RESUMEN

Fibrinolytic activity assay is particularly important for the detection, diagnosis, and treatment of cardiovascular disease and the development of fibrinolytic drugs. A novel efficacious strategy for real-time and label-free dynamic detection of fibrinolytic activity based on ordered porous layer interferometry (OPLI) was developed. Fibrin or a mixture of fibrin and plasminogen (Plg) was loaded into the highly ordered silica colloidal crystal (SCC) film scaffold to construct a fibrinolytic response interference layer to measure fibrinolytic activity with different mechanisms of action. Fibrinolytic enzyme-triggered fibrinolysis led to the migration of interference fringes in the interferogram, which could be represented by optical thickness changes (ΔOT) tracked in real time by the OPLI system. The morphology and optical property of the fibrinolytic response interference layer were characterized, and the Plg content in the fibrinolytic response interference layer and experimental parameters of the system were optimized. The method showed adequate sensitivity for the fibrinolytic activity of lumbrokinase and streptokinase, with wide linear ranges of 12-6000 and 10-2000 U/mL, respectively. Compared with the traditional fibrin plate method, it has a lower detection limit and higher linearity. The whole kinetic process of fibrinolysis by these two fibrinolytic drug models was recorded in real time, and the Michaelis constant and apparent kinetic parameters were calculated. Importantly, some other blood proteins were less interfering with this system, and it showed reliability in fibrin activity detection in real whole blood samples. This study established a better and more targeted research method of in vitro fibrinolysis and provided dynamic monitoring data for the analysis of fibrinolytic activity of whole blood.

Asunto(s)

Fibrina , Fibrinólisis , Interferometría , Interferometría/métodos , Fibrinólisis/efectos de los fármacos , Fibrina/metabolismo , Fibrina/química , Humanos , Plasminógeno/metabolismo , Plasminógeno/análisis , Estreptoquinasa , Dióxido de Silicio/química , Porosidad , Fibrinolíticos/farmacología , Fibrinolíticos/química , Cinética

RESUMEN

Enolase is a 47 kDa enzyme that functions within the glycolysis and gluconeogenesis pathways involved in the reversible conversion of D-2-phosphoglycerate (2PGA) to phosphoenolpyruvate (PEP). However, in the context of host-pathogen interactions, enolase from different species of parasites, fungi and bacteria have been shown to contribute to adhesion processes by binding to proteins of the host extracellular matrix (ECM), such as fibronectin (FN) or laminin (LM). In addition, enolase is a plasminogen (PLG)-binding protein and induces its activation to plasmin, the main protease of the host fibrinolytic system. These secondary 'moonlighting' functions of enolase are suggested to facilitate pathogen migration through host tissues. This study aims to uncover the moonlighting role of enolase from the parasite Fasciola hepatica, shedding light on its relevance to host-parasite interactions in fasciolosis, a global zoonotic disease of increasing concern. A purified recombinant form of F. hepatica enolase (rFhENO), functioning as an active homodimeric glycolytic enzyme of ~94 kDa, was successfully obtained, fulfilling its canonical role. Immunoblotting studies on adult worm extracts showed that the enzyme is present in the tegument and the excretory/secretory products of the parasite, which supports its key role at the host-parasite interface. Confocal immunolocalisation studies of the protein in newly excysted juveniles and adult worms also localised its expression within the parasite tegument. Finally, we showed by ELISA that rFhENO can act as a parasitic adhesin by binding host LM, but not FN. rFhENO also binds PLG and enhances its conversion to plasmin in the presence of the tissue-type and urokinase-type PLG activators (t-PA and u-PA). This moonlighting adhesion-like function of the glycolytic protein enolase could contribute to the mechanisms by which F. hepatica efficiently invades and migrates within its host and encourages further research efforts that are designed to impede this function by vaccination or drug design.

Asunto(s)

Matriz Extracelular , Fasciola hepatica , Interacciones Huésped-Parásitos , Fosfopiruvato Hidratasa , Fosfopiruvato Hidratasa/metabolismo , Fosfopiruvato Hidratasa/genética , Fasciola hepatica/enzimología , Fasciola hepatica/metabolismo , Animales , Matriz Extracelular/metabolismo , Fibrinólisis , Plasminógeno/metabolismo , Glucólisis , Fascioliasis/parasitología , Fascioliasis/metabolismo , Laminina/metabolismo

RESUMEN

To effectively solve the problem of significant loss of transplanted cells caused by thrombosis during cell transplantation, this study simulates the human fibrinolytic system and combines metabolic oligosaccharide engineering with strain-promoted azide-alkyne cycloaddition (SPAAC) click chemistry to construct a cell surface with fibrinolytic activity. First, a copolymer (POL) of oligoethylene glycol methacrylate (OEGMA) and 6-amino-2-(2-methylamido)hexanoic acid (Lys) was synthesized by reversible addition-fragmentation chain transfer (RAFT) copolymerization, and the dibenzocyclooctyne (DBCO) functional group was introduced into the side chain of the copolymer through an active ester reaction, resulting in a functionalized copolymer DBCO-PEG4-POL with ε-lysine ligands. Then, azide functional groups were introduced onto the surface of HeLa model cells through metabolic oligosaccharide engineering, and DBCO-PEG4-POL was further specifically modified onto the surface of HeLa cells via the SPAAC "click" reaction. In vitro investigations revealed that compared with unmodified HeLa cells, modified cells not only resist the adsorption of nonspecific proteins such as fibrinogen and human serum albumin but also selectively bind to plasminogen in plasma while maintaining good cell viability and proliferative activity. More importantly, upon the activation of adsorbed plasminogen into plasmin, the modified cells exhibited remarkable fibrinolytic activity and were capable of promptly dissolving the primary thrombus formed on their surfaces. This research not only provides a novel approach for constructing transplantable cells with fibrinolytic activity but also offers a new perspective for effectively addressing the significant loss of transplanted cells caused by thrombosis.

