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Abstract

Extracellular soluble hemoglobins (Hbs) have long been studied for their possible use as safe and effective alternatives to blood transfusion. While remarkable progress has been made in the use of cell-free Hb as artificial oxygen carrier, significant problems remain, including susceptibility to oxidative inactivation and propensity to induce vasoconstriction. Hemarina-M101 is a natural giant extracellular hemoglobin (3600 kDa) derived from marine invertebrate (polychaete annelid). Hemarina-M101 is a biopolymer composed of 156 globins and 44 non-globin linker chains and formulated in a product called HEMOXYCarrier®. Prior work has shown Hemarina-M101 to possess unique anti-oxidant activity and a high oxygen affinity. Topload experiment with this product into rats did not revealed any effect on heart rate (HR) and mean arterial pressure (MAP). A pilot study with the hamster dorsal skinfold window chamber model showed absence of microvascular vasoconstriction and no significant impact on mean arterial blood pressure. In vitro nitric oxide (NO) and carbon monoxide (CO) reaction kinetics measurements show that Hemarina-M101 has different binding rates as compared to human Hb. These results revealed for the first time that the presence of this marine hemoglobin appears to have no vasoactivity at the microvascular level in comparison to others hemoglobin based oxygen carriers (HBOCs) developed so far and merits further investigation.

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... For instance, Arenicola marina-derived natural extracellular Hb has been studied in the recent past [35][36][37]. Arenicola marina's Hb (M101, commercially manufactured as HEMO2life ® by Hemarina (Morlaix, France) has been shown to effectively deliver O2 in vivo [38,39] without any signs of oxidation, vasoconstriction, or hypertension [35,40]. Large heme pockets of Arenicola marina Hb allow O2 to easily escape in passive way [41][42][43]. ...
... After successive steps of solid/liquid extraction, purification, filtration and gamma irradiation, the final result is a class III medical device containing M101 [51]. Preclinical studies in rats and hamsters using M101 have shown reduced microvascular vasoconstriction and no significant impact on mean arterial blood pressure compared with other HBOCs that contain bovine or human Hb [40]. Moreover, the biocompatibility and absence of toxicity of HEMO2life ® has also been evaluated according to international standards (ISO 10993). ...
... M101 has different binding rates for gases as compared to human Hb. [40] Note:  decrease;  increase. Heart function in Celsior solution, either alone (control) or with the addition of M101 was measured by intra-ventricular balloon before arrest with cold (7.5 °C). ...
Article
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Oxygen (O2) is indispensable for aerobic respiration and cellular metabolism. In case of injury, reactive oxygen species are produced, causing oxidative stress, which triggers cell damaging chemical mediators leading to ischemic reperfusion injuries (IRI). Sufficient tissue oxygenation is necessary for optimal wound healing. In this context, several hemoglobin-based oxygen carriers have been developed and tested, especially as graft preservatives for transplant procedures. However , most of the commercially available O2 carriers increase oxidative stress and show some adverse effects. Interestingly, the hemoglobin derived from the marine lugworm Arenicola marina (M101) has been presented as an efficient therapeutic O2 carrier with potential anti-inflammatory, anti-bacterial , and antioxidant properties. Furthermore, it has demonstrated promise as a supplement to conventional organ preservatives by reducing IRI. This review summarizes the properties and various applications of M101. M101 is an innovative oxygen carrier with several beneficial therapeutic properties, and further research must be carried out to determine its efficacy in the management of different pathologies.
... Hemarina-M101 is a therapeutic oxygen carrier that made headlines in 2020 for its possible application in critical COVID-19 patients in a severe respiratory state. It is manufactured from the extracellular hemoglobin of marine animal, Arenicola marina, and is part of the formulated HemoxyCarrier® as a therapeutic oxygen carrier (AOC) [2,3]. A Phase I clinical trial named MONACO was aimed to take place in April 2020 in France for use in 10 critically ill COVID-19 patients to see if injection of Hemarina-M101 can improve the oxygenation in these patients. ...
... Each globin chain surrounds and protects its own O 2 -binding heme group consisting of a protoporphyrin ring with an iron atom in the center reversibly bound to one O 2 molecule [6]. One hemoglobin molecule of this large complex can carry up to 156 O 2 molecules when fully saturated compared to a single human hemoglobin molecule that can carry a maximum of 4 O 2 molecules [2,3]. It was found that the p50 of Arenicola marina hemoglobin (AmHb) is around 7.05 ± 0.93 mmHg compared to human blood which is between 26− 30 mmHg. ...
