2âµg/ml) were determined in the absence or presence of tPA (0-100âIU/ml). Then, either separately or in combination, plasma was exposed to iron (ferric chloride, 10âµmol/l) or CO (carbon monoxide-releasing molecule-2, 100âµmol/l) prior to incubation with venom; the plasma sample was subsequently subjected to thrombelastographic analysis with addition of tPA. Venom exposure in the absence of tPA did not result in detectable fibrinolysis. In the presence of tPA, venom markedly enhanced fibrinolysis. Iron and CO, markedly attenuated venom enhancement of fibrinolysis. C. atrox venom enhances tPA-mediated fibrinolysis, and interventions that enhance/protect alpha-2-antiplasmin activity significantly attenuate venom-enhanced fibrinolysis. Future preclinical investigation is required to determine if iron and CO can attenuate venom-mediated degradation of alpha-2-antiplasmin-dependent fibrinolytic resistance.
Asunto(s)
Monóxido de Carbono/farmacología , Cloruros/farmacología , Venenos de Crotálidos/antagonistas & inhibidores , Compuestos Férricos/farmacología , Fibrinólisis/efectos de los fármacos , Fibrinolíticos/farmacología , Hemostáticos/farmacología , Activador de Tejido Plasminógeno/antagonistas & inhibidores , Animales , Monóxido de Carbono/química , Venenos de Crotálidos/farmacología , Crotalus/metabolismo , Tiempo de Lisis del Coágulo de Fibrina , Fibrinógeno/metabolismo , Humanos , Compuestos Organometálicos/química , Compuestos Organometálicos/farmacología , Tromboelastografía , Activador de Tejido Plasminógeno/farmacología , alfa 2-Antiplasmina/metabolismo , alfa 2-Antiplasmina/farmacologíaRESUMEN
Tissue plasminogen activator (tPA) mediates a number of processes that are pivotal for synaptogenesis and remodeling of synapses, including proteolysis of the brain extracellular matrix, degradation of adhesion molecules, activation of neurotrophins, and activation of the N-methyl-d-aspartate receptor. Abnormalities in these processes have been consistently described in psychotic disorders. In this paper, we review the physiological roles of tPA, focusing on conditions characterized by low tPA activity, which are prevalent in schizophrenia. We then describe how tPA activity is influenced by lifestyle interventions and nutritional supplements that may ameliorate psychotic symptoms. Next, we analyze the role of tPA in the mechanism of action of hormones and medications effective in mitigating psychotic symptoms, such as pregnenolone, estrogen, oxytocin, dopamine D3 receptor antagonists, retinoic acid, valproic acid, cannabidiol, sodium nitroprusside, N-acetyl cysteine, and warfarin. We also review evidence that tPA participates in the mechanism by which electroconvulsive therapy and cigarette smoking may reduce psychotic symptoms.