Asunto(s)

Química Clic , Reacción de Cicloadición , Fibrinólisis , Oligosacáridos , Humanos , Células HeLa , Oligosacáridos/química , Fibrinólisis/efectos de los fármacos , Ingeniería Metabólica , Azidas/química , Polietilenglicoles/química , Metacrilatos/química , Alquinos/química , Animales , Supervivencia Celular/efectos de los fármacos , Plasminógeno/química , Plasminógeno/metabolismo , Propiedades de Superficie

RESUMEN

A new family of antifibrinolytic drugs has been recently discovered, combining a triazole moiety, an oxadiazolone, and a terminal amine. Two of the molecules of this family have shown activity that is greater than or similar to that of tranexamic acid (TXA), the current antifibrinolytic gold standard, which has been associated with several side effects and whose use is limited in patients with renal impairment. The aim of this work was to thoroughly examine the mechanism of action of the two ideal candidates of the 1,2,3-triazole family and compare them with TXA, to identify an antifibrinolytic alternative active at lower dosages. Specifically, the antifibrinolytic activity of the two compounds (1 and 5) and TXA was assessed in fibrinolytic isolated systems and in whole blood. Results revealed that despite having an activity pathway comparable to that of TXA, both compounds showed greater activity in blood. These differences could be attributed to a more stable ligand-target binding to the pocket of plasminogen for compounds 1 and 5, as suggested by molecular dynamic simulations. This work presents further evidence of the antifibrinolytic activity of the two best candidates of the 1,2,3-triazole family and paves the way for incorporating these molecules as new antifibrinolytic therapies.

Asunto(s)

Antifibrinolíticos , Ácido Tranexámico , Triazoles , Triazoles/química , Triazoles/farmacología , Antifibrinolíticos/farmacología , Antifibrinolíticos/química , Humanos , Ácido Tranexámico/farmacología , Ácido Tranexámico/química , Simulación de Dinámica Molecular , Plasminógeno/metabolismo , Plasminógeno/química , Fibrinólisis/efectos de los fármacos

RESUMEN

Staphylokinase (Sak), a small 15 kDa globular protein that is secreted by certain strains of Staphylococcus aureus, shows a potent fibrin-selective thrombolytic activity. Earlier work has shown that Sak could potentially become a low-cost alternative to currently used thrombolytic agents, such as tissue plasminogen activator (tPA). In attempts to improve its potential for clinical applications, numerous modifications of Sak have already been investigated. Here, we have characterized a novel Sak modification, cyclized Sak (cyc-Sak), which was prepared through split-intein mediated protein backbone cyclization. We have characterized the structure, stability and the activity of cyc-Sak using biophysical techniques, limited proteolysis studies and plasminogen (PG)-activation assays. Our results show that cyc-Sak possesses an identical structure, enhanced stability, resistance to proteolysis by exoproteases and improved PG-activation properties compared to its linear counterpart. It can be over-expressed with high yield in the cytoplasm of Escherichia coli and is easily purified in a two-step process. The intein-mediated cyclization occurs spontaneously in vivo during protein expression and does not necessitate further modification steps after purification of the protein. Furthermore, covalent Sak cyclization could be readily combined with other Sak modifications previously proposed, to generate an effective thrombolytic agent with lower immunogenicity and improved stability and activity.

Asunto(s)

Fibrina , Inteínas , Metaloendopeptidasas , Ciclización , Metaloendopeptidasas/química , Metaloendopeptidasas/metabolismo , Fibrina/química , Fibrina/metabolismo , Estabilidad de Enzimas , Proteolisis , Activadores Plasminogénicos/química , Activadores Plasminogénicos/metabolismo , Activadores Plasminogénicos/farmacología , Escherichia coli/efectos de los fármacos , Staphylococcus aureus/efectos de los fármacos , Humanos , Plasminógeno/metabolismo , Plasminógeno/química , Fibrinolíticos/farmacología , Fibrinolíticos/química

