RESUMEN
Disseminated intravascular coagulation (DIC) is a pathologic state that follows systemic injury and other diseases. Often a complication of sepsis or trauma, DIC causes coagulopathy associated with paradoxical thrombosis and hemorrhage. DIC upregulates the thrombotic pathways while simultaneously downregulating the fibrinolytic pathways that cause excessive fibrin deposition, microcirculatory thrombosis, multiorgan dysfunction, and consumptive coagulopathy with excessive bleeding. Given these opposing disease phenotypes, DIC management is challenging and includes treating the underlying disease and managing the coagulopathy. Currently, no therapies are approved for DIC. We have developed clot-targeted therapeutics that inhibit clot polymerization and activate clot fibrinolysis to manage DIC. We hypothesize that delivering both an anticoagulant and a fibrinolytic agent directly to clots will inhibit active clot polymerization while also breaking up pre-existing clots; therefore, reversing consumptive coagulopathy and restoring hemostatic balance. To test this hypothesis, we single- and dual-loaded fibrin-specific nanogels (FSNs) with antithrombinIII (ATIII) and/or tissue plasminogen activator (tPA) and evaluated their clot preventing and clot lysing abilities in vitro and in a rodent model of DIC. In vivo, single-loaded ATIII-FSNs decreased fibrin deposits in DIC organs and reduced blood loss when DIC rodents were injured. We also observed that the addition of tPA in dual-loaded ATIII-tPA-FSNs intensified the antithrombotic and fibrinolytic mechanisms, which proved advantageous for clot lysis and restoring platelet counts. However, the addition of tPA may have hindered wound healing capabilities when an injury was introduced. Our data supports the benefits of delivering both anticoagulants and fibrinolytic agents directly to clots to reduce the fibrin load and restore hemostatic balance in DIC.
Asunto(s)
Coagulación Intravascular Diseminada , Activador de Tejido Plasminógeno , Activador de Tejido Plasminógeno/farmacología , Activador de Tejido Plasminógeno/administración & dosificación , Activador de Tejido Plasminógeno/química , Animales , Coagulación Intravascular Diseminada/tratamiento farmacológico , Nanogeles/química , Fibrinolíticos/farmacología , Fibrinolíticos/química , Fibrinolíticos/administración & dosificación , Humanos , Ratas , Fibrina/metabolismo , Fibrina/química , Antitrombinas/farmacología , Antitrombinas/química , Antitrombinas/administración & dosificación , Ratones , Masculino , Trombosis/tratamiento farmacológico , Sistemas de Liberación de Medicamentos , Coagulación Sanguínea/efectos de los fármacosRESUMEN
Antithrombin (AT) is a critical regulator of the coagulation cascade by inhibiting multiple coagulation factors including thrombin and FXa. Binding of heparinoids to this serpin enhances the inhibition considerably. Mutations located in the heparin binding site of AT result in thrombophilia in affected individuals. Our aim was to study 10 antithrombin mutations known to affect their heparin binding in a heparin pentasaccharide bound state using two molecular dynamics (MD) based methods providing enhanced sampling, GaMD and LiGaMD2. The latter provides an additional boost to the ligand and the most important binding site residues. From our GaMD simulations we were able to identify four variants (three affecting amino acid Arg47 and one affecting Lys114) that have a particularly large effect on binding. The additional acceleration provided by LiGaMD2 allowed us to study the consequences of several other mutants including those affecting Arg13 and Arg129. We were able to identify several conformational types by cluster analysis. Analysis of the simulation trajectories revealed the causes of the impaired pentasaccharide binding including pentasaccharide subunit conformational changes and altered allosteric pathways in the AT protein. Our results provide insights into the effects of AT mutations interfering with heparin binding at an atomic level and can facilitate the design or interpretation of in vitro experiments.
