Anticoagulation With an Inhibitor of Factors XIa and XIIa During Cardiopulmonary Bypass

peer reviewedBackground: Exposure of blood to polyanionic artificial surfaces, for example, during cardiopulmonary bypass (CPB), induces a highly procoagulant condition requiring strong anticoagulation. Unfractionated heparin (UFH) is currently used during CPB but can lead to serious bleeding complications or development of a hypercoagulable state culminating in life-threatening thrombosis, highlighting the need for safer antithrombotics. Ixodes ricinus contact phase inhibitor (Ir-CPI) is a protein expressed by I. ricinus ticks, which specifically inhibits both factors XIIa and XIa, 2 factors contributing to thrombotic disease while playing a limited role in hemostasis. Objectives: This study assessed the antithrombotic activity of Ir-CPI in animal contact phase-initiated thrombosis models, including CPB. The safety of Ir-CPI also was evaluated. Methods: The authors evaluated the antithrombotic activity of Ir-CPI by using in vitro catheter-induced clotting assays and rabbit experimental models of catheter occlusion and arteriovenous shunt. During CPB with cardiac surgery in sheep, the clinical applicability of Ir-CPI was investigated and its efficacy compared to that of UFH using an uncoated system suitable for adult therapy. Taking advantage of the similar hemostatic properties of pigs and humans, the authors performed pig liver bleeding assays to evaluate the safety of Ir-CPI. Results: Ir-CPI prevented clotting in catheter and arteriovenous shunt rabbit models. During CPB, Ir-CPI was as efficient as UFH in preventing clot formation within the extracorporeal circuit and maintained physiological parameters during and post-surgery. Unlike UFH, Ir-CPI did not promote bleeding. Conclusions: Preclinical animal models used in this study showed that Ir-CPI is an effective and safe antithrombotic agent that provides a clinically relevant approach to thrombosis prevention in bypass systems, including highly thrombogenic CPB. © 2019 The Author

Modulatory activity of NADPH oxidase from equine neutrophils: in vitro tests on whole cells and development of a "cell-free system" assay

audience: researcher, professional, studentChez le cheval, la réponse inflammatoire causée par certaines pathologies (obstruction intestinale, fourbure,…) implique une stimulation excessive des polymorphonucléaires neutrophiles (PMNs) libérant de grandes quantités d’espèces activées de l’azote et de l’oxygène (RNOS). La première enzyme impliquée dans la formation de ces espèces activées est la NADPH oxydase, produisant l’anion superoxyde (O2.-) d’où dérivera la formation de diverses RNOS. Actuellement, la NADPH oxydase est considérée comme une cible privilégiée pour la modulation de l’inflammation excessive. Le but de ce travail a été d’étudier l’effet de molécules modulant la réponse inflammatoire (la curcumine, la quercétine et l’acépromazine : ACP) ou l’activité de la NADPH oxydase (le diphénylène iodonium : DPI) sur la production de RNOS par les PMNs stimulés. L’étude a été menée sur des PMNs équins isolés où la production de RNOS a été mise en évidence par trois techniques différentes : la chimiluminescence assistée par la lucigénine et la réduction du ferricytochrome C, ciblant essentiellement la production d’O2.- ; et une technique de fluorescence utilisant la 2’,7’-dichlorofluorescine-diacétate (DCFH-DA), ciblant la production intra et extracellulaire de RNOS. L’effet de ces molécules a pu être testé de façon plus spécifique sur la NADPH oxydase par une mesure en « cell-free system » que nous avons mise au point et consistant en la reconstitution in vitro de la NADPH oxydase. Nous avons constaté que les molécules polyphénoliques testées (quercétine et curcumine) agissaient surtout comme des antioxydants stoechiométriques en neutralisant les RNOS intracellulaires et extracellulaires sans pour autant être des inhibiteurs de la NADPH oxydase. Nous avons également démontré que certaines molécules (l’ACP et le DPI) utilisées à forte concentration pouvaient amplifier la réponse oxydante des PMNs. Enfin, grâce à la technique du « cell free system » nous avons démontré, pour la première fois, l’effet anti-catalytique du DPI sur la NADPH oxydase équine. La complémentarité de ces techniques permettra de faire la distinction entre un effet antioxydant, pro-oxydant ou anti-catalytique d’une molécule biologique pouvant moduler la réponse inflammatoire. Enfin, ce travail présente les premiers résultats de l’isolement des sous-unités membranaires de la NADPH oxydase équine. Les résultats sont encourageants et la purification à plus long terme du complexe enzymatique entier avec la production d’anticorps spécifiques devrait permettre la mise au point d’un dosage original de la NADPH oxydase consistant en l’immuno-capture de l’enzyme active afin de cibler l’effet de substances biologiques sur son assemblage et son activité spécifique