Asunto(s)
Fenotipo , Trastornos Psicóticos/diagnóstico , Trastornos Psicóticos/metabolismo , Activador de Tejido Plasminógeno/metabolismo , Animales , Antipsicóticos/uso terapéutico , Encéfalo/metabolismo , Suplementos Dietéticos , Terapia Electroconvulsiva , Hormonas/metabolismo , Humanos , Estilo de Vida , Neuropéptidos/metabolismo , Inhibidor 1 de Activador Plasminogénico/metabolismo , Unión Proteica , Trastornos Psicóticos/terapia , Esquizofrenia/diagnóstico , Esquizofrenia/dietoterapia , Esquizofrenia/metabolismo , Esquizofrenia/terapia , Serpinas/metabolismo , Fumar , Activador de Tejido Plasminógeno/antagonistas & inhibidores , NeuroserpinaRESUMEN
BACKGROUND: Although recent studies have assessed tranexamic acid (TXA) pharmacokinetics in different subgroups, the effective concentration of TXA required to completely inhibit fibrinolysis remains to be determined. OBJECTIVE: An in-vitro determination of the effective TXA concentration needed for 95% inhibition (EC95) of tissue-type plasminogen activator (t-PA) activated fibrinolysis, using an experimental model designed for thromboelastometry (ROTEM). DESIGN: A prospective interventional study. SETTING: Department of Anaesthesiology, Queen Fabiola Children's University Hospital and Laboratory of Haematology and Haemostasis, Brugmann University Hospital. Patients were enrolled between June 2013 and October 2014. PATIENTS AND VOLUNTEERS: Twenty children, aged between 1 and 10 years, undergoing elective cardiac catheterisation were included (10 with cyanotic and 10 with noncyanotic diseases). Exclusion criteria were child requiring a procedure in a moribund state. Ten adult volunteers were also included as controls. INTERVENTION: Citrated whole blood samples were obtained from children and volunteers. MAIN OUTCOMES MEASURES: The extrinsic coagulation pathway was activated by tissue factor using the EXTEM test on ROTEM. The degree of lysis measured 30 min (LI30) after the clotting time (CT), and clot amplitudes measured at different times were recorded at baseline, after addition of 1535 units t-PA ml(-1), and following the addition of increasing TXA concentrations in t-PA activated samples. RESULTS: The concentration-effect analysis performed with lysis index after 30 min (LI30) allowed the determination of TXA efficacy concentration 50% (EC50), and calculation of the EC95, which was significantly lower in cardiac surgery children than in adults [8.6 µg ml(-1); 95% confidence interval (95% CI) 6.9 to 14.9 versus 11.3 µg ml(-1); 95% CI 10.6 to 12.9, P < 0.001]. CONCLUSION: In this in-vitro study, we observed that the EC95 TXA concentration that completely inhibited t-PA induced hyperfibrinolysis in children with congenital heart was significantly lower than the concentration required in healthy adult volunteers. Further studies are needed to confirm that this plasma concentration can effectively inhibit fibrinolysis activation in children undergoing cardiac surgery.
Asunto(s)
Antifibrinolíticos/uso terapéutico , Fibrinólisis/efectos de los fármacos , Cardiopatías Congénitas/tratamiento farmacológico , Cardiopatías Congénitas/cirugía , Activador de Tejido Plasminógeno/antagonistas & inhibidores , Ácido Tranexámico/uso terapéutico , Adulto , Antifibrinolíticos/farmacología , Niño , Preescolar , Femenino , Fibrinólisis/fisiología , Cardiopatías Congénitas/sangre , Humanos , Lactante , Masculino , Estudios Prospectivos , Activador de Tejido Plasminógeno/sangre , Ácido Tranexámico/farmacología , Resultado del TratamientoRESUMEN
A hexapeptide derived from an enzyme inhibitor was used as an affinity ligand for the conjugation of a hydrophilic polymer to the enzyme. The peptide targeted the polymer to the "berth" of the inhibitor in the enzyme, affording the enzyme resistance to the inhibitor without affecting the enzymatic activity.
Asunto(s)
Oligopéptidos/química , Inhibidor 1 de Activador Plasminogénico/química , Polímeros/química , Activador de Tejido Plasminógeno/antagonistas & inhibidores , Activador de Tejido Plasminógeno/química , Animales , Interacciones Hidrofóbicas e Hidrofílicas , Ligandos , Ratones , Modelos Moleculares , Inhibidor 1 de Activador Plasminogénico/farmacología , Conformación Proteica , RatasRESUMEN
Hypoxic-ischemic brain injury is an important cause of neurodevelopmental deficits in neonates. Intrauterine infection and the ensuing fetal inflammatory responses augment hypoxic-ischemic brain injury and attenuate the efficacy of therapeutic hypothermia. Here, we review evidences from preclinical studies suggesting that the induction of brain parenchymal tissue-type plasminogen activator (tPA) plays an important pathogenic role in these conditions. Moreover, administration of a stable-mutant form of plasminogen activator inhibitor-1 called CPAI confers potent protection against hypoxic-ischemic injury with and without inflammation via different mechanisms. Besides intracerebroventricular injection, CPAI can also be administered into the brain using a noninvasive intranasal delivery strategy, adding to its applicability in clinical use. In sum, the therapeutic potential of CPAI in neonatal care merits further investigation with large-animal models of hypoxia-ischemia and cerebral palsy.