Asunto(s)
Antitrombinas , Heparina , Simulación de Dinámica Molecular , Mutación , Heparina/metabolismo , Heparina/química , Sitios de Unión , Humanos , Antitrombinas/química , Antitrombinas/metabolismo , Unión Proteica , Oligosacáridos/química , Oligosacáridos/metabolismoRESUMEN
Thrombin is a crucial enzyme in the coagulation cascade, and inhibitors of thrombin have been extensively studied as potential antithrombotic agents. The objective of this study was to identify natural inhibitors of thrombin from Panax notoginseng and evaluate their biological activity in vitro and binding characteristics. A combined approach involving molecular docking, thrombin inhibition assays, surface plasmon resonance, and molecular dynamics simulation was utilized to identify natural thrombin inhibitors. The results demonstrated that panaxatriol directly inhibits thrombin, with an IC50 of 10.3 µM. Binding studies using surface plasmon resonance revealed that panaxatriol interacts with thrombin, with a KD value of 7.8 µM. Molecular dynamics analysis indicated that the thrombin-panaxatriol system reached equilibrium rapidly with minimal fluctuations, and the calculated binding free energy was - 23.8 kcal/mol. The interaction between panaxatriol and thrombin involves the amino acid residues Glu146, Glu192, Gly216, Gly219, Tyr60A, and Trp60D. This interaction provides a mechanistic basis for further optimizing panaxatriol as a thrombin inhibitor. Our study has shown that panaxatriol serves as a direct thrombin inhibitor, laying the groundwork for further research and development of novel thrombin inhibitors.
Asunto(s)
Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Panax notoginseng , Trombina , Panax notoginseng/química , Trombina/antagonistas & inhibidores , Ginsenósidos/química , Ginsenósidos/farmacología , Humanos , Antitrombinas/farmacología , Antitrombinas/química , Resonancia por Plasmón de SuperficieRESUMEN
Low molecular weight heparin and synthetic mimetics such as fondaparinux show different binding kinetics, protease specificity, and clinical effects. A combination of allosteric and template-mediated bridging mechanisms have been proposed to explain the differences in rate acceleration and specificity. The difficulty in working with heterogeneous heparin species has rendered a crystallographic interpretation of the differences in antithrombin activation between mimetics and natural heparin inaccessible. In this study, we examine the allosteric changes in antithrombin caused by binding fondaparinux, enoxaparin and depolymerized natural heparins using millisecond hydrogen deuterium exchange mass spectrometry (TRESI-HDX MS) and relate these conformational changes to complex stability in the gas phase using collision induced unfolding (CIU). This exploration reveals that in addition to the dynamic changes caused by fondaparinux, long chain heparins reduce structural flexibility proximal to Arg393, the cleavable residue in the reactive centre loop of the protein. These local changes in protein dynamics are associated with an increase in overall complex stability that increases with heparin chain length. Ultimately, these results shed light on the molecular mechanisms underlying differences in activity and specificity between heparin mimetics and natural heparins.
Asunto(s)
Antitrombinas , Fondaparinux , Heparina , Fondaparinux/química , Heparina/química , Antitrombinas/química , Antitrombinas/farmacología , Desplegamiento Proteico/efectos de los fármacos , Medición de Intercambio de Deuterio , Humanos , Cinética , Unión Proteica , Polisacáridos/química , Polisacáridos/farmacología , Modelos MolecularesRESUMEN
The interaction of heparin with antithrombin (AT) involves a specific sequence corresponding to the pentasaccharide GlcNAc/NS6S-GlcA-GlcNS3S6S-IdoA2S-GlcNS6S (AGA*IA). Recent studies have revealed that two AGA*IA-containing hexasaccharides, which differ in the sulfation degree of the iduronic acid unit, exhibit similar binding to AT, albeit with different affinities. However, the lack of experimental data concerning the molecular contacts between these ligands and the amino acids within the protein-binding site prevents a detailed description of the complexes. Differential epitope mapping (DEEP)-STD NMR, in combination with MD simulations, enables the experimental observation and comparison of two heparin pentasaccharides interacting with AT, revealing slightly different bound orientations and distinct affinities of both glycans for AT. We demonstrate the effectiveness of the differential solvent DEEP-STD NMR approach in determining the presence of polar residues in the recognition sites of glycosaminoglycan-binding proteins.