Activity of NADPH oxydase: a new target for curcumin ?

La NADPH oxydase (Nox2) des neutrophiles (PMNs) est une enzyme multi-protéique responsable de la production d'espèce activées de l'oxygène (ROS) pour la destruction des pathogènes. L'activation excessive des PMNs est souvent associée à des pathologies mortelles chez le cheval, faisant de l'activité de la Nox2 une cible thérapeutique privilégiée. Le premier but de ce travail, a été de développer une méthode appelée "cell-free system" (CSF) pour mesurer l'activité in vitro de la Nox2 équine. Un inconvénient de cette technique sont les interférences possibles entre les inhibiteurs et la sonde utilisée pour la mesure de l'activité. Sur base de notre CFS, nous avons créé l'EquiNox2, un nouvel outil pharmacologique, pour étudier les interactions entre des inhibiteurs et la Nox2 et leurs effets sur l'assemblage et l'activité de l'enzyme. Cette méthode consiste en la mesure in vitro de l'activité de la Nox2 fixée sur un support solide, ce qui permet d'éliminer les inhibiteurs avant la mesure de l'activité et réduit les interférences citées pour le CFS. Le CFS et l'EquiNox2 ont été validés avec le diphénylène iodonium et le Gp91ds-tat, deux inhibiteurs connus de la Nox2 humaine. Le deuxième but de ce travail, était l'étude du NDS27, un complexe de lysinate de curcumine avec l'hydroxypropyl-β-cyclodextrine (HPβCD) sur la réponse oxydante des PMNs. Le NDS27, n'est pas toxique et est capable d'entrer et d'interagir avec les membranes des PMNs et d'inhiber l'activité de la myéloperoxydase, la Nox2 et la PKCδ (un activateur de Nox2) impliquées dans la production de ROS. Nous avons montré en CFS et EquiNox2 que le NDS27 se fixe fortement à la Nox2 pour empêcher son assemblage et que l'HPβCD, l'excipient du NDS27, en plus de solubiliser et transporter la curcumine, augmente l'action de celle-ci sur les activités de la PKC et la Nox2. L'effet modulateur du NDS27 sur l'activation de la Nox2 ouvre des perspectives thérapeutiques pour traitement des pathologies accompagnées de réactions inflammatoires excessives.Purification de la NADPH oxydase des neutrophiles équins : mise au point de nouveaux outils pour la quantification et la mesure spécifique de son activité et pour la recherche d'inhibiteurs de l'enzym

Etude de l'effet intracellulaire d'une nouvelle forme soluble de la curcumine (NDS27) sur la réponse oxydante des neutrophiles stimulés