RESUMEN

Burkitt's lymphoma (BL) is a rare and highly aggressive B-cell non-Hodgkin lymphoma. Although the outcomes of patients with BL have greatly improved, options for patients with relapsed and refractory BL are limited. Therefore, there is an urgent need to improve BL therapeutics and to develop novel drugs with reduced toxicity. In this study, we demonstrated that enolase 1 (ENO1) is a potential novel drug target for BL treatment. We determined that ENO1 was aberrantly upregulated in BL, which was closely related to its invasiveness and poor clinical outcomes. Furthermore, using RNA interference, we demonstrated that ENO1 depletion significantly inhibited cell proliferation and invasion both in vitro and in vivo. Mechanistically, we established that ENO1 knockdown suppressed the PI3K-AKT and epithelial-mesenchymal transition (EMT) signaling pathways by reducing plasminogen (PLG) recruitment, plasmin (PL) generation, and TGF-ß1 activation. Addition of activated TGF-ß1 protein to the culture medium of shENO1 cells reversed the inhibitory effects on cell proliferation and invasion, as well as those on the PI3K-AKT and EMT signaling pathways. Notably, our research led to the discovery of a novel ENO1-PLG interaction inhibitor, Ciwujianoside E (L-06). L-06 effectively disrupts the interaction between ENO1 and PLG, consequently reducing PL generation and suppressing TGF-ß1 activation. In both in vitro and in vivo experiments, L-06 exerted impressive antitumor effects. In summary, our study elucidated the critical role of ENO1 in BL cell proliferation and invasion and introduced a novel ENO1 inhibitor, which holds promise for improving the treatment of patients with BL in the future.

Asunto(s)

Linfoma de Burkitt , Proliferación Celular , Proteínas de Unión al ADN , Transición Epitelial-Mesenquimal , Invasividad Neoplásica , Fosfopiruvato Hidratasa , Plasminógeno , Factor de Crecimiento Transformador beta1 , Proteínas Supresoras de Tumor , Fosfopiruvato Hidratasa/metabolismo , Humanos , Proliferación Celular/efectos de los fármacos , Línea Celular Tumoral , Animales , Linfoma de Burkitt/tratamiento farmacológico , Linfoma de Burkitt/patología , Linfoma de Burkitt/metabolismo , Transición Epitelial-Mesenquimal/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Plasminógeno/metabolismo , Transducción de Señal/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Masculino , Ratones Desnudos , Femenino , Fosfatidilinositol 3-Quinasas/metabolismo , Ratones , Ratones Endogámicos BALB C , Movimiento Celular/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto , Biomarcadores de Tumor

RESUMEN

The pivotal role of human endometrial stromal cells (hESCs) in the development of endometriosis lies in their ability to adopt a pro-invasive and proinflammatory profile upon migration to areas outside the uterus. However, the molecular mechanisms involved in these events remain unclear. In this study, we investigated how angiotensin II (Ang II) affects the plasminogen-plasmin system in hESCs, and the mechanisms underlying cell proliferation, migration, matrix degradation, and inflammation. Precursors, receptors, and peptidases involved in angiotensin metabolism increased significantly in Ang II-treated hESCs. The expression and activity of tissue (tPA)- and urokinase (uPA)- type plasminogen activators and the receptor for uPA (uPAR) were induced in the presence of Ang II. The up-regulation of tPA-uPA/uPAR pathway significantly contributes to heightened plasmin production both on the surface of hESCs and in their conditioned media. As a result, the plasmin generation induced by Ang II enhances the degradation of fibrin and matrix proteins, while also boosting hESC viability, proliferation, and migration through the up-regulation of growth factor expression. Notably, Ang II-induced hESC migration was dependent on the generation of active plasmin on cell surface. Ang II regulates oxidative and inflammatory signalling in hESCs primarily via NADPH oxidase and through the up-regulation of proinflammatory cytokines and adhesion molecules. Interestingly, Ang II receptor (AT1R) blockage, decreased plasmin generation, tPA-uPA/uPAR expression and hESC migration. Our results suggest that Ang II/AT1R axis regulates hESC proliferation and migration through tPA-uPA/uPAR pathway activation and plasmin generation. We propose the Ang II/AT1R axis as a potential target for endometriosis treatment.

Asunto(s)

Angiotensina II , Movimiento Celular , Endometrio , Matriz Extracelular , Fibrinolisina , Plasminógeno , Receptor de Angiotensina Tipo 1 , Transducción de Señal , Células del Estroma , Humanos , Femenino , Endometrio/metabolismo , Endometrio/citología , Endometrio/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Fibrinolisina/metabolismo , Células del Estroma/metabolismo , Células del Estroma/efectos de los fármacos , Angiotensina II/farmacología , Transducción de Señal/fisiología , Transducción de Señal/efectos de los fármacos , Matriz Extracelular/metabolismo , Matriz Extracelular/efectos de los fármacos , Receptor de Angiotensina Tipo 1/metabolismo , Plasminógeno/metabolismo , Células Cultivadas , Inflamación/metabolismo