Asunto(s)
Antitrombinas , Heparina , Oligosacáridos , Humanos , Antitrombinas/química , Antitrombinas/metabolismo , Sitios de Unión , Mapeo Epitopo/métodos , Heparina/química , Heparina/metabolismo , Espectroscopía de Resonancia Magnética/métodos , Simulación de Dinámica Molecular , Oligosacáridos/química , Oligosacáridos/metabolismo , Unión Proteica , Solventes/químicaRESUMEN
During inflammation and situations of cellular stress protein disulfide isomerase (PDI) is released in the blood plasma from the platelet and endothelial cells to influence thrombosis. The addition of exogenous PDI makes the environment pro-thrombotic by inducing disulfide bond formation in specific plasma protein targets like vitronectin, factor V, and factor XI. However, the mechanistic details of PDI interaction with its target remain largely unknown. A decrease in the coagulation time was detected in activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT) on addition of the purified recombinant PDI (175 nM). The coagulation time can be controlled using an activator (quercetin penta sulfate, QPS) or an inhibitor (quercetin 3-rutinoside, Q3R) of PDI activity. Likewise, the PDI variants that increase the PDI activity (H399R) decrease, and the variant with low activity (C53A) increases the blood coagulation time. An SDS-PAGE and Western blot analysis showed that the PDI does not form a stable complex with either thrombin or antithrombin (ATIII) but it uses the ATIII-thrombin complex as a template to bind and maintain its activity. A complete inhibition of thrombin activity on the formation of ATIII-thrombin-PDI complex, and the complex-bound PDI-catalyzed disulfide bond formation of the target proteins may control the pro- and anti-thrombotic role of PDI.
Asunto(s)
Coagulación Sanguínea , Proteína Disulfuro Isomerasas , Trombina , Humanos , Proteína Disulfuro Isomerasas/metabolismo , Trombina/metabolismo , Antitrombina III/metabolismo , Unión Proteica , Antitrombinas/metabolismo , Antitrombinas/química , Quercetina/farmacología , Quercetina/análogos & derivadosRESUMEN
Antithrombin (AT) is the major plasma inhibitor of thrombin (FIIa) and activated factor X (FXa), and antithrombin deficiency (ATD) is one of the most severe thrombophilic disorders. In this study, we identified nine novel AT mutations and investigated their genotype-phenotype correlations. Clinical and laboratory data from patients were collected, and the nine mutant AT proteins (p.Arg14Lys, p.Cys32Tyr, p.Arg78Gly, p.Met121Arg, p.Leu245Pro, p.Leu270Argfs*14, p.Asn450Ile, p.Gly456delins_Ala_Thr and p.Pro461Thr) were expressed in HEK293 cells; then, Western blotting, N-Glycosidase F digestion, and ELISA were used to detect wild-type and mutant AT. RT-qPCR was performed to determine the expression of AT mRNA from the transfected cells. Functional studies (AT activity in the presence and in the absence of heparin and heparin-binding studies with the surface plasmon resonance method) were carried out. Mutations were also investigated by in silico methods. Type I ATD caused by altered protein synthesis (p.Cys32Tyr, p.Leu270Argfs*14, p.Asn450Ile) or secretion disorder (p.Met121Arg, p.Leu245Pro, p.Gly456delins_Ala_Thr) was proved in six mutants, while type II heparin-binding-site ATD (p.Arg78Gly) and pleiotropic-effect ATD (p.Pro461Thr) were suggested in two mutants. Finally, the pathogenic role of p.Arg14Lys was equivocal. We provided evidence to understand the pathogenic nature of novel SERPINC1 mutations through in vitro expression studies.
