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1.
Int J Mol Sci ; 22(23)2021 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-34884431

RESUMO

Ticks, lice, flees, mosquitos, leeches and vampire bats need to prevent the host's blood coagulation during their feeding process. This is primarily achieved by injecting potent anticoagulant proteins. Basophils frequently accumulate at the site of tick feeding. However, this occurs only after the second encounter with the parasite involving an adaptive immune response and IgE. To study the potential role of basophils and mast cells in the defense against ticks and other ectoparasites, we produced anticoagulant proteins from three blood-feeding animals; tick, mosquito, and leech. We tested these anticoagulant proteins for their sensitivity to inactivation by a panel of hematopoietic serine proteases. The majority of the connective tissue mast cell proteases tested, originating from humans, dogs, rats, hamsters, and opossums, efficiently cleaved these anticoagulant proteins. Interestingly, the mucosal mast cell proteases that contain closely similar cleavage specificity, had little effect on these anticoagulant proteins. Ticks have been shown to produce serpins, serine protease inhibitors, upon a blood meal that efficiently inhibit the human mast cell chymase and cathepsin G, indicating that ticks have developed a strategy to inactivate these proteases. We show here that one of these tick serpins (IRS-2) shows broad activity against the majority of the mast cell chymotryptic enzymes and the neutrophil proteases from human to opossum. However, it had no effect on the mast cell tryptases or the basophil specific protease mMCP-8. The production of anticoagulants, proteases and anti-proteases by the parasite and the host presents a fascinating example of an arms race between the blood-feeding animals and the mammalian immune system with an apparent and potent role of the connective tissue mast cell chymases in the host defense.


Assuntos
Proteínas Antitrombina/química , Basófilos/enzimologia , Quimases/metabolismo , Mastócitos/enzimologia , Parasitos/metabolismo , Imunidade Adaptativa , Animais , Quimiocina CCL19/química , Culicidae/metabolismo , Humanos , Imunoglobulina E/metabolismo , Sanguessugas/metabolismo , Camundongos , Proteólise , Proteínas Proto-Oncogênicas c-sis/química , Carrapatos/metabolismo
2.
Int J Mol Sci ; 22(2)2021 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-33419227

RESUMO

N-linked glycosylation is a crucial post-translational modification involved in protein folding, function, and clearance. N-linked glycosylation is also used therapeutically to enhance the half-lives of many proteins. Antithrombin, a serpin with four potential N-glycosylation sites, plays a pivotal role in hemostasis, wherein its deficiency significantly increases thrombotic risk. In this study, we used the introduction of N-glycosylation sites as a tool to explore what effect this glycosylation has on the protein folding, secretion, and function of this key anticoagulant. To accomplish this task, we introduced an additional N-glycosylation sequence in each strand. Interestingly, all regions that likely fold rapidly or were surrounded by lysines were not glycosylated even though an N-glycosylation sequon was present. The new sequon in the strands of the A- and B-sheets reduced secretion, and the B-sheet was more sensitive to these changes. However, the mutations in the strands of the C-sheet allowed correct folding and secretion, which resulted in functional variants. Therefore, our study revealed crucial regions for antithrombin secretion and could potentially apply to all serpins. These results could also help us understand the functional effects of natural variants causing type-I deficiencies.


Assuntos
Proteínas Antitrombina/química , Proteínas Antitrombina/metabolismo , Conformação Proteica , Processamento de Proteína Pós-Traducional , Antitrombina III/química , Antitrombina III/genética , Antitrombina III/metabolismo , Proteínas Antitrombina/genética , Dicroísmo Circular , Glicosilação , Humanos , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação , Trombose
3.
Cell Chem Biol ; 28(1): 26-33.e8, 2021 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-33096052

RESUMO

Despite possessing only 32 residues, the tsetse thrombin inhibitor (TTI) is among the most potent anticoagulants described, with sub-picomolar inhibitory activity against thrombin. Unexpectedly, TTI isolated from the fly is 2000-fold more active and 180 Da heavier than synthetic and recombinant variants. We predicted the presence of a tyrosine O-sulfate post-translational modification of TTI, prompting us to investigate the effect of the modification on anticoagulant activity. A combination of chemical synthesis and functional assays was used to reveal that sulfation significantly improved the inhibitory activity of TTI against thrombin. Using X-ray crystallography, we show that the N-terminal sulfated segment of TTI binds the basic exosite II of thrombin, establishing interactions similar to those of physiologic substrates, while the C-terminal segment abolishes the catalytic activity of thrombin. This non-canonical mode of inhibition, coupled with its potency and small size, makes TTI an attractive scaffold for the design of novel antithrombotics.