... In 2012, animal studies using rats and hamsters investigated Hemarina-M101 ′ s effects inside the body. A top-load experiment giving 10 % total blood volume (estimated as 7% of total body weight) using Hemarina-M101 in a hamster skinfold window chamber model showed no effect on both the heart rate and mean arterial pressure [2]. Injection of Hemarina-M101 (600 mg/kg over 5 min) did not significantly increase the heart rate or myocardial contractility in the rat model compared to the saline treated control group [2]. ...
Article
Therapeutic Oxygen Carriers (TOCs) have been studied in the past for utilization in resuscitation fluid, treatment of organ ischemia, and as an alternative to red blood cell transfusion. One TOC, Hemarina-M101, seems promising in transplantation and oxygenation due its capability as a non-immunogenic, nontoxic, high-oxygen-carrying capacity TOC with little to no side effects. This mini-review focuses on Hemarina-M101 and explores its characteristics and possible utilities through past and recent studies.
... Natural acellular Hb of terrestrial and marine worms is configured in this manner. 83 Hemoglobin from the marine worm Arenicola marina is being developed as an HBOC 84 and has properties similar to the synthesized zero linked polymeric Hb OxiVita 85 developed by Prof. Bucci's group. 86 The rheology of hemoglobin vesicles A different means for introducing Hb in the circulation is to encapsulate it in vesicles (HbV). ...
... 148 Its NO binding rate is somewhat lower than human Hb while CO binding rate is somewhat greater. 84 Evaluation of HEMOXYCarrier® in rodent preparations showed that administration of 40 mg/mL Hb solution in saline caused a transient increase of blood pressure not statistically different from that due to administering saline. 84 This HBOC is reported to be stable over a wide range conditions (ionic compositions, osmolarities, and oxidative environments) and to have natural antioxidant properties in the presence of natural superoxide dismutase like enzymes. ...
... 84 Evaluation of HEMOXYCarrier® in rodent preparations showed that administration of 40 mg/mL Hb solution in saline caused a transient increase of blood pressure not statistically different from that due to administering saline. 84 This HBOC is reported to be stable over a wide range conditions (ionic compositions, osmolarities, and oxidative environments) and to have natural antioxidant properties in the presence of natural superoxide dismutase like enzymes. 149 ...
Article
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The development of oxygen (O2)-carrying blood substitutes has evolved from the goal of replicating blood O2 transport properties to that of preserving microvascular and organ function, reducing the inherent or potential toxicity of the material used to carry O2, and treating pathologies initiated by anemia and hypoxia. Furthermore, the emphasis has shifted from blood replacement fluid to "O2 therapeutics" that restore tissue oxygenation to specific tissues regions. This review covers the different alternatives, potential and limitations of hemoglobin-based O2 carriers (HBOCs) and perfluorocarbon-based O2 carriers (PFCOCs), with emphasis on the physiologic conditions disturbed in the situation that they will be used. It describes how concepts learned from plasma expanders without O2-carrying capacity can be applied to maintain O2 delivery and summarizes the microvascular responses due to HBOCs and PFCOCs. This review also presents alternative applications of HBOCs and PFCOCs namely: 1) How HBOC O2 affinity can be engineered to target O2 delivery to hypoxic tissues; and 2) How the high gas solubility of PFCOCs provides new opportunities for carrying, dissolving, and delivering gases with biological activity. It is concluded that the development of current blood substitutes has amplified their applications horizon by devising therapeutic functions for O2 carriers requiring limited O2 delivery capacity restoration. Conversely, full, blood-like O2-carrying capacity reestablishment awaits the control of O2 carrier toxicity.
... Over the last years, several studies have focused on the Hb found in the marine invertebrate Arenicola marina (Rousselot et al., 2006a;Tsai et al., 2012). This natural extracellular respiratory pigment, named M101, is a high molecular weight protein (∼3,600 kDa). ...
... significant vasoconstriction effect nor impact on heart rate are induced after M101 top load i.v. injection in rodents (Tsai et al., 2012). ...
... 2.1. M101 production M101 (HEMARINA SA, France) is manufactured using GMP ("good manufacturing practices") standards governing medicinal products (Tsai et al., 2012). Briefly, extracellular Hb is extracted from Arenicola marina frozen lugworms under gentle agitation at 4 • C, then purified and conditioned in 20 mL bags. ...