Asunto(s)
Deficiencia de Antitrombina III , Antitrombinas , Humanos , Antitrombinas/química , Células HEK293 , Anticoagulantes , Heparina/metabolismo , Mutación , Deficiencia de Antitrombina III/genéticaRESUMEN
Antithrombin is an essential protein that acts as a natural anticoagulant in the human body. It is synthesized by the liver and belongs to the serine protease inhibitors, which are commonly referred to as the SERPINS superfamily. The antithrombin molecule comprises 432 amino acids and has a molecular weight of approximately 58â200âD. It consists of three domains, including an amino-terminal domain, a carbohydrate-rich domain, and a carboxyl-terminal domain. The amino-terminal domain binds with heparin, whereas the carboxyl-terminal domain binds with serine protease. Antithrombin is a crucial natural anticoagulant that contributes approximately 60-80% of plasma anticoagulant activities in the human body. Moreover, antithrombin has anti-inflammatory effects that can be divided into coagulation-dependent and coagulation-independent effects. Furthermore, it exhibits antitumor activity and possesses a broad range of antiviral properties. Inherited type I antithrombin deficiency is a quantitative disorder that is characterized by low antithrombin activity due to low plasma levels. On the other hand, inherited type II antithrombin deficiency is a qualitative disorder that is characterized by defects in the antithrombin molecule. Acquired antithrombin deficiencies are more common than hereditary deficiencies and are associated with various clinical conditions due to reduced synthesis, increased loss, or enhanced consumption. The purpose of this review was to provide an update on the structure, functions, clinical implications, and methods of detection of antithrombin.
Asunto(s)
Deficiencia de Antitrombina III , Antitrombinas , Humanos , Antitrombinas/uso terapéutico , Antitrombinas/química , Antitrombina III , Anticoagulantes , Heparina , Coagulación Sanguínea , Deficiencia de Antitrombina III/tratamiento farmacológicoRESUMEN
Unusual blood clots can cause serious health problems, such as lung embolism, stroke, and heart attack. Inhibiting thrombin activity was adopted as an effective strategy for preventing blood clots. In this study, we explored computational-based method for designing peptide inhibitors of human thrombin therapeutic peptides to prevent platelet aggregation. The random peptides and their 3-dimentional structures were generated to build a virtual peptide library. The generated peptides were docked into the binding pocket of human thrombin. The designed strong binding peptides were aligned with the native binder by comparative study, and we showed the top 5 peptide binders display strong binding affinity against human thrombin. The 5 peptides were synthesized and validated their inhibitory activity. Our result showed the 5-mer peptide AEGYA, EVVNQ, and FASRW with inhibitory activity against thrombin, range from 0.53 to 4.35 µM. In vitro anti-platelet aggregation assay was carried out, suggesting the 3 peptides can inhibit the platelet aggregation induced by thrombin. This study showed computer-aided peptide inhibitor design can be a robust method for finding potential binders for thrombin, which provided solutions for anticoagulation.
Asunto(s)
Antitrombinas , Trombina , Trombosis , Humanos , Anticoagulantes/farmacología , Plaquetas/metabolismo , Péptidos/farmacología , Péptidos/metabolismo , Agregación Plaquetaria , Inhibidores de Agregación Plaquetaria/farmacología , Trombina/metabolismo , Antitrombinas/química , Antitrombinas/farmacologíaRESUMEN
Antithrombin (AT) deficiency increases the risk for venous thromboembolism, therefore, a highly sensitive assay to identify this condition is crucial. The aim of this paper was to perform a meta-analysis comparing AT activities measured by different AT activity assays in patients with heparin binding site AT deficiency. In addition, the diagnostic sensitivity of selected assays was compared depending on the available data. An extensive literature search was performed considering results with publication date up to July 10, 2021. Seven relevant English-language observational studies, comparing AT activity measured by different AT activity assays in Caucasian Europeans with either the AT Budapest III or AT Padua I mutation were included in meta-analyses. There was no significant difference in AT activity between Labexpert and Innovance in patients with AT Budapest III (P = 0.567) and AT Padua I (P = 0.265), while AT activity determined by HemosIL was significantly higher compared to Innovance for both mutations (AT Budapest III: P < 0.001; AT Padua I: P < 0.001). These results are in line with the results of comparison of diagnostic sensitivity. In patients with AT Budapest III, the AT activity was also higher when measured with Berichrom compared to Innovance (P = 0.002), however, the results of comparison of diagnostic sensitivity across studies were variable. No significant difference (P = 0.117) in AT activity as well as diagnostic sensitivity was observed between Sta-Stachrom and Innovance. The results of our study suggest that Innovance, Labexpert and Sta-Stachrom are the most sensitive activity assays for detection of AT Budapest III and AT Padua I, whereas HemosIL showed considerably lower sensitivity for these two variants. As revealed in our study, the diagnostic sensitivity of AT activity assays to type II heparin binding site AT deficiency is different, and in some assays mutation dependent.