RESUMEN

Introduction: Relapsing fever (RF) remains a neglected human disease that is caused by a number of diverse pathogenic Borrelia (B.) species. Characterized by high cell densities in human blood, relapsing fever spirochetes have developed plentiful strategies to avoid recognition by the host defense mechanisms. In this scenario, spirochetal lipoproteins exhibiting multifunctional binding properties in the interaction with host-derived molecules are known to play a key role in adhesion, fibrinolysis and complement activation. Methods: Binding of CihC/FbpC orthologs to different human proteins and conversion of protein-bound plasminogen to proteolytic active plasmin were examined by ELISA. To analyze the inhibitory capacity of CihC/FbpC orthologs on complement activation, a microtiter-based approach was performed. Finally, AlphaFold predictions were utilized to identified the complement-interacting residues. Results and discussion: Here, we elucidate the binding properties of CihC/FbpC-orthologs from distinct RF spirochetes including B. parkeri, B. hermsii, B. turicatae, and B. recurrentis to human fibronectin, plasminogen, and complement component C1r. All CihC/FbpC-orthologs displayed similar binding properties to fibronectin, plasminogen, and C1r, respectively. Functional studies revealed a dose dependent binding of plasminogen to all borrelial proteins and conversion to active plasmin. The proteolytic activity of plasmin was almost completely abrogated by tranexamic acid, indicating that lysine residues are involved in the interaction with this serine protease. In addition, a strong inactivation capacity toward the classical pathway could be demonstrated for the wild-type CihC/FbpC-orthologs as well as for the C-terminal CihC fragment of B. recurrentis. Pre-incubation of human serum with borrelial molecules except CihC/FbpC variants lacking the C-terminal region protected serum-susceptible Borrelia cells from complement-mediated lysis. Utilizing AlphaFold2 predictions and existing crystal structures, we mapped the putative key residues involved in C1r binding on the CihC/FbpC orthologs attempting to explain the relatively small differences in C1r binding affinity despite the substitutions of key residues. Collectively, our data advance the understanding of the multiple binding properties of structural and functional highly similar molecules of relapsing fever spirochetes proposed to be involved in pathogenesis and virulence.

Asunto(s)

Proteínas Bacterianas , Borrelia , Fibrinólisis , Interacciones Huésped-Patógeno , Plasminógeno , Humanos , Adhesión Bacteriana , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Borrelia/inmunología , Borrelia/metabolismo , Activación de Complemento , Proteínas del Sistema Complemento/inmunología , Proteínas del Sistema Complemento/metabolismo , Fibrinolisina/metabolismo , Fibronectinas/metabolismo , Interacciones Huésped-Patógeno/inmunología , Evasión Inmune , Plasminógeno/metabolismo , Unión Proteica , Fiebre Recurrente/inmunología , Fiebre Recurrente/microbiología

RESUMEN

Lipoprotein(a) (Lp(a)), an independent, causal cardiovascular risk factor, is a lipoprotein particle that is formed by the interaction of a low-density lipoprotein (LDL) particle and apolipoprotein(a) (apo(a))1,2. Apo(a) first binds to lysine residues of apolipoprotein B-100 (apoB-100) on LDL through the Kringle IV (KIV) 7 and 8 domains, before a disulfide bond forms between apo(a) and apoB-100 to create Lp(a) (refs. 3-7). Here we show that the first step of Lp(a) formation can be inhibited through small-molecule interactions with apo(a) KIV7-8. We identify compounds that bind to apo(a) KIV7-8, and, through chemical optimization and further application of multivalency, we create compounds with subnanomolar potency that inhibit the formation of Lp(a). Oral doses of prototype compounds and a potent, multivalent disruptor, LY3473329 (muvalaplin), reduced the levels of Lp(a) in transgenic mice and in cynomolgus monkeys. Although multivalent molecules bind to the Kringle domains of rat plasminogen and reduce plasmin activity, species-selective differences in plasminogen sequences suggest that inhibitor molecules will reduce the levels of Lp(a), but not those of plasminogen, in humans. These data support the clinical development of LY3473329-which is already in phase 2 studies-as a potent and specific orally administered agent for reducing the levels of Lp(a).

Asunto(s)

Descubrimiento de Drogas , Lipoproteína(a) , Macaca fascicularis , Animales , Femenino , Humanos , Masculino , Ratones , Administración Oral , Kringles , Lipoproteína(a)/antagonistas & inhibidores , Lipoproteína(a)/sangre , Lipoproteína(a)/química , Lipoproteína(a)/metabolismo , Ratones Transgénicos , Bibliotecas de Moléculas Pequeñas/farmacología , Bibliotecas de Moléculas Pequeñas/química , Plasminógeno/química , Plasminógeno/metabolismo , Especificidad de la Especie , Ensayos Clínicos Fase II como Asunto , Apolipoproteínas A/química , Apolipoproteínas A/metabolismo

RESUMEN

The potent angiogenesis inhibitor known as human plasminogen Kringle 5 has shown promise in the treatment of vascular disorders and malignancies. The study aimed to investigate the recognition and interaction between Kringle 5 and the A2M domain of human complement component C5 using bio-specific methodologies and molecular dynamics (MD) simulation. Initially, the specific interaction between Kringle 5 and A2M was confirmed and characterized through Ligand Blot and ELISA, yielding the dissociation constant (Kd) of 1.70 × 10-7 mol/L. Then, Kringle 5 showcased a dose-dependent inhibition of the production of C5a in lung cancer A549 cells, consequently impeding their proliferation and migration. Following the utilization of frontal affinity chromatography (FAC), it was revealed that there exists a singular binding site with the binding constant (Ka) of 3.79 × 105 L/mol. Following the implementation of homology modeling and MD optimization, the detailed results indicate that only a specific segment of the N-terminal structure of the A2M molecule engages in interaction with Kringle 5 throughout the binding process and the principal driving forces encompass electrostatic force, hydrogen bonding, and van der Waals force. In conclusion, the A2M domain of human complement C5 emerges as a plausible binding target for Kringle 5 in vivo.