Assuntos
Anticoagulantes/farmacologia , Proteínas Antitrombina/farmacologia , Proteínas de Insetos/farmacologia , Trombina/antagonistas & inibidores , Tirosina/análogos & derivados , Animais , Anticoagulantes/síntese química , Anticoagulantes/química , Proteínas Antitrombina/síntese química , Proteínas Antitrombina/química , Linhagem Celular , Humanos , Proteínas de Insetos/síntese química , Proteínas de Insetos/química , Estrutura Molecular , Trombina/metabolismo , Moscas Tsé-Tsé , Tirosina/síntese química , Tirosina/química , Tirosina/farmacologia
4.
Biotechnol Lett ; 42(1): 103-114, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31686286

RESUMO

OBJECTIVE: Thrombin, platelets, and plasmin are three key factors involved in hemostasis and thrombolysis. Thrombolytic therapy with clinically approved drugs is often followed by recurrent thrombosis caused by thrombin-induced platelet aggregation from the clot debris. In order to minimize these problems, new constructs were designed for the expression of recombinant staphylokinase (rSAK) and also a fusion protein composed of staphylokinase, 20 amino acids containing 2 RGD followed by tsetse thrombin Inhibitor (SAK-2RGD-TTI) in Pichia pastoris. RESULT: Modeling the tertiary structure of SAK-2RGD-TTI showed that the linker containing RGD and TTI did not interfere with proper folding of SAK. In laboratory testing, the purified SAK-2RGD-TTI (420 µg/mL) dissolved an average of 45% of the blood clot. The activity of the SAK-2RGD-TTI was also confirmed in various tests including human plasminogen activation assay, fibrin clot lysis assay, well diffusion method, activated partial thromboplastin time and platelet rich clot lysis assay. CONCLUSION: Our findings suggest that SAK-2RGD-TTI has improved therapeutic properties preventing reocclussion. It further confirms that it is practicable to assemble and produce a hybrid multifunctional protein that targets hemostatic process at various stages.


Assuntos
Metaloendopeptidases/metabolismo , Pichia/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Terapia Trombolítica/métodos , Proteínas Antitrombina/química , Proteínas Antitrombina/genética , Proteínas Antitrombina/metabolismo , Humanos , Proteínas de Insetos/química , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Metaloendopeptidases/química , Metaloendopeptidases/genética , Simulação de Dinâmica Molecular , Oligopeptídeos/química , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Pichia/genética , Conformação Proteica , Proteínas Recombinantes de Fusão/genética
5.
Biotechnol Prog ; 35(4): e2819, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30972956

RESUMO

Staphylokinase (SAK) is a promising thrombolytic agent for the treatment of patients suffering from blood-clotting disorders. To increase the potency of SAK and to minimize vessel reocclusion, a new construct bearing SAK motif fused to tsetse thrombin inhibitor (TTI) via a 20-amino acid linker with 2 RGD (2 × arginine-glycine-aspartic acid inhibiting platelet aggregation via attachment to integrin receptors of platelet) was codon optimized and expressed comparatively in Pichia pastoris GS115 as a Mut+ strain and KM71H as a Muts strain. Fusion protein was optimized in terms of best expression condition and fibrinolytic activity and compared with the rSAK. Expression level of the designed construct reached up to 175 mg/L of the culture medium after 72-hr stimulation with 2.5% methanol and remained steady for 3-4 days. The highest expression was obtained at the range of 2-3% methanol. The SAK-2RGD-TT (relative activity >82%) was more active at 25-37 °C than rSAK (relative activity of 93%). Further, it showed relative activity >80% at pH ranges of 7-9. Western blot analysis showed two bands of nearly 27 and 24 kDa at ratio of 5 to 3, respectively. The specific fibrinolytic activity of the SAK-2RGD-TTI was measured as 8,269 U/mg, and 19,616 U/mg for the nonpurified and purified proteins, respectively. Deglycosylation by using tunicamycin in culture medium resulted in higher fibrinolytic activity of SAK-2RGD-TTI (2.2 fold). Consequently, compared to the rSAK, at the same equimolar proportion, addition of RGD and TTI fragments could increase fibrinolytic activity. Also, P. pastoris can be considered as an efficient host for overexpression of the soluble SAK-2RGD-TTI with high activity without requiring a complicated purification procedure.