Asunto(s)
Deficiencia de Antitrombina III , Heparina , Humanos , Heparina/metabolismo , Anticoagulantes , Pruebas de Coagulación Sanguínea/métodos , Deficiencia de Antitrombina III/diagnóstico , Deficiencia de Antitrombina III/genética , Sitios de Unión , Antitrombinas/químicaRESUMEN
The interaction between coagulation factors Xa and IXa and the activated state of their inhibitor, antithrombin (AT),have been investigated using X-ray diffraction studies. However, only mutagenesis data are available for non-activated AT. Our aim was to propose a model based on docking and advanced-sampling molecular dynamics simulations that can reveal the conformational behavior of the systems when AT is not binding a pentasaccharide. We built the initial structure for non-activated AT-FXa and AT-FIXa complexes using HADDOCK 2.4. The conformational behavior was studied using Gaussian accelerated molecular dynamics simulations. In addition to the docked complexes, two systems based on the X-ray structures were also simulated, with and without the ligand. The simulations revealed large variability in conformation for both factors. In the docking-based complex of AT-FIXa, conformations with stable Arg150-AT interactions can exist for longer time periods but the system also has a higher tendency for reaching states with very limited interaction with the "exosite" of AT. By comparing simulations with or without the pentasaccharide, we were able to gain insights into the effects of conformational activation on the Michaelis complexes. RMSF analysis and correlation calculations for the alpha-carbon atoms revealed important details of the allosteric mechanisms. Our simulations provide atomistic models for better understanding the conformational activation mechanism of AT against its target factors.
Asunto(s)
Antitrombinas , Factor Xa , Antitrombinas/química , Antitrombinas/metabolismo , Factor Xa/química , Factor Xa/metabolismo , Heparina/química , Simulación de Dinámica Molecular , Conformación Proteica , Antitrombina III/química , Oligosacáridos , CinéticaRESUMEN
Antithrombin is a key protein of the coagulation system belonging to the serine protease inhibitor family. Antithrombin preparations are used as a therapeutic treatment for patients with decreased antithrombin activity. Elucidating the structural features of this protein is an important part of the control strategy to assure a high quality. This study presents an ion exchange chromatographic method coupled to mass spectrometry capable of characterizing antithrombin post-translational modifications such as N-glycosylation, phosphorylation or deamidation. Furthermore, the method was successfully used to evidence irreversible/inactive conformers of antithrombin which are commonly observed for serine protease inhibitors and referred to as latent forms.