Asunto(s)

Simulación de Dinámica Molecular , Plasminógeno , Unión Proteica , Humanos , Plasminógeno/química , Plasminógeno/metabolismo , Sitios de Unión , Complemento C5a/química , Complemento C5a/metabolismo , Células A549 , Dominios Proteicos , Proliferación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Fragmentos de Péptidos

RESUMEN

Aim: Streptokinase has poor selectivity and provokes the immune response. In this study, we used in silico studies to design a fusion protein to achieve targeted delivery to the thrombus. Materials & methods: Streptokinase was analyzed computationally for mapping. The fusion protein modeling and quality assessment were carried out on several servers. The enzymatic activity and the stability of the fusion protein and its complex with plasminogen were assessed through molecular docking analysis and molecular dynamics simulation respectively. Results: Physicochemical properties analysis, protein quality assessments, protein-protein docking and molecular dynamics simulations predicted that the designed fusion protein is functionally active. Conclusion: Our results showed that this fusion protein might be a prospective candidate as a novel thrombolytic agent with better selectivity.

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Asunto(s)

Fibrinolíticos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Proteínas Recombinantes de Fusión , Estreptoquinasa , Trombosis , Estreptoquinasa/química , Estreptoquinasa/administración & dosificación , Estreptoquinasa/metabolismo , Estreptoquinasa/genética , Trombosis/tratamiento farmacológico , Fibrinolíticos/química , Fibrinolíticos/administración & dosificación , Humanos , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/genética , Sistemas de Liberación de Medicamentos/métodos , Fibrina/metabolismo , Fibrina/química , Plasminógeno/metabolismo , Plasminógeno/química , Simulación por Computador , Unión Proteica

RESUMEN

ADAMTS13, a disintegrin and metalloprotease with a thrombospondin type 1 motif, member 13, regulates the length of Von Willebrand factor (VWF) multimers and their platelet-binding activity. ADAMTS13 is constitutively secreted as an active protease and is not inhibited by circulating protease inhibitors. Therefore, the mechanisms that regulate ADAMTS13 protease activity are unknown. We performed an unbiased proteomics screen to identify ligands of ADAMTS13 by optimizing the application of BioID to plasma. Plasma BioID identified 5 plasma proteins significantly labeled by the ADAMTS13-birA* fusion, including VWF and plasminogen. Glu-plasminogen, Lys-plasminogen, mini-plasminogen, and apo(a) bound ADAMTS13 with high affinity, whereas micro-plasminogen did not. None of the plasminogen variants or apo(a) bound to a C-terminal truncation variant of ADAMTS13 (MDTCS). The binding of plasminogen to ADAMTS13 was attenuated by tranexamic acid or ε-aminocaproic acid, and tranexamic acid protected ADAMTS13 from plasmin degradation. These data demonstrate that plasminogen is an important ligand of ADAMTS13 in plasma by binding to the C-terminus of ADAMTS13. Plasmin proteolytically degrades ADAMTS13 in a lysine-dependent manner, which may contribute to its regulation. Adapting BioID to identify protein-interaction networks in plasma provides a powerful new tool to study protease regulation in the cardiovascular system.

Asunto(s)

Fibrinolisina , Ácido Tranexámico , Fibrinolisina/metabolismo , Factor de von Willebrand/metabolismo , Proteína ADAMTS13 , Proteínas ADAM/metabolismo , Ligandos , Plasminógeno/metabolismo

RESUMEN

Parkinson's disease (PD) is the second most frequently diagnosed neurodegenerative disease, and it is characterized by the intracellular and extracellular accumulation of α-synuclein (α-syn) and Tau, which are major components of cytosolic protein inclusions called Lewy bodies, in the brain. Currently, there is a lack of effective methods that preventing PD progression. It has been suggested that the plasminogen activation system, which is a major extracellular proteolysis system, is involved in PD pathogenesis. We investigated the functional roles of plasminogen in vitro in an okadaic acid-induced Tau hyperphosphorylation NSC34 cell model, ex vivo using brains from normal controls and methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice, and in vivo in a widely used MPTP-induced PD mouse model and an α-syn overexpression mouse model. The in vitro, ex vivo and in vivo results showed that the administered plasminogen crossed the blood‒brain barrier (BBB), entered cells, and migrated to the nucleus, increased plasmin activity intracellularly, bound to α-syn through lysine binding sites, significantly promoted α-syn, Tau and TDP-43 clearance intracellularly and even intranuclearly in the brain, decreased dopaminergic neurodegeneration and increased the tyrosine hydroxylase levels in the substantia nigra and striatum, and improved motor function in PD mouse models. These findings indicate that plasminogen plays a wide range of pivotal protective roles in PD and therefore may be a promising drug candidate for PD treatment.