Assuntos
Proteínas Antitrombina/farmacologia , Fibrinolíticos/farmacologia , Proteínas de Insetos/farmacologia , Metaloendopeptidases/metabolismo , Inibidores da Agregação Plaquetária/farmacologia , Proteínas Antitrombina/química , Fibrinolíticos/química , Humanos , Concentração de Íons de Hidrogênio , Proteínas de Insetos/química , Metaloendopeptidases/química , Metaloendopeptidases/genética , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/química , Temperatura
6.
J Biol Chem ; 292(40): 16513-16520, 2017 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-28743742

RESUMO

Antithrombin mainly inhibits factor Xa and thrombin. The reactive center loop (RCL) is crucial for its interactions with its protease targets and is fully inserted into the A-sheet after its cleavage, causing translocation of the covalently linked protease to the opposite end of the A-sheet. Antithrombin variants with altered RCL hinge residues behave as substrates rather than inhibitors, resulting in stoichiometries of inhibition greater than one. Other antithrombin residues have been suggested to interfere with RCL insertion or the stability of the antithrombin-protease complex, but available crystal structures or mutagenesis studies have failed to identify such residues. Here, we characterized two mutations, S365L and I207T, present in individuals with type II antithrombin deficiency and identified a new antithrombin functional domain. S365L did not form stable complexes with thrombin or factor Xa, and the I207T/I207A variants inhibited both proteases with elevated stoichiometries of inhibition. Close proximity of Ile-207 and Ser-365 to the inserted RCL suggested that the preferred reaction of these mutants as protease substrates reflects an effect on the rate of the RCL insertion and protease translocation. However, both residues lie within the final docking site for the protease in the antithrombin-protease complex, supporting the idea that the enhanced substrate reactions may result from an increased dissociation of the final complexes. Our findings demonstrate that the distal end of the antithrombin A-sheet is crucial for the last steps of protease inhibition either by affecting the rate of RCL insertion or through critical interactions with proteases at the end of the A-sheet.


Assuntos
Proteínas Antitrombina/química , Transtornos Herdados da Coagulação Sanguínea , Fator Xa/química , Simulação de Acoplamento Molecular , Trombina/química , Substituição de Aminoácidos , Proteínas Antitrombina/genética , Proteínas Antitrombina/metabolismo , Domínio Catalítico , Fator Xa/genética , Fator Xa/metabolismo , Feminino , Humanos , Masculino , Mutação de Sentido Incorreto , Domínios Proteicos , Estrutura Secundária de Proteína , Trombina/genética , Trombina/metabolismo
7.
Bull Exp Biol Med ; 162(6): 718-721, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28429220

RESUMO

Coagulation and optical (based on chromogenic substrate) methods were employed to examine antithrombin activity of erythrocytes and erythrocyte-derived microvesicles isolated days 7, 14, 21, and 28 on erythrocyte storage. The erythrocyte-derived microvesicles decelerated fibrin clot formation from fibrinogen in the presence of exogenous thrombin both with and without heparin. Microvesicles reduced optical density of chromogenic substrate. These data suggest that erythrocyte-derived microvesicles display a prominent antithrombin activity, which significantly increases during erythrocyte storage.


Assuntos
Proteínas Antitrombina/química , Preservação de Sangue/métodos , Micropartículas Derivadas de Células/química , Eritrócitos/química , Fibrinogênio/química , Trombina/química , Adenina/química , Coagulação Sanguínea , Testes de Coagulação Sanguínea , Células Cultivadas , Compostos Cromogênicos/análise , Citratos/química , Eritrócitos/citologia , Fibrina/química , Glucose/química , Heparina/química , Humanos , Fosfatos/química , Refrigeração/métodos , Espectrofotometria
8.
Anal Chim Acta ; 947: 58-65, 2016 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-27846990

RESUMO

Antithrombin (AT) is a plasma glycoprotein which possesses anticoagulant and anti-inflammatory properties. AT exhibits various forms, among which are native, latent and heterodimeric ones. We studied the potential of capillary electrophoresis-mass spectrometry (CE-MS) using a sheath liquid interface, electrospray ionization (ESI), and a quadrupole-time-of-flight (Q-TOF) mass spectrometer to separate and quantify the different AT forms. For CE separation, a neutral polyvinyl alcohol (PVA) coated capillary was employed. The protein conformation was preserved by using a background electrolyte (BGE) at physiological pH. A sheath liquid of isopropanol-water 50:50 (v/v) with 14 mM ammonium acetate delivered at a flow rate of 120 µL h-1 resulted in optimal signal intensities. Each AT form exhibited a specific mass spectrum, allowing unambiguous distinction. Several co-injection experiments proved that latent AT had a higher electrophoretic mobility (µep) than native AT, and that these conformers could associate to form a heterodimer during the CE analysis. The developed CE-MS method enabled the detection and quantitation of latent and heterodimeric forms in a commercial AT preparation stored at room temperature for three weeks.