Asunto(s)
Antitrombinas , Inhibidores de Serina Proteinasa , Humanos , Antitrombinas/química , Antitrombinas/metabolismo , Isoformas de Proteínas , Espectrometría de Masas/métodos , Cromatografía por Intercambio Iónico/métodosRESUMEN
Biomaterials used for blood contacting devices are inherently thrombogenic. Antithrombotic agents can be used as surface modifiers on biomaterials to reduce thrombus formation on the surface and to maintain device efficacy. For quality control and to assess the effectiveness of immobilization strategies, it is necessary to quantify the surface-immobilized antithrombotic agent directly. There are limited methods that allow direct quantification on device surfaces such as catheters. In this study, an enzyme immunoassay (EIA) has been developed to measure the density of a synthetic antithrombin-heparin (ATH) covalent complex immobilized on a catheter surface. The distribution of the immobilized ATH was further characterized by an immunohistochemical assay. This analyte-specific EIA is relatively simple and has high throughput, thus providing a tool for quantitative analysis of biomaterial surface modifications. These methods may be further modified to evaluate plasma proteins adsorbed and immobilized on various biomaterial surfaces of complex shapes, with a range of bioactive functionalities, as well as to assess conformational changes of proteins using specific antibodies.
Asunto(s)
Heparina , Proteínas de la Membrana , Antitrombinas/química , Materiales Biocompatibles , Fibrinolíticos , Heparina/química , Proteínas Inmovilizadas , Propiedades de SuperficieRESUMEN
In this study; a spectrum-effect relationship analysis combined with a high-performance liquid chromatography-mass spectrometry (LC-MS) analysis was established to screen and identify active components that can inhibit thrombin and factor Xa (THR and FXa) in Salviae Miltiorrhizae Radix et Rhizoma-Chuanxiong Rhizoma (Danshen-Chuanxiong) herbal pair. Ten potential active compounds were predicted through a canonical correlation analysis (CCA), and eight of them were tentatively identified through an LC-MS analysis. Furthermore; the enzyme inhibitory activity of six available compounds; chlorogenic acid; Z-ligustilide; caffeic acid; ferulic acid; tanshinone I and tanshinone IIA; were tested to verify the feasibility of the method. Among them; chlorogenic acid was validated to possess a good THR inhibitory activity with IC50 of 185.08 µM. Tanshinone I and tanshinone IIA are potential FXa inhibitors with IC50 of 112.59 µM and 138.19 µM; respectively. Meanwhile; molecular docking results show that tanshinone I and tanshinone IIA; which both have binding energies of less than -7.0 kcal·mol-1; can interact with FXa by forming H-bonds with residues of SER214; GLY219 and GLN192. In short; the THR and FXa inhibitors in the Danshen-Chuanxiong herbal pair have been successfully characterized through a spectrum-effect relationship analysis and an LC-MS analysis.
Asunto(s)
Antitrombinas/farmacología , Medicamentos Herbarios Chinos/farmacología , Inhibidores del Factor Xa/farmacología , Trombina/antagonistas & inhibidores , Antitrombinas/química , Evaluación Preclínica de Medicamentos , Medicamentos Herbarios Chinos/química , Inhibidores del Factor Xa/química , Humanos , Simulación del Acoplamiento Molecular , Salvia miltiorrhiza/químicaRESUMEN
Tyrosine sulfation is an important post-translational modification of peptides and proteins which underpins and modulates many protein-protein interactions. In order to overcome the inherent instability of the native modification, we report the synthesis of two sulfonate analogues and their incorporation into two thrombin-inhibiting sulfopeptides. The effective mimicry of these sulfonate analogues for native sulfotyrosine was validated in the context of their thrombin inhibitory activity and binding mode, as determined by X-ray crystallography.