Asunto(s)

Enfermedades Neurodegenerativas , Enfermedad de Parkinson , Plasminógeno , Animales , Ratones , alfa-Sinucleína , Modelos Animales de Enfermedad , Proteínas de Unión al ADN/metabolismo , Dopamina , Enfermedades Neurodegenerativas/metabolismo , Enfermedad de Parkinson/metabolismo , Plasminógeno/metabolismo , Serina Proteasas , Proteínas tau/metabolismo , Neuronas Dopaminérgicas/patología

RESUMEN

Joint injury is associated with risk for development of osteoarthritis (OA). Increasing evidence suggests that activation of fibrinolysis is involved in OA pathogenesis. However, the role of the fibrinolytic pathway is not well understood. Here, we showed that the fibrinolytic pathway, which includes plasminogen/plasmin, tissue plasminogen activator, urokinase plasminogen activator (uPA), and the uPA receptor (uPAR), was dysregulated in human OA joints. Pharmacological inhibition of plasmin attenuated OA progression after a destabilization of the medial meniscus in a mouse model whereas genetic deficiency of plasmin activator inhibitor, or injection of plasmin, exacerbated OA. We detected increased uptake of uPA/uPAR in mouse OA joints by microPET/CT imaging. In vitro studies identified that plasmin promotes OA development through multiple mechanisms, including the degradation of lubricin and cartilage proteoglycans and induction of inflammatory and degradative mediators. We showed that uPA and uPAR produced inflammatory and degradative mediators by activating the PI3K, 3'-phosphoinositide-dependent kinase-1, AKT, and ERK signaling cascades and activated matrix metalloproteinases to degrade proteoglycan. Together, we demonstrated that fibrinolysis contributes to the development of OA through multiple mechanisms and suggested that therapeutic targeting of the fibrinolysis pathway can prevent or slow development of OA.

Asunto(s)

Modelos Animales de Enfermedad , Fibrinolisina , Fibrinólisis , Osteoartritis , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Activador de Plasminógeno de Tipo Uroquinasa , Animales , Ratones , Humanos , Fibrinolisina/metabolismo , Osteoartritis/metabolismo , Osteoartritis/patología , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Receptores del Activador de Plasminógeno Tipo Uroquinasa/metabolismo , Masculino , Femenino , Ratones Endogámicos C57BL , Plasminógeno/metabolismo , Transducción de Señal , Ratones Noqueados

RESUMEN

AIMS: Alteplase is a cornerstone thrombolytic agent in clinical practice but presents a potential bleeding risk. Stroke patients need pre-screening to exclude haemorrhagic stroke before using alteplase. In this study, we develop a new thrombolytic agent citPA5, characterized by an enhanced safety profile and minimal bleeding tendency. METHODS AND RESULTS: A clot lysis agent, named citPA5, is developed based on rtPA with point mutations to completely suppress its proteolytic activity in the absence of fibrin. In the presence of fibrin, citPA5 exhibited significantly higher fibrinolytic activity (a 15.8-fold increase of kcat/Km). Furthermore, citPA5 showed resistance to endogenous fibrinolysis inhibitor, PAI-1, resulting in enhanced potency. In a series of safety evaluation experiments, including thrombelastography assay, mice tail bleeding assay, and a murine intracerebral haemorrhage (ICH) model, citPA5 did not cause systemic bleeding or worsen ICH compared with alteplase. This highlights the low risk of bleeding associated with citPA5. Finally, we found that citPA5 effectively improved cerebral blood flow and reduced infarct volume in a carotid embolism-induced stroke model. CONCLUSION: This clot lysis agent, citPA5, not only exhibits a low risk of bleeding but also demonstrates highly effective thrombolysis capabilities. As a result, citPA5 shows great potential for administration prior to the classification of stroke types, making it possible for use in ambulances at the onset of stroke when symptoms are identified. The findings presented in this study also suggest that this strategy could be applied to develop a new generation of fibrinolytic drugs that offer greater safety and specificity in targeting fibrin.

Asunto(s)

Modelos Animales de Enfermedad , Fibrinolíticos , Ratones Endogámicos C57BL , Terapia Trombolítica , Animales , Fibrinolíticos/efectos adversos , Fibrinolíticos/farmacología , Terapia Trombolítica/efectos adversos , Humanos , Activador de Tejido Plasminógeno/efectos adversos , Factores de Riesgo , Masculino , Fibrinólisis/efectos de los fármacos , Circulación Cerebrovascular/efectos de los fármacos , Medición de Riesgo , Plasminógeno/metabolismo , Hemorragia Cerebral/inducido químicamente , Mutación Puntual , Hemorragia/inducido químicamente , Proteínas Recombinantes , Coagulación Sanguínea/efectos de los fármacos , Activadores Plasminogénicos

