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1.
Haemophilia ; 27(2): 321-328, 2021 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-33550714

RESUMEN

INTRODUCTION/AIM: Eptacog beta is a recombinant activated human factor VII approved to treat and control bleeding in haemophilia A and B patients with inhibitors. Emicizumab is a factor VIIIa mimetic antibody approved for prophylactic treatment of haemophilia A with and without inhibitors (HAI and HA, respectively). Inhibitor patients treated with emicizumab should expect breakthrough bleeding that requires bypassing agent treatment to restore haemostasis. The aim of this study is to quantify the in vitro thrombin generation induced by the addition of eptacog beta to HAI and HA plasma containing emicizumab. METHODS: Thrombin generation assays were performed using HAI and HA plasma. Thrombin generation parameters were examined using a fixed effects model with inhibitor titre, eptacog beta concentration and emicizumab concentration as main effects, and eptacog beta concentration with inhibitor and emicizumab concentration with inhibitor as interaction effects. RESULTS: A significant increase in peak thrombin, ETP and velocity was observed when combinations of eptacog beta (0, 1, 2 or 5 µg/ml) and emicizumab (0, 50 or 100 µg/ml) were evaluated in HA and HAI plasma; the effect remained below that observed in Normal Plasma (NP). A small shortening of lag time below that of NP was observed. CONCLUSIONS: Eptacog beta and emicizumab induced thrombin generation in haemophilia A plasma (with and without inhibitors) with the thrombin generation parameters remaining below those of normal plasma. These data provide in vitro proof of concept supporting the concept of use of eptacog beta for the treatment and control of breakthrough bleeding in patients on emicizumab prophylaxis.


Asunto(s)
Hemofilia A , Anticuerpos Biespecíficos , Anticuerpos Monoclonales Humanizados , Factor VIIa , Hemofilia A/tratamiento farmacológico , Humanos , Proteínas Recombinantes , Trombina
2.
Haematologica ; 105(9): 2335-2340, 2020 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-33054058

RESUMEN

Rendering coagulation factor X sensitive to thrombin was proposed as a strategy that can bypass the need for factor VIII. In this paper, this non-replacement strategy was evaluated in vitro and in vivo in its ability to correct factor VIII but also factor IX, X and XI deficiencies. A novel modified factor X, named Actiten, was generated and produced in the HEK293F cell line. The molecule possesses the required post-translational modifications, partially keeps its ability to be activated by RVV-X, factor VIIa/tissue factor, factor VIIIa/factor IXa and acquires the ability to be activated by thrombin. The potency of the molecule was evaluated in respective deficient plasmas or hemophilia A plasmas, for some with inhibitors. Actiten corrects dose dependently all the assayed deficient plasmas. It is able to normalize the thrombin generation at 20 µg/mL showing however an increased lagtime. It was then assayed in a rabbit antibody-induced model of hemophilia A where, in contrast to recombinant factor X wild-type, it normalized the bleeding time and the loss of hemoglobin. No sign of thrombogenicity was observed and the generation of activated factor X was controlled by the anticoagulation pathway in all performed coagulation assays. This data indicates that Actiten may be considered as a possible non replacement factor to treat hemophilia's with the advantage of being a zymogen correcting bleedings only when needed.


Asunto(s)
Factor X , Hemofilia A , Animales , Factor IX , Factor VIII/genética , Factor VIIa , Factor X/genética , Hemofilia A/tratamiento farmacológico , Hemofilia A/genética , Conejos , Trombina
3.
Br J Haematol ; 180(5): 715-720, 2018 03.
Artículo en Inglés | MEDLINE | ID: mdl-29363751