Neutrophils (PMNs) are involved in host defense against infections through the production of reactive oxygen species (ROS) to kill pathologic agents. But, an excessive ROS production, called “oxidative stress” is associated with tissue damages and development of chronic or acute inflammatory diseases. PMNs are prime therapeutic targets to control inflammatory events associated to ROS production. Nowadays, there is a growing interest for the use of polyphenolic molecules to modulate the inflammatory response. The aim of this work was to study the antioxidant effect of NDS27 (1), a new highly water-soluble form of the polyphenolic molecule curcumin, on in vitro stimulated equine PMNs. NDS27 (10-6 to 10-4 M) was pre-incubated with cells and eliminated before their activation. The ability of NDS27 to enter into the cells was checked by HPLC from the cellular extracts. The intracellular ROS production by phorbol myristate acetate (PMA) stimulated PMNs was measured by fluorescence using 2’,7’-dichlorofluorescin diacetate. Lucigenin dependent chemiluminescence was used to measure extracellular ROS production. Additionally, the effect of NDS27 was tested on the activity of myeloperoxidase (MPO), a hemic enzyme contributing to the oxidant response of neutrophils. The activity of the released MPO by cytochalazine B (CB) and N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulated PMNs was measured by SIEFED (“Specific Immunologic Extraction Followed by Enzymatic Detection”) (2). The HPLC results showed that NDS27 enters into PMNs and interacts with their membrane. NDS27 significantly and dose-dependently inhibited the ROS production in neutrophils without affecting their viability. Likewise, the activity of MPO released by PMNs was lowered by NDS27. Overall, our findings demonstrate that the membrane of neutrophils is permeable to NDS27 or interacts with the drug, suggesting that its inhibitory effect on ROS production is mainly associated to an intracellular effect probably by acting on the enzymes implied in respiratory burst like NADPH oxidase and MPO. The modulatory effect of NDS27 towards the oxidant activity of cells involved in immune and inflammatory response open therapeutic perspectives to control equine or human pathologies with excessive inflammatory reactions. 1. Neven et al. 2011, Patent Application Publication: US2011/0257126 A1 2. Serteyn et al. 2005, European Patent Specification : EP1711817 B

Effect of different kinds of anoxia/reoxygenation on the mitochondrial function and the free radicals production of cultured primary equine skeletal myoblasts.

Horses are outstanding athletes, performing in many different disciplines involving different kinds of efforts and metabolic responses. Depending on exercise intensity, their skeletal muscle oxygenation decreases, and the reperfusion at cessation of the exercise can cause excessive production of free radicals. This study on cultured primary equine myoblasts investigated the effect of different kinds of anoxia/reoxygenation (A/R) on routine respiration, mitochondrial complex I specific activity and free radicals production. Our data revealed that short cycles of A/R caused a decrease of all the parameters, opposite to what a single long period of anoxia did. A preconditioning-like effect could explain our first pattern of results whereas mild uncoupling could be more appropriate for the second one. Anyway, it seems that mitochondrial complex I could play a major role in the regulation of the balance between metabolic and antioxidant protection of the muscular function of athletic horses

La nouvelle forme soluble de la curcumine (NDS27) inhibe t-elle la réponse oxydante des neutrophiles et des HL-60 ?

Neutrophils (PMNs) are involved in host defense against infections through the production of reactive oxygen species (ROS) and the release of an oxidant enzyme, myeloperoxidase (MPO), to kill pathologic agents. But, an excessive stimulation of PMNs is associated with development of inflammatory diseases. Neutrophils are prime targets to control inflammatory events and the therapeutic use of polyphenols is proposed to lower oxidative stress. The aim of this work was to study antioxidant effect of NDS27, a water-soluble form of curcumin, on stimulated equine PMNs and human promyelocytic leukemia cells (HL-60) which are less differenciated. 2',7'-Dichlorofluorescin diacetate and lucigenin were used to measure ROS production by activated HL-60 cells or PMNs. NDS27 (10-6 to 10-4 M) was pre-incubated with cells and eliminated before their activation to study its intracellular effects on ROS production. The effect of NDS27 on MPO activity released by the cells was determined by SIEFED. Likewise, the ability of NDS27 to enter into the cells was checked by HPLC on the cellular extracts.NDS27 significantly and dose-dependently inhibited the ROS production in both cell types without affecting their viability. Its intracellular effect showed higher efficiency for PMNs while its interaction with HL-60 cells remained better. The activity of MPO released by PMNs and HL-60 cells was decreased by NDS27 with a more efficient effect for PMNs. Our findings suggest that the greater efficiency of NDS27 in mature PMNs is not due to a better membrane permeability or a better interaction between membrane and NDS27, but rather to an inhibitory effect on the ROS production by the more mature cells, probably by targeting the enzymes implied in respiratory burst like MPO and NADPH oxidase. The modulatory effect of NDS27 towards oxidant activity of cells involved in immune and inflammatory response opens therapeutic perspectives to control pathologies with excessive inflammatory reactions.Purification de la NADPH oxydase des neutrophiles équins : mise au point de nouveaux outils pour la quantification et la mesure spécifique de son activité et pour la recherche d'inhibiteurs de l'enzym