Asunto(s)
Antitrombinas/química , Péptidos/química , Trombina/antagonistas & inhibidores , Tirosina/análogos & derivados , Antitrombinas/síntesis química , Antitrombinas/metabolismo , Cristalografía por Rayos X , Pruebas de Enzimas , Humanos , Péptidos/síntesis química , Péptidos/metabolismo , Unión Proteica , Trombina/metabolismo , Tirosina/químicaRESUMEN
The salivary glands of the flea Xenopsylla cheopis, a vector of the plague bacterium, Yersinia pestis, express proteins and peptides thought to target the hemostatic and inflammatory systems of its mammalian hosts. Past transcriptomic analyses of salivary gland tissue revealed the presence of two similar peptides (XC-42 and XC-43) having no extensive similarities to any other deposited sequences. Here we show that these peptides specifically inhibit coagulation of plasma and the amidolytic activity of α-thrombin. XC-43, the smaller of the two peptides, is a fast, tight-binding inhibitor of thrombin with a dissociation constant of less than 10 pM. XC-42 exhibits similar selectivity as well as kinetic and binding properties. The crystal structure of XC-43 in complex with thrombin shows that despite its substrate-like binding mode, XC-43 is not detectably cleaved by thrombin and that it interacts with the thrombin surface from the enzyme catalytic site through the fibrinogen-binding exosite I. The low rate of hydrolysis was verified in solution experiments with XC-43, which show the substrate to be largely intact after 2 h of incubation with thrombin at 37 °C. The low rate of XC-43 cleavage by thrombin may be attributable to specific changes in the catalytic triad observable in the crystal structure of the complex or to extensive interactions in the prime sites that may stabilize the binding of cleavage products. Based on the increased arterial occlusion time, tail bleeding time, and blood coagulation parameters in rat models of thrombosis XC-43 could be valuable as an anticoagulant.
Asunto(s)
Anticoagulantes/química , Antitrombinas/química , Proteínas de Insectos/química , Glándulas Salivales/química , Proteínas y Péptidos Salivales/química , Trombina , Xenopsylla/química , Animales , Humanos , Ratas , Trombina/antagonistas & inhibidores , Trombina/química , Xenopsylla/metabolismoRESUMEN
Cardiovascular disease (CVD) poses serious health concerns worldwide. The lack of transplantable vascular grafts is an unmet clinical need in the surgical treatment of CVD. Although expanded polytetrafluoroethylene (ePTFE) vascular grafts have been used in clinical practice, a low long-term patency rate in small-diameter transplantation application is still the biggest challenge. Thus, surface modification of ePTFE is sought after. In this study, polydopamine (PDA) was used to improve the hydrophilia and provide immobilization sites in ePTFE. Bivalirudin (BVLD), a direct thrombin inhibitor, was used to enhance the anti-thrombotic activity of ePTFE. The peptides derived from extracellular matrix proteins were used to elevate the bioactivity of ePTFE. The morphology, chemical composition, peptide modified strength, wettability, and hemocompatibility of modified ePTFE vascular grafts were investigated. Then, an endothelial cell proliferation assay was used to evaluate the best co-modification strategy of the ePTFE vascular graft in vitro. Since a large animal could relatively better mimic human physiology, we chose a porcine carotid artery replacement model in the current study. The results showed that the BVLD/REDV co-modified ePTFE vascular grafts had a satisfactory patency rate (66.7%) and a higher endothelial cell coverage ratio (70%) at 12 weeks after implantation. This may offer an opportunity to produce a multi-biofunctional ePTFE vascular graft, thereby yielding a potent product to meet the clinical needs.
Asunto(s)
Prótesis Vascular , Materiales Biocompatibles Revestidos/química , Politetrafluoroetileno/química , Animales , Antitrombinas/química , Antitrombinas/uso terapéutico , Arterias Carótidas/patología , Arterias Carótidas/cirugía , Traumatismos de las Arterias Carótidas/terapia , Proliferación Celular/efectos de los fármacos , Materiales Biocompatibles Revestidos/farmacología , Materiales Biocompatibles Revestidos/uso terapéutico , Modelos Animales de Enfermedad , Células Endoteliales/citología , Células Endoteliales/metabolismo , Hirudinas/química , Indoles/química , Masculino , Fragmentos de Péptidos/química , Fragmentos de Péptidos/uso terapéutico , Polímeros/química , Proteínas Recombinantes/química , Proteínas Recombinantes/uso terapéutico , Porcinos , Porcinos Enanos , Trombosis/tratamiento farmacológico , HumectabilidadRESUMEN
Aberrant protein citrullination is associated with many pathologies; however, the specific effects of this modification remain unknown. We have previously demonstrated that serine protease inhibitors (SERPINs) are highly citrullinated in rheumatoid arthritis (RA) patients. These citrullinated SERPINs include antithrombin, antiplasmin, and t-PAI, which regulate the coagulation and fibrinolysis cascades. Notably, citrullination eliminates their inhibitory activity. Here, we demonstrate that citrullination of antithrombin and t-PAI impairs their binding to their cognate proteases. By contrast, citrullination converts antiplasmin into a substrate. We recapitulate the effects of SERPIN citrullination using in vitro plasma clotting and fibrinolysis assays. Moreover, we show that citrullinated antithrombin and antiplasmin are increased and decreased in a deep vein thrombosis (DVT) model, accounting for how SERPIN citrullination shifts the equilibrium toward thrombus formation. These data provide a direct link between increased citrullination and the risk of thrombosis in autoimmunity and indicate that aberrant SERPIN citrullination promotes pathological thrombus formation.