RESUMEN

Heparin anticoagulation followed by protamine reversal is commonly used in cardiopulmonary bypass (CPB). As an alternative to protamine, a recombinant inactive antithrombin (riAT) was designed as an antidote to heparin and was previously shown to be as potent as protamine in-vitro. In the present study, riAT was assessed for its ability to neutralize heparin after CPB in a rat model. After 60 min of CPB under heparin, rats received 5 mg/kg protamine, 37.5 mg/kg riAT or phosphate buffered saline (PBS) as placebo. Residual anticoagulant activity was assessed using the activated partial thromboplastin time assay before, and 10-30 min after reversion. Haemodynamic monitoring was performed and plasma histamine concentration was also measured. In this model, riAT appeared to be as efficient as protamine in neutralizing heparin. Ten minutes after injection, riAT and protamine both decreased heparin activity, to 1.8 ± 1.3 and 4.5 ± 1.4 u/ml, respectively (23.1 ± 5.1 u/ml in placebo group). Furthermore, evolution of mean carotid arterial pressure, heart rate and plasma histamine levels was comparable in rats treated with PBS or riAT, while protamine exhibited haemodynamic side effects and increased histamine plasma concentration. Thus, riAT could represent an advantage over protamine in CPB because it efficiently reverses heparin activity without negative effects on haemodynamic parameters and plasma histamine level.


Asunto(s)
Anticoagulantes/farmacología , Puente Cardiopulmonar , Antagonistas de Heparina/farmacología , Heparina/farmacología , Protaminas/farmacología , Animales , Antitrombinas/farmacología , Hemodinámica/efectos de los fármacos , Histamina/metabolismo , Masculino , Ratas Wistar
4.
Electrophoresis ; 37(12): 1696-703, 2016 07.
Artículo en Inglés | MEDLINE | ID: mdl-26989842

RESUMEN

Antithrombin (AT) is a human plasma glycoprotein that possesses anticoagulant and anti-inflammatory properties. However, the native (active) form of AT is unstable and undergoes conformational changes, leading to latent, cleaved, and heterodimeric forms. The presence of these alternative forms mostly inactive can highly impact the quality and therapeutic activity of pharmaceutical AT preparations. We developed a capillary zone electrophoresis method, based on a neutral polyethylene oxide-coated capillary and a buffer close to physiological conditions, enabling the separation of more than eight forms of AT. Several peaks were identified as native, latent, and heterodimeric forms. The CZE method was reproducible with intraday relative standard deviations less than 0.5 and 2% for migration times and peak areas, respectively. The method was applied to the comparison of AT preparations produced by five competitive pharmaceutical companies, and statistical tests were performed. Important differences in the proportion of each form were highlighted. In particular, one AT preparation was shown to contain a high quantity of heterodimer, and two preparations contained high quantities of latent form. In addition, one AT preparation exhibited additional forms, not yet identified.


Asunto(s)
Antitrombina III/aislamiento & purificación , Electroforesis Capilar/métodos , Tampones (Química) , Dimerización , Composición de Medicamentos , Electroforesis Capilar/instrumentación , Polietilenglicoles , Conformación Proteica , Isoformas de Proteínas/aislamiento & purificación
5.
Biologicals ; 44(4): 226-233, 2016 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-27156143

RESUMEN

Albumin displays several important functions for homeostasis amongst which the maintenance of the plasma redox-state. The study aim was to compare the redox state of pharmaceutical human albumin preparations since it reflects the oxidation-reduction status of the surrounding environment. Using an array of analytical methods, four commercially available albumins were compared with respect to their structural characteristics (cobalt ion binding, glycation, spectrophotometric and fluorometric profiles) and their ability to scavenge hydroxyl, peroxyl or free radicals. The different albumins exhibited a similar structural profile as well as hydroxyl and peroxyl scavenging activities. By contrast, the albumin from LFB (Vialebex(®)) possessed a significantly higher capacity to transfer electrons to DPPH, as compared with other albumins that was correlated with the level of free cysteine-34. Commercially available albumins differed for some of their antioxidant properties. The albumin preparation possessing the highest level of free cysteine-34 exhibited the highest antioxidant potential.