Le NDS27, une forme hydrosoluble de la curcumine, inhibe l'activité de la myéloperoxidase et de la NADPH oxydase, deux enzymes majeures des neutrophiles

Neutrophils (PMNs) produce reactive oxygen species (ROS) to kill pathogenic agents. After appropriate stimulation, leading to the activation of protein kinase C (PKC), the cytosolic subunits of the NADPH oxidase (Nox2) are phosphorylated and translocated to the membrane flavocytochrome b558, forming the active enzyme which produces superoxide anion (O2●-). From O2●- derives H2O2 used by the PMNs myeloperoxidase (MPO) to form strong oxidant species. Many human and animal pathologies with fatal issue are associated with uncontrolled activation of PMNs. The modulation of enzymes implied in ROS production is thus a primary target to manage excessive inflammatory events. For this purpose, we evaluated the effects of NDS27, a water-soluble salt of curcumin combined with hydroxypropyl-β-cyclodextrin, on the activities of PKC, Nox2 and MPO. PKC activation was determined by western blotting with specific antibodies against phosphorylated PKC in extracts from PMNs after their incubation or not with NDS27. A cell-free assay was used to evaluate the effect of NDS27 before or after the assembly of Nox2 subunits. MPO activity was tested by the SIEFED technique in which NDS27 was pre-incubated with the enzyme and discarded before its activity measurement. An inhibition of PKC phosphorylation and Nox2 activity were observed at respectively 10-4 and 10-5 M of NDS27. The Nox2 inhibition was more pronounced when NDS27 was added before the assembly stimulation, suggesting a direct action of NDS27 on the subunits translocation. NDS27 also dose-dependently decreased the activity of MPO (21 % at 10-5 M), indicating an interaction with the enzyme structure. Our results demonstrated that NDS27 is a potent inhibitor of the two major enzymes responsible for ROS production in PMNs, and also acts on the activation cascade of Nox2. The modulatory effect of NDS27 towards the oxidant activity of PMNs opens therapeutic perspectives to control pathologies with excessive inflammatory reactions.Purification de la NADPH oxydase des neutrophiles équins : mise au point de nouveaux outils pour la quantification et la mesure spécifique de son activité et pour la recherche d'inhibiteurs de l'enzym

AN IMMUNOLOGICAL METHOD TO COMBINE THE MEASUREMENT OF ACTIVE AND TOTAL HUMAN MYELOPEROXIDASE ON THE SAME SAMPLE FROM A COMPLEX MEDIUM

Active neutrophil myeloperoxidase (MPO) is a powerful producer of oxidant molecules in acute or chronic inflammation, and it is essential to measure its activity in biological samples. We combined two immunological techniques, the SIEFED (specific immunologic extraction followed by enzymatic detection) and an ELISA, to measure the active and total contents of human MPO on the same sample and with the same calibration curve, to define an accurate ratio between the active and total enzyme. After the extraction of MPO from aqueous or biological samples by immobilized anti-MPO antibodies followed by a washing to eliminate unbound material, the active and the total contents of the enzyme were sequentially measured without interferences (patent: EP2017351-B1, 2010). Compared to a classical sandwich ELISA, there is one additional step corresponding to the in situ measurement of MPO activity, but this step does not affect the following measurement of the total MPO content. After validation, the combined technique was applied to a whole blood model of in vitro stimulation with phorbol-myristate-acetate (PMA), cytochalasin B/N-formyl-methionyl-leucyl-phenyl-alanine (CB/fMLP) or lipopolysaccharide/ tumor necrosis factor-alpha (LPS/TNF-α) (n=9). The active/total MPO ratio in whole blood reached 0.267±0.126 for non-stimulated condition and was significantly (p<0.05) higher for PMA (0.360±0.106), CB/fMLP (0.380±0.113) and LPS/TNF-α (0.432±0.124) stimulated conditions. These different ratios highlight the real oxidant potential of MPO, which depends on the stimulating conditions, witness of what could happen in pathological situations with diagnostic purpose. The combined SIEFED/ELISA method also appeared as a powerful tool to screen potential inhibitors that could interact directly with the enzyme, either on its active site or on another key position