Asunto(s)
Antifibrinolíticos/farmacología , Antitrombinas/farmacología , Inactivadores Plasminogénicos/farmacología , Inhibidores de Serina Proteinasa/farmacología , Trombosis de la Vena/tratamiento farmacológico , Animales , Antifibrinolíticos/química , Antitrombinas/química , Modelos Animales de Enfermedad , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Péptido Hidrolasas/metabolismo , Inactivadores Plasminogénicos/química , Inhibidores de Serina Proteinasa/química , Trombosis de la Vena/metabolismoRESUMEN
Antithrombin is unique among serpin family protein protease inhibitors with respect to the major reactive center loop (RCL) and core conformational changes that mediate allosteric activation of its anticoagulant function by heparin. A critical role for expulsion of the RCL hinge from a native stabilizing interaction with the hydrophobic core in the activation mechanism has been proposed from reports that antithrombin variants that block this change through engineered disulfide bonds block activation. However, the sufficiency of core conformational changes for activation without expulsion of the RCL from the core is suggested by variants that are activated without the need for heparin and retain the native RCL-core interaction. To resolve these apparently conflicting findings, we engineered variants in which disulfides designed to block the RCL conformational change were combined with constitutively activating mutations. Our findings demonstrate that while a reversible constitutive activation can be engineered in variants that retain the native RCL-core interaction, engineered disulfides that lock the RCL native conformation can also block heparin allosteric activation. Such findings support a three-state allosteric activation model in which constitutive activating mutations stabilize an intermediate-activated state wherein core conformational changes and a major activation have occurred without the release of the RCL from the core but with a necessary repositioning of the RCL to allow productive engagement with an exosite. Rigid disulfide bonds that lock the RCL native conformation block heparin activation by preventing both RCL repositioning in the intermediate-activated state and the release of the RCL from the core in the fully activated state.
Asunto(s)
Antitrombinas/química , Antitrombinas/metabolismo , Heparina/metabolismo , Regulación Alostérica , Humanos , Cinética , Modelos Moleculares , Mutación , Unión Proteica , Conformación ProteicaRESUMEN
The exposure-response relationship of direct acting oral anti-coagulants (DOACs) for bleeding risk is steep relative to ischemic stroke reduction. As a result, small changes in exposure may lead to bleeding events. The overall goal of this project was to determine the effect of critical formulation parameters on the pharmacokinetics (PKs) and thus safety and efficacy of generic DOACs. In this first installment of our overall finding, we developed and verified a physiologically-based PK (PBPK) model for dabigatran etexilate (DABE) and its metabolites. The model was developed following a middle out approach leveraging available in vitro and in vivo data. External validity of the model was confirmed by overlapping predicted and observed PK profiles for DABE as well as free and total dabigatran for a dataset not used during model development. The verified model was applied to interrogate the impact of modulating the microenvironment pH on DABE systemic exposure. The PBPK exploratory analyses highlighted the high sensitivity of DABE exposure to supersaturation ratio and precipitation kinetics.