Assuntos
Proteínas Antitrombina/química , Eletroforese Capilar/métodos , Espectrometria de Massas/métodos , Multimerização Proteica , Proteínas Antitrombina/isolamento & purificação , Modelos Moleculares , Estrutura Quaternária de Proteína , Temperatura
9.
Glycobiology ; 26(5): 482-92, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26747427

RESUMO

The structure of the N-linked oligosaccharides attached to antithrombin (AT) has been shown to affect its anticoagulant activity and pharmacokinetics. Human AT has biantennary complex-type oligosaccharides with the unique feature of lacking a core fucose, which affects its biological activities by changing its heparin-binding affinity. In human plasma, AT circulates as a mixture of the α-form bearing four oligosaccharides and the ß-form lacking an oligosaccharide at Asn135. However, it remains unclear how the immature high-mannose-type oligosaccharides produced by mammalian cells affect biological activities of AT. Here, we succeeded in directly comparing the activities between the high-mannose and complex types. Interestingly, although there were no substantial differences in thrombin inhibitory activity, the high-mannose type showed higher heparin-binding affinity. The anticoagulant activities were increased by heparin and correlated with the heparin-binding affinity, resulting in the strongest anticoagulant activity being displayed in the ß-form with the high-mannose type. In pharmacokinetic profiling, the high-mannose type showed a much shorter plasma half-life than the complex type. The ß-form was found to have a prolonged plasma half-life compared with the α-form for the high-mannose type; conversely, the α-form showed a longer half-life than the ß-form for the complex-type. The present study highlights that AT physiological activities are strictly controlled not only by a core fucose at the reducing end but also by the high-mannose-type structures at the nonreducing end. The ß-form with the immature high-mannose type appears to function as a more potent anticoagulant than the AT typically found in human plasma, once it emerges in the blood.


Assuntos
Proteínas Antitrombina/metabolismo , Heparina/metabolismo , Manose/metabolismo , Oligossacarídeos/metabolismo , Proteínas Antitrombina/química , Glicosilação , Heparina/química , Humanos , Manose/química , Oligossacarídeos/química , Ligação Proteica
10.
Biomed Res Int ; 2015: 630482, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25866798

RESUMO

Physiological hemostatic balance is a coordinated outcome of counteracting coagulation and fibrinolytic systems. An imbalance of procoagulant and anticoagulant factors may result in life threatening thromboembolism. Presently, anticoagulant administration is the first line of therapy for the treatment of these conditions and several anticoagulants have been approved, including various forms of heparin. However, the polyanionic nature and multispecificity of heparin pose several complications. Generally, the polysulfated compounds with antithrombotic potential are thought to have feasible synthetic procedures with much less bleeding, thus having favourable safety profiles. Here we report the synthesis of a novel compound, trehalose octasulfate and the assessment of its anticoagulation potential. Molecular docking of trehalose and trehalose octasulfate with antithrombin showed a specificity switch in binding affinity on sulfation, where trehalose octasulfate interacts with critical residues of AT that are either directly involved in heparin binding or in the conformational rearrangement of AT on heparin binding. An in vitro analysis of trehalose octasulfate demonstrated prolonged clotting time. Lead compound when intravenously injected in occlusion induced thrombotic rats showed remarkable reduction in the size and weight of the clot at a low dose. Delay in coagulation time was observed by analysing blood plasma isolated from rats preinjected with trehalose octasulfate. A decrease in Adenosine 5'-Diphosphate (ADP) induced platelet aggregation indicated a probable dual anticoagulant and antiplatelet mechanism of action. To summarize, this study presents trehalose octasulfate as a novel, effective, dual acting antithrombotic agent.


Assuntos
Anticoagulantes , Proteínas Antitrombina/química , Inibidores da Agregação Plaquetária , Ésteres do Ácido Sulfúrico , Trealose , Animais , Feminino , Masculino , Simulação de Acoplamento Molecular , Inibidores da Agregação Plaquetária/química , Inibidores da Agregação Plaquetária/farmacologia , Ratos , Ratos Sprague-Dawley , Ésteres do Ácido Sulfúrico/química , Ésteres do Ácido Sulfúrico/farmacologia , Trombose/tratamento farmacológico , Trealose/química , Trealose/farmacologia
11.
Protein Pept Lett ; 22(5): 410-8, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25687119

RESUMO

Antithrombin inhibits blood coagulation through the interaction with serine proteases in both intrinsic and extrinsic pathways. In addition, antithrombin also shows anti-inflammatory properties, which are independent of its effects on coagulation. This work shows for the first time the cloning and sequencing of antithrombin from a snake species. This predicted protein is composed by 430 amino acids and presents about 64.5% sequence identity to human antithrombin. Biacore experiments revealed that the binding affinity of Bothrops jararaca snake antithrombin to heparin was ~30 times higher than that of human antithrombin. Furthermore, Bothrops jararaca antithrombin is more effective in preventing acute inflammation induced by carrageenan when compared to human antithrombin. Hence, the results showed herein suggest that Bothrops jararaca antithrombin can play a key role in the control of acute inflammation and that this molecule might be used as a pharmacological tool and as a prototype for drug development.