RESUMEN

RATIONALE & OBJECTIVE: Glomerular disorders have a highly variable clinical course, and biomarkers that reflect the molecular mechanisms underlying their progression are needed. Based on our previous work identifying plasminogen as a direct cause of podocyte injury, we designed this study to test the association between urine plasmin(ogen) (ie, plasmin and its precursor plasminogen) and end-stage kidney disease (ESKD). STUDY DESIGN: Multicenter cohort study. SETTING & PARTICIPANTS: 1,010 patients enrolled in the CureGN Cohort with biopsy-proven glomerular disease (focal segmental glomerulosclerosis, membranous nephropathy, and immunoglobulin A nephropathy). PREDICTORS: The main predictor was urine plasmin(ogen) at baseline. Levels were measured by an electrochemiluminescent immunoassay developed de novo. Traditional clinical and analytical characteristics were used for adjustment. The ratio of urine plasmin(ogen)/expected plasmin(ogen) was evaluated as a predictor in a separate model. OUTCOME: Progression to ESKD. ANALYTICAL APPROACH: Cox regression was used to examine the association between urinary plasmin(ogen) and time to ESKD. Urinary markers were log2 transformed to approximate normal distribution and normalized to urinary creatinine (Log2uPlasminogen/cr, Log2 urinary protein/cr [UPCR]). Expected plasmin(ogen) was calculated by multiple linear regression. RESULTS: Adjusted Log2uPlasminogen/cr was significantly associated with ESKD (HR per doubling Log2 uPlasminogen/cr 1.31 [95% CI, 1.22-1.40], P<0.001). Comparison of the predictive performance of the models including Log2 uPlasminogen/cr, Log2 UPCR, or both markers showed the plasmin(ogen) model superiority. The ratio of measured/expected urine plasmin(ogen) was independently associated with ESKD: HR, 0.41 (95% CI, 0.22-0.77) if ratio<0.8 and HR 2.42 (95% CI, 1.54-3.78) if ratio>1.1 (compared with ratio between 0.8 and 1.1). LIMITATIONS: Single plasmin(ogen) determination does not allow for the study of changes over time. The use of a cohort of mostly white patients and the restriction to patients with 3 glomerular disorders limits the external validity of our analysis. CONCLUSIONS: Urinary plasmin(ogen) and the ratio of measured/expected plasmin(ogen) are independently associated with ESKD in a cohort of patients with glomerular disease. Taken together with our previous experimental findings, urinary plasmin(ogen) could be a useful biomarker in prognostic decision making and a target for the development of novel therapies in patients with proteinuria and glomerular disease. PLAIN-LANGUAGE SUMMARY: Glomerular diseases are an important cause of morbidity and mortality in patients of all ages. Knowing the individual risk of progression to dialysis or transplantation would help to plan the follow-up and treatment of these patients. Our work studies the usefulness of urinary plasminogen as a marker of progression in this context, since previous studies indicate that plasminogen may be involved in the mechanisms responsible for the progression of these disorders. Our work in a sample of 1,010 patients with glomerular disease demonstrates that urinary plasminogen (as well as the ratio of measured to expected plasminogen) is associated with the risk of progression to end-stage kidney disease. Urine plasminogen exhibited good performance and, if further validated, could enable risk stratification for timely interventions in patients with proteinuria and glomerular disease.

Asunto(s)

Biomarcadores , Progresión de la Enfermedad , Fallo Renal Crónico , Plasminógeno , Humanos , Masculino , Femenino , Biomarcadores/orina , Plasminógeno/orina , Plasminógeno/metabolismo , Persona de Mediana Edad , Adulto , Fallo Renal Crónico/orina , Estudios de Cohortes , Glomeruloesclerosis Focal y Segmentaria/orina , Glomeruloesclerosis Focal y Segmentaria/diagnóstico , Glomerulonefritis por IGA/orina , Glomerulonefritis por IGA/diagnóstico , Glomerulonefritis Membranosa/orina , Glomerulonefritis Membranosa/diagnóstico , Fibrinolisina/orina , Fibrinolisina/metabolismo

RESUMEN

Streptococcus suis is an emerging zoonotic pathogen that can cause invasive disease commonly associated with meningitis in pigs and humans. To cause meningitis, S. suis must cross the blood-brain barrier (BBB) comprising blood vessels that vascularize the central nervous system (CNS). The BBB is highly selective due to interactions with other cell types in the brain and the composition of the extracellular matrix (ECM). Purified streptococcal surface enolase, an essential enzyme participating in glycolysis, can bind human plasminogen (Plg) and plasmin (Pln). Plg has been proposed to increase bacterial traversal across the BBB via conversion to Pln, a protease which cleaves host proteins in the ECM and monocyte chemoattractant protein 1 (MCP1) to disrupt tight junctions. The essentiality of enolase has made it challenging to unequivocally demonstrate its role in binding Plg/Pln on the bacterial surface and confirm its predicted role in facilitating translocation of the BBB. Here, we report on the CRISPR/Cas9 engineering of S. suis enolase mutants eno261, eno252/253/255, eno252/261, and eno434/435 possessing amino acid substitutions at in silico predicted binding sites for Plg. As expected, amino acid substitutions in the predicted Plg binding sites reduced Plg and Pln binding to S. suis but did not affect bacterial growth in vitro compared to the wild-type strain. The binding of Plg to wild-type S. suis enhanced translocation across the human cerebral microvascular endothelial cell line hCMEC/D3 but not for the eno mutant strains tested. To our knowledge, this is the first study where predicted Plg-binding sites of enolase have been mutated to show altered Plg and Pln binding to the surface of S. suis and attenuation of translocation across an endothelial cell monolayer in vitro.