Article
Natural giant extracellular hemoglobins (Hbs) from polychaete annelids are currently actively investigated as promising oxygen carriers. Their powerful oxygenating ability and their safety have been demonstrated in preclinical studies, motivating their development for therapeutic and industrial applications. HEMARINA-M101 (M101) is derived from the marine invertebrate Arenicola marina. It is formulated as a manufactured product designated HEMOXYCarrier(®) (HEMARINA SA, France). The aim of the present study was to unveil the fate of M101 after a single intravenous (i.v.) injection in mice. For this purpose, M101 was tagged with a far-red fluorescent dye. Repeated non-invasive fluorescent imaging revealed a rapid diffusion of M101 in the whole body of animals, reaching all the examined organs such as brain, liver, lungs and ovaries. Functional M101 was circulating in bloodstream for several hours, without inducing any obvious side-effects. Last, a single i.v. injection of M101 in mice bearing human-derived subcutaneous tumors demonstrated the ability of this Hb to reduce hypoxia in poorly vascularized tissues, thus supporting the biological relevance of M101 oxygen release to vertebrate tissues. Altogether, these results further encourage the development of M101 as an oxygen carrying therapeutic.
... The qua ter nary struc ture of this mol e cule is a hexag o nal-bi layer with a di men sion of 25 nm (face view) and 15 nm (pro fil view) [13]. Each glo bin chain has a heme group sim i lar to hu man and the linker chains pos sess an anti-ox ida tive prop erty due to a Su per ox ide Dis mu tase Ac tiv ity (SOD) ac tiv ity-like based on cop per and zinc [15]. ...
... O re leas ing is just done in a par tial oxy gen gra di ent, when p is be low the p , the oxy gen is re leased pas sively to the tis sues, and con sumed by cells or tis sues, avoid ing ox ida tive dam ages. Two im por tant points, the p of the myo glo bin is 2.6 mm of Hg, so be low the p of this oxy gen car rier and even this mol e cule is high oxy gen affin ity the p is sim i lar of the he mo glo bin A (HbA) in side the red blood cell [15]. HEMO Life® has a red color, it is ster ile, is py ro gen-free and frozen at 20° C (+/ 3° C). ...
... This hy poth e sis is also sup ported by the fact that we have shown in a rat model af fected with trau matic brain in jury [10] and there fore highly sus cep ti ble to in travas cu lar mi crothrom bo sis [60] that our oxy gen car rier could rapidly re duce acute brain hy poxia tis sue, by over com ing clas sic, post-trau matic vas cu lar size re duc tion with out in duc ing vaso con stric tion it self. In deed, HEMO Life® does not have a vaso con stric tion ef fect com pared to with other HBOC of first or sec ond gen er a tion de vel oped so far [15,19]. Fi nally, the hy poth e sis that the COVID-19 could re move the heme on -glo bin chain and re moved its iron has been put for ward [36] at least on red blood cell prog en i tors with the nu cleus. ...
Article
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Background Infection with SARS-CoV-2 is responsible for the COVID-19 crisis affecting the whole world. This virus can provoke acute respiratory distress syndrome (ARDS) leading to overcrowed the intensive care unit (ICU). Over the last months, worldwide experience demonstrated that the ARDS in COVID-19 patients are in many ways “atypical”. The mortality rate in ventilated patients is high despite the application of the gold standard treatment (protective ventilation, curare, prone position, inhaled NO). Several studies suggested that the SARS-CoV-2 could interact negatively on red blood cell homeostasis. Furthermore, SarsCov2 creates Reactive Oxygen Species (ROS), which are toxic and generate endothelial dysfunction. Hypothesis/Objective(s) We hypothesis that HEMO2Life® administrated intravenously is safe and could help symptomatically the patient condition. It would increase arterial oxygen content despite lung failure and allow better tissue oxygenation control. The use of HEMO2Life® is also interesting due to its anti-oxidative effect preventing cytokine storm induced by the SARS-CoV-2. Evaluation of the hypothesis: Hemarina is based on the properties of the hemoglobin of the Arenicola marina sea-worm (HEMO2Life®). This extracellular hemoglobin has an oxygen capacity 40 times greater than the hemoglobin of vertebrates. Furthermore, the size of this molecule is 250 times smaller than a human red blood cell, allowing it to diffuse in all areas of the microcirculation, without diffusing outside the vascular sector. It possesses an antioxidative property du a Superoxide Dismutase Activity. This technology has been the subject of numerous publications and HEMO2Life® was found to be well-tolerated and did not induce toxicity. It was administered intravenously to hamsters and rats, and showed no acute effect on heart rate and blood pressure and did not cause microvascular vasoconstriction. In preclinical in vivo models (mice, rats, and dogs), HEMO2Life® has enabled better tissue oxygenation, especially in the brain. This molecule has already been used in humans in organ preservation solutions and the patients showed no abnormal clinical signs. Consequences of the hypothesis The expected benefits of HEMO2Life® for COVID-19 patients are improved survival, avoidance of tracheal intubation, shorter oxygen supplementation, and the possibility of treating a larger number of patients as molecular respirator without to use an invasive machine.