Assuntos
Anti-Inflamatórios/uso terapêutico , Proteínas Antitrombina/uso terapêutico , Bothrops/genética , Inflamação/tratamento farmacológico , Proteínas de Répteis/uso terapêutico , Sequência de Aminoácidos , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/metabolismo , Proteínas Antitrombina/química , Proteínas Antitrombina/genética , Carragenina , Clonagem Molecular , Edema/induzido quimicamente , Edema/tratamento farmacológico , Humanos , Inflamação/induzido quimicamente , Masculino , Camundongos , Dados de Sequência Molecular , Proteínas de Répteis/química , Proteínas de Répteis/genética , Alinhamento de Sequência
12.
Protein Pept Lett ; 20(4): 403-11, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23016581

RESUMO

Endogenous angiogenesis inhibitor that specifically decreases tumor cell proliferation can be used to treat cancer since angiogenesis is required at every step of tumor progression and metastasis. Endothelial cells are the main target for the antiangiogenic therapy because they are non-transformed and easily accessible to angiogenic inhibitors. Antithrombin functions as a principal plasma protein inhibitor of blood coagulation proteinases and belongs to the family of serine protease inhibitors (serpins) which have common mechanism of inhibition. Antithrombin acquires a potent antiangiogenic activity upon conversion of the native molecule to cleaved or latent conformation. Cleaved and latent preparations of bovine and human plasma derived antithrombin inhibits capillary endothelial cell proliferation and the growth of human SK-NAS neuroblastoma and Lewis lung carcinoma tumors in mice but not the native antithrombin's. The native form of antithrombin binds with high affinity to vascular heparan sulfate proteoglycans containing a specific pentasaccharide sequence and it is this cofactor interaction that activates antithrombin to maximal rate of thrombin inhibition. Upon inhibitory complex formation with target proteinases the antithrombin undergoes stressed to relaxed transformation and lose their high affinity for pentasacchride. Low affinity relaxed conformation with reduced heparin binding like cleaved and latent are antiangiogenic but native high affinity heparin binding stressed conformation is not, indicating the critical importance of heparin affinity in antithrombin antiangiogenic function. Based on evidence of interactions of the endothelial cell growth factors bFGF (basic fibroblast growth factor) and VEGF (vascular endothelial cell growth factor) with heparin like molecule in matrix, the possibility of antiangiogenic antithrombin to interfere with endothelial cell growth and angiogenesis through heparin mediated mechanism deserves serious consideration and investigation. It is also possible that cleaved and latent conformations with reduced affinity for heparins can also induce conformational change in the antithrombin which can open an epitope on the antithrombin surface for appropriate interactions on the endothelial surface for better antiangiogenic activity. This review illustrates the potential of antithrombin and other serpin family members as endogenous antiangiogenic proteins.


Assuntos
Inibidores da Angiogênese/farmacologia , Proteínas Antitrombina/química , Proteínas Antitrombina/metabolismo , Antitrombinas/farmacologia , Animais , Antitrombinas/química , Antitrombinas/metabolismo , Bovinos , Proliferação de Células/efeitos dos fármacos , Células Endoteliais , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/patologia , Fator 2 de Crescimento de Fibroblastos/metabolismo , Heparina/metabolismo , Humanos , Conformação Proteica , Inibidores de Serina Proteinase/farmacologia , Serpinas/química , Serpinas/farmacologia , Relação Estrutura-Atividade
13.
J Biomol Struct Dyn ; 30(6): 684-700, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22812415

RESUMO

The comparison between two protein structures is important for understanding a molecular function. In particular, the comparison of protein surfaces to measure their similarity provides another challenge useful for studying molecular evolution, docking, and drug design. This paper presents an algorithm, called the BetaSuperposer, which evaluates the similarity between the surfaces of two structures using the beta-shape which is a geometric structure derived from the Voronoi diagram of molecule. The algorithm performs iterations of mix-and-match between the beta-shapes of two structures for the optimal superposition from which a similarity measure is computed, where each mix-and-match step attempts to solve an NP-hard problem. The devised heuristic algorithm based on the assignment problem formulation quickly produces a good superposition and an assessment of similarity. The BetaSuperposer was fully implemented and benchmarked against popular programs, the Dali and the Click, using the SCOP models. The BetaSuperposer is freely available to the public from the Voronoi Diagram Research Center ( http://voronoi.hanyang.ac.kr ).