Asunto(s)
Encefalitis/tratamiento farmacológico , Encefalitis/fisiopatología , Hipoxia-Isquemia Encefálica/tratamiento farmacológico , Hipoxia-Isquemia Encefálica/fisiopatología , Fármacos Neuroprotectores/administración & dosificación , Inactivadores Plasminogénicos/administración & dosificación , Animales , Encéfalo/efectos de los fármacos , Encéfalo/fisiopatología , Humanos , Activador de Tejido Plasminógeno/antagonistas & inhibidoresRESUMEN
Plasminogen activator inhibitor-1 (PAI-1) is the main inhibitor of plasminogen activators, such as tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA), and a major regulator of the fibrinolytic system. PAI-1 plays a pivotal role in acute thrombotic events such as deep vein thrombosis (DVT) and myocardial infarction (MI). The biological effects of PAI-1 extend far beyond thrombosis including its critical role in fibrotic disorders, atherosclerosis, renal and pulmonary fibrosis, type-2 diabetes, and cancer. The conversion of PAI-1 from the active to the latent conformation appears to be unique among serpins in that it occurs spontaneously at a relatively rapid rate. Latency transition is believed to represent a regulatory mechanism, reducing the risk of thrombosis from a prolonged antifibrinolytic action of PAI-1. Thus, relying solely on plasma concentrations of PAI-1 without assessing its function may be misleading in interpreting the role of PAI-1 in many complex diseases. Environmental conditions, interaction with other proteins, mutations, and glycosylation are the main factors that have a significant impact on the stability of the PAI-1 structure. This review provides an overview on the current knowledge on PAI-1 especially importance of PAI-1 level and stability and highlights the potential use of PAI-1 inhibitors for treating cardiovascular disease.
Asunto(s)
Infarto del Miocardio/genética , Inhibidor 1 de Activador Plasminogénico/genética , Trombosis de la Vena/genética , Humanos , Riñón/metabolismo , Riñón/patología , Infarto del Miocardio/sangre , Infarto del Miocardio/patología , Inhibidor 1 de Activador Plasminogénico/sangre , Inhibidor 1 de Activador Plasminogénico/uso terapéutico , Estabilidad Proteica , Activador de Tejido Plasminógeno/antagonistas & inhibidores , Activador de Tejido Plasminógeno/genética , Activador de Plasminógeno de Tipo Uroquinasa/antagonistas & inhibidores , Activador de Plasminógeno de Tipo Uroquinasa/genética , Trombosis de la Vena/sangre , Trombosis de la Vena/patologíaRESUMEN
Although tissue plasminogen activator (tPA) is known to promote neuronal remodeling in the CNS, no mechanism of how this plastic function takes place has been reported so far. We provide here in vitro and in vivo demonstrations that this serine protease neutralizes inhibitory chondroitin sulfate proteoglycans (CSPGs) by promoting their degradation via the direct activation of endogenous type 4 disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS-4). Accordingly, in a model of compression-induced spinal cord injury (SCI) in rats, we found that administration of either tPA or its downstream effector ADAMTS-4 restores the tPA-dependent activity lost after the SCI and thereby, reduces content of CSPGs in the spinal cord, a cascade of events leading to an improved axonal regeneration/sprouting and eventually long term functional recovery. This is the first study to reveal a tPA-ADAMTS-4 axis and its function in the CNS. It also raises the prospect of exploiting such cooperation as a therapeutic tool for enhancing recovery after acute CNS injuries.