Asunto(s)
Antioxidantes/farmacología , Radicales Libres/antagonistas & inhibidores , Radical Hidroxilo/antagonistas & inhibidores , Albúmina Sérica/farmacología , Antioxidantes/química , Antioxidantes/uso terapéutico , Compuestos de Bifenilo/química , Cisteína/química , Depuradores de Radicales Libres/química , Depuradores de Radicales Libres/farmacología , Depuradores de Radicales Libres/uso terapéutico , Radicales Libres/metabolismo , Glicosilación , Humanos , Radical Hidroxilo/metabolismo , Oxidación-Reducción/efectos de los fármacos , Picratos/química , Albúmina Sérica/química , Albúmina Sérica/uso terapéutico , Espectrofotometría
6.
Thromb Res ; 167: 88-95, 2018 07.
Artículo en Inglés | MEDLINE | ID: mdl-29800795

RESUMEN

Antithrombin (AT) binds in vitro and in vivo to endothelial cells through various receptors, including heparan sulphate glycosaminoglycan (HSPG) that could modulate the AT activity. A thrombin generation assay (TGA) was set up at the surface of HUVEC and HMVEC evaluating their participation in the coagulation-anticoagulation processes. TGA induced by 0.5 pM Tissue Factor was performed in normal or AT-deficient plasma spiked with various amounts of recombinant or plasma-derived AT (0, 0.1, 0.5 and 1.0 U/ml). To evaluate the role of HSPG or cellular anticoagulant receptors, cells were treated or not with heparin, a mix of heparanase I, II and III, a neutralizing anti-Endothelial Protein C Receptor (EPCR) or with an anti-Tissue Factor Pathway Inhibitor (TFPI) antibody. The presence of the cells diminished the TG in normal plasma and maintained anticoagulation in AT-deficient plasma. Spiking the AT-deficient plasma with different doses of AT demonstrated that the cells did not amplify the anticoagulant activity of AT. The recombinant AT binds the cells with a higher avidity than the plasma-derived one but this did not affect its anticoagulant potency. Moreover both bindings are independent of the HSPG. The antithrombotic activity kept in absence of AT was not inhibited by blocking antibodies directed against EPCR or TFPI. Our data did not reveal a major co-factor activity for AT from endothelial cells that could have been mediated by HSPG. In contrast, it reveals the presence of alternative anti-coagulant system(s) in two venous cell types that maintain an antithrombotic activity.


Asunto(s)
Antitrombinas/uso terapéutico , Coagulación Sanguínea/efectos de los fármacos , Células Endoteliales/metabolismo , Antitrombinas/farmacología , Humanos
7.
Hum Mutat ; 27(7): 676-85, 2006 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-16786531

RESUMEN

Hemophilia A (HA) is an X-linked hereditary bleeding disorder defined by a qualitative and/or quantitative factor VIII (FVIII) deficiency. The molecular diagnosis of HA is challenging because of the high number of different causative mutations that are distributed throughout the large F8 gene. The putative role of the novel mutations, especially missense mutations, may be difficult to interpret as causing HA. We identified 95 novel mutations out of 180 different mutations responsible for HA in 515 patients from 406 unrelated families followed up at a single hemophilia treatment center of the Bicêtre university hospital (Assistance Publique-Hôpitaux de Paris [AP-HP], Le Kremlin-Bicêtre). These 95 novel mutations comprised 55 missense mutations, 12 nonsense mutations, 11 splice site mutations, and 17 small insertions/deletions. We therefore developed a mutation analysis based on a body of proof that combines the familial segregation of the mutation, the resulting biological and clinical HA phenotype, and the molecular consequences of the amino acid (AA) substitution. For the latter, we studied the putative biochemical modifications: its conservation status with cross-species FVIII and homologous proteins, its putative location in known FVIII functional regions, and its spatial position in the available FVIII 3D structures. The usefulness of such a strategy in interpreting the causality of novel F8 mutations is emphasized.


Asunto(s)
Análisis Mutacional de ADN/métodos , Factor VIII/genética , Hemofilia A/genética , Mutación , Factor VIII/química , Hemofilia A/diagnóstico , Humanos , Masculino , Estructura Terciaria de Proteína , Análisis de Secuencia de Proteína
8.
Anal Chim Acta ; 947: 58-65, 2016 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-27846990

RESUMEN

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.