Assuntos
Algoritmos , Modelos Moleculares , Software , Sequência de Aminoácidos , Proteínas Antitrombina/química , Humanos , Dados de Sequência Molecular , Dinâmica não Linear , Estrutura Terciária de Proteína , Análise de Regressão , Homologia Estrutural de Proteína , Propriedades de Superfície
14.
Mol Med ; 18: 762-70, 2012 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-22481271

RESUMO

Mutations affecting mobile domains of antithrombin induce conformational instability resulting in protein polymerization that associates with a severe clinical phenotype, probably by an unknown gain of function. By homology with other conformational diseases, we speculated that these variants might infect wild-type (WT) monomers reducing the anticoagulant capacity. Infective polymerization of WT polymers and different P1 mutants (p.R425del, p.R425C and p.R425H) were evaluated by using native gels and radiolabeled WT monomers and functional assays. Human embryonic kidney cells expressing the Epstein-Barr nuclear antigen 1 (HEK-EBNA) cells expressing inducible (p.R425del) or two novel constitutive (p.F271S and p.M370T) conformational variants were used to evaluate intracellular and secreted antithrombin under mild stress (pH 6.5 and 39°C for 5 h). We demonstrated the conformational sensitivity of antithrombin London (p.R425del) to form polymers under mild heating. Under these conditions purified antithrombin London recruited WT monomers into growing polymers, reducing the anticoagulant activity. This process was also observed in the plasma of patients with p.R425del, p.R425C and p.R425H mutations. Under moderate stress, coexpression of WT and conformational variants in HEK-EBNA cells increased the intracellular retention of antithrombin and the formation of disulfide-linked polymers, which correlated with impaired secretion and reduction of anticoagulant activity in the medium. Therefore, mutations inducing conformational instability in antithrombin allow its polymerization with the subsequent loss of function, which under stress could sequestrate WT monomers, resulting in a new prothrombotic gain of function, particularly relevant for intracellular antithrombin. The in vitro results suggest a temporal and severe plasma antithrombin deficiency that may contribute to the development of the thrombotic event and to the clinical severity of these mutations.


Assuntos
Deficiência de Antitrombina III/metabolismo , Proteínas Antitrombina/química , Proteínas Antitrombina/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Proteínas Antitrombina/genética , Linhagem Celular , Humanos , Conformação Proteica , Multimerização Proteica , Estabilidade Proteica , Estresse Fisiológico
15.
Anim Biotechnol ; 23(2): 89-100, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22537058

RESUMO

Expression of recombinant pharmaceutical proteins in the mammalian mammary gland is of great interest for the medical industry. This study was designed to express recombinant human antithrombin (rhAT) in the mammary gland of rabbits by adenovirus vectors infection. Replication-defective adenovirus encoding human antithrombin complementary DNA (cDNA) was constructed and directly infused into the mammary gland of rabbits via the teat canal. The milk serum was collected from the infected mammary gland 48 h post-infection and subjected to Western blot analysis, Enzyme-linked immunosorbent assay (ELISA), and antithrombotic activity assay. In this way, the target protein was verified, and a high expression level of rhAT up to 4.8 g/L was obtained, and antithrombotic activity of the rhAT was not different than that of a standard human antithrombin protein (p > 0.05). Compared to previous attempts to produce human antithrombin in the mammary gland of transgenic animals or fractionation the plasma of blood donors, the method for rhAT expression we established would reduce production cost and further increase production efficacy.


Assuntos
Adenoviridae/genética , Proteínas Antitrombina/biossíntese , Glândulas Mamárias Animais/metabolismo , Proteínas Recombinantes/biossíntese , Animais , Animais Geneticamente Modificados , Proteínas Antitrombina/análise , Proteínas Antitrombina/química , Western Blotting , Ensaio de Imunoadsorção Enzimática , Células Epiteliais , Escherichia coli , Feminino , Vetores Genéticos/genética , Células HEK293 , Humanos , Glândulas Mamárias Animais/citologia , Leite/química , Coelhos , Proteínas Recombinantes/análise , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Transfecção/métodos
16.
Thromb Haemost ; 107(3): 468-76, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22318644

RESUMO

The natural anticoagulant protein S contains a so-called thrombin- sensitive region (TSR), which is susceptible to proteolytic cleavage. We have previously shown that a platelet-associated protease is able to cleave protein S under physiological plasma conditions in vitro . The aim of the present study was to investigate the relation between platelet-associated protein S cleaving activity and in vivo protein S cleavage, and to evaluate the impact of in vivo protein S cleavage on its anticoagulant activity. Protein S cleavage in healthy subjects and in thrombocytopenic and thrombocythaemic patients was evaluated by immunological techniques. Concentration of cleaved and intact protein S was correlated to levels of activated protein C (APC)-dependent and APC-independent protein S anticoagulant activity. In plasma from healthy volunteers 25% of protein S is cleaved in the TSR. While in plasma there was a clear positive correlation between levels of intact protein S and both APC-dependent and APC-independent protein S anticoagulant activities, these correlations were absent for cleaved protein S. Protein S cleavage was significantly increased in patients with essential thrombocythaemia (ET) and significantly reduced in patients with chemotherapy-induced thrombocytopenia. In ET patients on cytoreductive therapy, both platelet count and protein S cleavage returned to normal values. Accordingly, platelet transfusion restored cleavage of protein S to normal values in patients with chemotherapy-induced thrombocytopenia. In conclusion, proteases from platelets seem to contribute to the presence of cleaved protein S in the circulation and may enhance the coagulation response in vivo by down regulating the anticoagulant activity of protein S.