Asunto(s)

Meningitis , Streptococcus suis , Animales , Humanos , Porcinos , Plasminógeno/metabolismo , Barrera Hematoencefálica , Streptococcus suis/genética , Streptococcus suis/metabolismo , Traslocación Bacteriana , Fibrinolisina/metabolismo , Sitios de Unión , Fosfopiruvato Hidratasa/química

RESUMEN

An intriguing effect of short-term caloric restriction (CR) is the expansion of certain stem cell populations, including muscle stem cells (satellite cells), which facilitate an accelerated regenerative program after injury. Here, we utilized the MetRSL274G (MetRS) transgenic mouse to identify liver-secreted plasminogen as a candidate for regulating satellite cell expansion during short-term CR. Knockdown of circulating plasminogen prevents satellite cell expansion during short-term CR. Furthermore, loss of the plasminogen receptor KT (Plg-RKT) is also sufficient to prevent CR-related satellite cell expansion, consistent with direct signaling of plasminogen through the plasminogen receptor Plg-RKT/ERK kinase to promote proliferation of satellite cells. Importantly, we are able to replicate many of these findings in human participants from the CALERIE trial. Our results demonstrate that CR enhances liver protein secretion of plasminogen, which signals directly to the muscle satellite cell through Plg-RKT to promote proliferation and subsequent muscle resilience during CR.

Asunto(s)

Plasminógeno , Receptores de Superficie Celular , Ratones , Animales , Humanos , Plasminógeno/metabolismo , Receptores de Superficie Celular/metabolismo , Restricción Calórica , Hígado/metabolismo , Ratones Transgénicos , Serina Proteasas , Proliferación Celular , Músculos/metabolismo

RESUMEN

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a moonlighting enzyme. Apart from its primary role in the glycolytic pathway, in many bacterial species it is found in the extracellular milieu and also on the bacterial surface. Positioning on the bacterial surface allows the GAPDH molecule to interact with many host molecules such as plasminogen, fibrinogen, fibronectin, laminin and mucin etc. This facilitates the bacterial colonization of the host. Helicobacter pylori is a major human pathogen that causes a number of gastrointestinal infections and is the main cause of gastric cancer. The binding analysis of H. pylori GAPDH (HpGAPDH) with host molecules has not been carried out. Hence, we studied the interaction of HpGAPDH with holo-transferrin, lactoferrin, haemoglobin, fibrinogen, fibronectin, catalase, plasminogen and mucin using biolayer interferometry. Highest and lowest binding affinity was observed with lactoferrin (4.83 ± 0.70 × 10-9 M) and holo-transferrin (4.27 ± 2.39 × 10-5 M). Previous studies established GAPDH as a heme chaperone involved in intracellular heme trafficking and delivery to downstream target proteins. Therefore, to get insights into heme binding, the interaction between HpGAPDH and hemin was analyzed. Hemin binds to HpGAPDH with an affinity of 2.10 µM while the hemin bound HpGAPDH does not exhibit activity. This suggests that hemin most likely binds at the active site of HpGAPDH, prohibiting substrate binding. Blind docking of hemin with HpGAPDH also supports positioning of hemin at the active site. Metal ions were found to inhibit the activity of HpGAPDH, suggesting that it also possibly occupies the substrate binding site. Furthermore, with metal-bound HpGAPDH, hemin binding was not observed, suggesting metal ions act as an inhibitor of hemin binding. Since GAPDH has been identified as a heme chaperone, it will be interesting to analyse the biological consequences of inhibition of heme binding to GAPDH by metal ions.

Asunto(s)

Helicobacter pylori , Hemina , Humanos , Hemina/metabolismo , Helicobacter pylori/metabolismo , Fibronectinas/metabolismo , Lactoferrina/metabolismo , Unión Proteica , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Hemo/metabolismo , Fibrinógeno , Plasminógeno/metabolismo , Iones/metabolismo , Mucinas/metabolismo

RESUMEN

We modify a three-dimensional multiscale model of fibrinolysis to study the effect of plasmin-mediated degradation of fibrin on tissue plasminogen activator (tPA) diffusion and fibrinolysis. We propose that tPA is released from a fibrin fiber by simple kinetic unbinding, as well as by "forced unbinding," which occurs when plasmin degrades fibrin to which tPA is bound. We show that, if tPA is bound to a small-enough piece of fibrin that it can diffuse into the clot, then plasmin can increase the effective diffusion of tPA. If tPA is bound to larger fibrin degradation products (FDPs) that can only diffuse along the clot, then plasmin can decrease the effective diffusion of tPA. We find that lysis rates are fastest when tPA is bound to fibrin that can diffuse into the clot, and slowest when tPA is bound to FDPs that can only diffuse along the clot. Laboratory experiments confirm that FDPs can diffuse into a clot, and they support the model hypothesis that forced unbinding of tPA results in a mix of FDPs, such that tPA bound to FDPs can diffuse both into and along the clot. Regardless of how tPA is released from a fiber, a tPA mutant with a smaller dissociation constant results in slower lysis (because tPA binds strongly to fibrin), and a tPA mutant with a larger dissociation constant results in faster lysis.

Asunto(s)

Fibrinolisina , Fibrinólisis , Fibrinolisina/metabolismo , Fibrinolisina/farmacología , Activador de Tejido Plasminógeno/metabolismo , Activador de Tejido Plasminógeno/farmacología , Fibrina/metabolismo , Cinética , Plasminógeno/metabolismo