Asunto(s)
Proteínas Antitrombina/química , Electroforesis Capilar/métodos , Espectrometría de Masas/métodos , Multimerización de Proteína , Proteínas Antitrombina/aislamiento & purificación , Modelos Moleculares , Estructura Cuaternaria de Proteína , Temperatura
9.
Thromb Haemost ; 93(5): 824-32, 2005 May.
Artículo en Inglés | MEDLINE | ID: mdl-15886795

RESUMEN

Factor VIII (FVIII) processing within mammalian cells is demonstrated to be much less efficient than proteins of similar size. The deletion of the B-domain from FVIII improves the level of production, due partly to the increase in mRNA synthesis. We aimed to characterise the cellular fate and the intracellular processing of the FVIII molecule devoid of B-domain. A B-domain deleted factor VIII (BDD-FVIII) possessing a furin consensus cleavage site in the connecting segment between the heavy and the light chain, was produced in CHO cell line. In such cells, FVIII was retained as two single chain products from which a majority was aggregated. The two species were located in Triton X-100 soluble (for 60-80%) and insoluble fractions (for 20-40%). The incubation of the expressing cells with tunicamycin (5 mug/ml) and the treatment of the intracellular species with a mixture of Neuraminidase and N-glycosidase-F revealed that both intracellular species were N-glycosylated. Furin over-expression neither diminished the intracellular FVIII contents nor improved its extracellular production. Intracellular FVIII was degraded through both lysosomal and proteasomal pathways as evidenced by inhibitor treatments (e.g. NH(4)Cl, leupeptin, clasto-Lactacystin beta-lactone and MG-132), pulse-chase analysis and confocal observations. This study demonstrates that a BDD-FVIII expressed in CHO cells is inefficiently processed consecutively to intracellular aggregation, proteasomal degradation, and routage to lysosomes.


Asunto(s)
Factor VIII/fisiología , Lisosomas/metabolismo , Fragmentos de Péptidos/fisiología , Complejo de la Endopetidasa Proteasomal/metabolismo , Animales , Antígenos CD/biosíntesis , Células CHO , Centrifugación por Gradiente de Densidad , Cricetinae , Detergentes/farmacología , Factor VIII/química , Furina/química , Glicosilación , Immunoblotting , Inmunoprecipitación , Leupeptinas/química , Proteínas de Membrana de los Lisosomas , Microscopía Confocal , Microscopía Fluorescente , Neuraminidasa/metabolismo , Octoxinol/farmacología , Fragmentos de Péptidos/química , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/metabolismo , Estructura Terciaria de Proteína , Sacarosa/farmacología , Transfección , Tunicamicina/farmacología
10.
J Pharm Biomed Anal ; 111: 64-70, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25863018

RESUMEN

With the aim to determine the binding affinity of a new generation of recombinant antithrombin (AT) toward heparin, we developed a dynamic equilibrium-affinity capillary electrophoresis (DE-ACE) method. This method allows the determination of an AT-heparin binding constant (Kd) directly from the cell culture supernatant used to produce the AT variants. Eight measurements per AT variant are sufficient to determine an accurate Kd (uncertainty ≤ 22%, regression coefficient ≥ 0.97), which is not significantly different from the value obtained from a higher number of measurements. Due to the relatively short time required to determine the Kd of one AT variant (2h), this method has the potential for being a low throughput screening method. The method was validated by analyzing five AT variants, whose Kd have been reported in the literature using fluorescence spectroscopy. Finally, the method was applied to estimate the Kd of one new AT variant and one AT conformer, a latent form, that exhibits a significant loss of affinity.


Asunto(s)
Antitrombinas/química , Heparina/química , Técnicas de Cultivo de Célula/métodos , Electroforesis Capilar/métodos , Humanos , Cinética , Espectrometría de Fluorescencia/métodos
11.
Thromb Haemost ; 107(2): 315-27, 2012 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-22234396

RESUMEN

Coagulation factor VIII (FVIII) is a multidomain glycoprotein in which the FVIII A2 domain is a key structural element. We aimed at identifying residues within FVIII A2 domain that are crucial for the maintenance of the cofactor function. A high number (n=206) of mutants were generated by substituting original residues with alanine. The mutants were expressed in COS-1 cells and their antigen levels and procoagulant activities were measured. The residues were classified in three categories: those with a non-detrimental alteration of their activities (activity >50 % of control FVIII; n=98), those with a moderate alteration (15 %