Assuntos
Proteínas Antitrombina/metabolismo , Plaquetas/metabolismo , Neoplasias Hematológicas/sangue , Fragmentos de Peptídeos/metabolismo , Proteína S/metabolismo , Trombocitemia Essencial/sangue , Trombocitopenia/sangue , Proteínas Antitrombina/química , Proteínas Antitrombina/genética , Coagulação Sanguínea/efeitos dos fármacos , Testes de Coagulação Sanguínea , Plaquetas/patologia , Domínio Catalítico/genética , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Feminino , Neoplasias Hematológicas/complicações , Neoplasias Hematológicas/tratamento farmacológico , Humanos , Masculino , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Transfusão de Plaquetas , Proteína C/metabolismo , Processamento de Proteína Pós-Traducional/genética , Proteína S/química , Proteína S/genética , Proteólise/efeitos dos fármacos , Trombocitemia Essencial/prevenção & controle , Trombocitopenia/etiologia , Trombocitopenia/prevenção & controle
17.
Blood ; 117(23): 6347-54, 2011 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-21511958

RESUMO

Histidine-rich protein II (HRPII) is an abundant protein released into the bloodstream by Plasmodium falciparum, the parasite that causes the most severe form of human malaria. Here, we report that HRPII binds tightly and selectively to coagulation-active glycosaminoglycans (dermatan sulfate, heparan sulfate, and heparin) and inhibits antithrombin (AT). In purified systems, recombinant HRPII neutralized the heparin-catalyzed inhibition of factor Xa and thrombin by AT in a Zn(2+)-dependent manner. The observed 50% inhibitory concentration (IC(50)) for the HRPII neutralization of AT activity is approximately 30nM for factor Xa inhibition and 90nM for thrombin inhibition. Zn(2+) was required for these reactions with a distribution coefficient (K(d)) of approximately 7µM. Substituting Zn(2+) with Cu(2+), but not with Ca(2+), Mg(2+), or Fe(2+), maintained the HRPII effect. HRPII attenuated the prolongation in plasma clotting time induced by heparin, suggesting that HRPII inhibits AT activity by preventing its stimulation by heparin. In the microvasculature, where erythrocytes infected with P falciparum are sequestered, high levels of released HRPII may bind cellular glycosaminoglycans, prevent their interaction with AT, and thereby contribute to the procoagulant state associated with P falciparum infection.


Assuntos
Antígenos de Protozoários/metabolismo , Proteínas Antitrombina/metabolismo , Malária Falciparum/metabolismo , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Anticoagulantes/farmacologia , Antígenos de Protozoários/química , Antígenos de Protozoários/genética , Proteínas Antitrombina/química , Proteínas Antitrombina/genética , Coagulação Sanguínea/efeitos dos fármacos , Coagulação Sanguínea/genética , Fator Xa/química , Fator Xa/genética , Fator Xa/metabolismo , Heparina/farmacologia , Humanos , Malária Falciparum/genética , Metais/química , Metais/metabolismo , Plasmodium falciparum/genética , Plasmodium falciparum/imunologia , Proteínas de Protozoários/química , Proteínas de Protozoários/genética
18.
PLoS One ; 6(3): e17519, 2011 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-21423730

RESUMO

UNLABELLED: Direct-acting fibrin(ogen)olytic agents such as plasmin have been proved to contain effective and safety thrombolytic potential. Unfortunately, plasmin is ineffective when administered by the intravenous route because it was neutralized by plasma antiplasmin. Direct-acting fibrin(ogen)olytic agents with resistance against antiplasmin will brighten the prospect of anti-thrombosis. As reported in 'Compendium of Materia Medica', the insect of Eupolyphaga sinensis Walker has been used as traditional anti-thrombosis medicine without bleeding risk for several hundreds years. Currently, we have identified a fibrin(ogen)olytic protein (Eupolytin1) containing both fibrin(ogen)olytic and plasminogen-activating (PA) activities from the beetle, E. sinensis. OBJECTIVES: To investigate the role of native and recombinant eupolytin1 in fibrin(ogen)olytic and plasminogen-activating processes. METHODS AND RESULTS: Using thrombus animal model, eupolytin1 was proved to contain strong and rapid thrombolytic ability and safety in vivo, which are better than that of urokinase. Most importantly, no bleeding complications were appeared even the intravenous dose up to 0.12 µmol/kg body weight (3 times of tested dose which could completely lyse experimental thrombi) in rabbits. It is the first report of thrombolytic agents containing both direct-acting fibrin(ogen)olytic and plasminogen-activating activities. CONCLUSIONS: The study identified novel thrombolytic agent with prospecting clinical potential because of its bi-functional merits containing both plasmin- and PA-like activities and unique pharmacological kinetics in vivo.