Asunto(s)
Factor VIII/metabolismo , Hemofilia A/genética , Proteínas Mutantes/metabolismo , Secuencia de Aminoácidos , Animales , Coagulación Sanguínea/efectos de los fármacos , Células COS , Chlorocebus aethiops , Cricetinae , Factor VIII/genética , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/genética , Mutación/genética , Fragmentos de Péptidos/genética , Estructura Terciaria de Proteína/genética
12.
Thromb Haemost ; 106(1): 121-31, 2011 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-21614407

RESUMEN

The factor VIII (FVIII) is a cofactor of the coagulation cascade. The FVIII C2 domain is a critical domain that participates in the interactions with the von Willebrand factor and the phospholipidic surfaces. To assess the importance of each residue of this domain in the maintenance of the structure and the function of FVIII, a number (n=139) of mutants were generated by substituting the original residues, from Ser2173 to Gly2325, by an alanine. Mutants were built within a complete B domain-deleted FVIII and expressed in COS-1 cells. Mutant antigen levels and procoagulant activities were measured. Two in silico analyses, a sliding average procedure and an analysis of the mutation energy cost were conducted in parallel on the FVIII structure. Both results were in agreement with the functional data, and illustrated the benefit of using such strategies prior to targeting specific residues in the aim of generating active recombinant molecules. The functional assays identify the residues that are important to maintaining the structure of the C2 domain, mainly those forming ß-sheet, and those that can afford substitution, establishing a detailed functional relation with the available crystallographic data. This study provided a comprehensive functional mapping of the FVIII C2 domain and discussed the implication of specific residues in respect to the maintenance in the activity and structure stability, the efficiency in secretion, the binding to phospholipids and the formation of epitope.


Asunto(s)
Factor VIII/metabolismo , Factor de von Willebrand/metabolismo , Alanina/genética , Alanina/metabolismo , Animales , Coagulación Sanguínea/genética , Pruebas de Coagulación Sanguínea , Células COS , Chlorocebus aethiops , Factor VIII/genética , Humanos , Mutagénesis Sitio-Dirigida , Mutación/genética , Unión Proteica/genética , Estructura Secundaria de Proteína/genética , Estructura Terciaria de Proteína/genética , Relación Estructura-Actividad
13.
Br J Haematol ; 127(5): 568-75, 2004 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-15566360

RESUMEN

Haemophilia A is an attractive target for gene therapy. We designed a haemophilia A gene therapy strategy involving the genetic modification of haematopoietic stem cells to achieve tissue-specific expression of a factor VIII (FVIII) transgene in the megakaryocytic lineage. Platelets would then serve as vehicles to store the expressed FVIII and deliver the coagulation factor at the site of vascular injury. A local correction of the haemostasis defect could, therefore, be expected following platelet activation and secretion. In this study, we demonstrated that a model of haematopoietic cell lines (Dami cells) could produce a correctly processed FVIII. FVIII transgenes were placed under the control of the human platelet glycoprotein IIb (GPIIb) promoter and used for stable transfection of the Dami megakaryocytic cell line. The highest FVIII production was obtained when the FVIII transgene contained a factor IX intron 1 gene sequence inserted in the FVIII intron 1 and 13 sites. Reverse transcription polymerase chain reaction demonstrated that the splicing of these introns was complete. Recombinant FVIII (rFVIII) produced in Dami cells was a biologically active molecule (specific activity: 5664 IU/mg) that was correctly glycosylated and sulphated. This recombinant FVIII protein exhibited biochemical characteristics after deglycosylation or thrombin activation that were comparable to a commercially available B-domainless rFVIII. These results demonstrate the advantages of a modified FVIII transgene and represent the first biochemical characterization of megakaryocyte-produced FVIII.


Asunto(s)
Factor VIII/biosíntesis , Terapia Genética/métodos , Hemofilia A/terapia , Megacariocitos/metabolismo , Reactores Biológicos , Western Blotting/métodos , Línea Celular , Cloratos/farmacología , Factor VIII/análisis , Factor VIII/genética , Factor VIIIa/análisis , Humanos , Inmunoprecipitación , Integrina alfa2 , Glicoproteínas de Membrana/genética , Regiones Promotoras Genéticas , Transfección/métodos , Transgenes
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