Assuntos
Proteínas Antitrombina/metabolismo , Fibrinólise , Ativadores de Plasminogênio/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Antitrombina/administração & dosagem , Proteínas Antitrombina/química , Proteínas Antitrombina/isolamento & purificação , Tempo de Sangramento , Besouros , Fibrinólise/efeitos dos fármacos , Trato Gastrointestinal/metabolismo , Hemostasia/efeitos dos fármacos , Humanos , Hidrólise/efeitos dos fármacos , Cinética , Camundongos , Dados de Sequência Molecular , Filogenia , Coelhos , Ativador de Plasminogênio Tipo Uroquinase/farmacologia
19.
Clin Appl Thromb Hemost ; 17(3): 273-8, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20211924

RESUMO

BACKGROUND: The reactive center loop (RCL) of native antithrombin is partially inserted in the main serpin body. It must be fully exposed for optimal inhibitory function. OBJECTIVE: To test the hypothesis that P(14)-s2B interaction affects loop insertion in antithrombin. By mutating Phe(274) to Tyr(274), the objective was to introduce P(14)-s2B interaction in antithrombin. METHODS: Site-directed mutagenesis and affinity chromatography were used to obtain purified recombinant protein. Antithrombin's ability to form sodium dodecyl sulfate (SDS)-stable complex with thrombin, stoichiometry of thrombin inhibition, second-order rate constant for thrombin and factor Xa (fXa) inhibition (M(-1) s(-1)), and heparin dissociation constant (K(D); tryptophan fluorescence emission spectra) were determined. RESULTS AND CONCLUSION: A marginal, but inconclusive, difference between the wild type and the mutant was observed. The result highlights the variable effect of P(14)-s2B interaction in different serpins. Alternate hypothesis for achieving loop expulsion is proposed.


Assuntos
Substituição de Aminoácidos , Proteínas Antitrombina/química , Domínio Catalítico , Mutação de Sentido Incorreto , Animais , Proteínas Antitrombina/genética , Proteínas Antitrombina/metabolismo , Linhagem Celular , Humanos , Mutagênese Sítio-Dirigida , Fenilalanina/química , Fenilalanina/genética , Fenilalanina/metabolismo , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Spodoptera , Tirosina/química , Tirosina/genética , Tirosina/metabolismo
20.
J Med Chem ; 53(22): 8030-40, 2010 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-21028827

RESUMO

Terminal 1,6-anhydro-aminosugars (1,6-anAS) are typical structural moieties of enoxaparin, a low-molecular-weight heparin (LMWH) widely used for prevention and treatment of thrombotic disorders. In the enoxaparin manufacturing process, these modified amino sugars are formed during the ß-eliminative cleavage of heparin. To investigate the effect of terminal anAS on antithrombin (AT) binding and on inhibition of factor Xa (FXa), two octasaccharides containing modified AT-binding pentasaccharide sequences were isolated from enoxaparin. The molecular conformation of the octasaccharides terminating with N-sulfo-1,6-anhydro-D-mannosamine and N-sulfo-1,6-anhydro-D-glucosamine, respectively, has been determined both in the absence and presence of AT by NMR experiments and docking simulations. Reduced overall contacts of the terminal anAS residues with the binding region of AT induce a decrease in affinity for AT as well as lower anti-FXa activity. The anti-FXa measured either in buffer or plasma milieu does not show any significant difference, suggesting that the inhibition of anti-FXa remains specific and biologically relevant.


Assuntos
Anticoagulantes/isolamento & purificação , Proteínas Antitrombina/química , Enoxaparina/química , Hexosaminas/química , Oligossacarídeos/isolamento & purificação , Anticoagulantes/química , Anticoagulantes/farmacologia , Proteínas Antitrombina/metabolismo , Sequência de Carboidratos , Fator Xa/química , Inibidores do Fator Xa , Hexosaminas/metabolismo , Humanos , Técnicas In Vitro , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação Molecular , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Oligossacarídeos/química , Oligossacarídeos/farmacologia , Ligação Proteica , Relação Estrutura-Atividade
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