... Hemarina (HEMARINA SA, Morlaix, France) developed the manufactured product HEMOXYCarrier® (HC), based on extracted and purified Arenicola marina haemoglobin. [12] This promising new HBOC caused no allergic reaction or kidney damage after injection in mice. [13,14] Animals showed no significant vasoconstriction, hypertension, or modification of heart rate. ...
... [13,14] Animals showed no significant vasoconstriction, hypertension, or modification of heart rate. [12] Preclinical trials have begun for the treatment of haemorrhagic shock and traumatic brain injury. [16] In the near future, this product could become an effective blood substitute and consequently be of interest to athletes for doping. ...
... Final concentrations (200 mg/kg and lower concentrations) were based on previous studies in mice. [12,13] ...
Article
Manipulation of blood and blood components is prohibited in sports by the World Anti-Doping Agency (WADA). This includes the use of blood substitutes to increase oxygen transport, like hemoglobin-based oxygen carriers (HBOCs), which are compounds derived from hemoglobin. Despite their medical interest, the first generation of HBOCs had serious adverse effects and was abandoned. However, new studies are now exploiting the properties of marine worm hemoglobins, which circulate as giant extracellular complexes with high oxygen-binding capacities. HEMOXYCarrier® (HC), developed by Hemarina, is one of the most advanced and promising HBOCs, and HC may become a tempting doping tool for athletes in the future. Here, HC detection in plasma/serum was evaluated with the method used to detect the first HBOCs, based on electrophoresis and heme peroxidase properties. An HC-derived product was identified in human plasma up to 72 h after in vitro incubation at 37 °C. HC degradation also induced methemalbumin formation. After injecting HC at the effective dose of 200 mg/kg into mice, the HC-derived product was detected only for a few hours and no accumulation of methemalbumin was observed. Due to this limited detection window in vivo, measuring specific worm globin degradation products by mass spectrometry might be an alternative for future anti-doping analyses.
... Many studies have been previously performed using M101 revealing a variety of potential applications. It has been widely studied for its ability to carry and transfer oxygen (Le Gall et al. 2014;Pape et al. 2015) and as a promising therapeutical oxygen carrier (Rousselot et al. 2006;Harnois et al. 2009;Tsai et al. 2012). Its function in organ preservation for transplantation of heart (Teh et al. 2017), lung (Glorion et al. 2018) and kidney (Thuillier et al. 2011;Mallet et al. 2014) is noteworthy. ...
... These compounds also face the challenges like potential toxicity, susceptibility to proteases, high production costs and unclear mode of action. M101 can be considered as an potential treatment against periodontal biofilms since it has no known side effects in connection with its applications in tissue repair (Zal and Rousselot 2009), ischemia or anemia pathologies (Rousselot et al. 2006;Harnois et al. 2009;Tsai et al. 2012), organ transplant (Thuillier et al. 2011;Mallet et al. 2014;Teh et al. 2017;Glorion et al. 2018). ...
Thesis
The oral bacteria form a complex community and their pathogenic switch can be influenced by host-related factors such as systemic iron and oxygen levels. Previous studies have suggested that genetic hemochromatosis, characterized by systemic iron overload, favours the occurrence of severe periodontitis but the mechanisms involved are unclear. Therefore, this thesis aimed to analyse the behaviour of multispecies oral biofilms at different iron levels composed of periodontal bacteria selected for their importance in the development of periodontal diseases: Streptococcus gordonii, Porphyromonas gingivalis and Treponema denticola. To realize this multispecies biofilm, an iron-controlled growth medium (Mixed Medium for Bacterial Community) was developed. This work includes applied research work and a fundamental part. In the framework of the development of a system for evaluating the anti biofilm activity of new products, methods for testing the effectiveness of antimicrobial liquids or gels were developed and used to challenge the 3-species biofilm. To better understand the effect of iron on the behaviour of three-species oral biofilm, the interspecies associations in biofilm initiation/attachment and followed by biofilm growth and development in response to iron levels (0.8, 8, 80 μM FeSO4) were investigated. This study, for the first time, showed the interactions between S. gordonii and T. denticola in biofilms and the effect of iron on these biofilms. The most striking result was the lower initial attachment and growth of this dual species biofilm at the lower iron concentrations. Moreover, the presence of the three-species altogether favoured the attachment of both pathogens P. gingivalis and T. denticola compared to mono-species biofilms and displayed a preferential growth of all the three species at 80 μM iron at 144 hours. Such preference for high iron levels for biofilm maturation may lead to oral dysbiosis and the progression of periodontitis during iron overload diseases. To further evaluate the inter-bacterial associations, the effect of iron in the evolution of the oral biofilm is being studied in the form of a new mathematical model with T. denticola in a previously established model for P. gingivalis and S. gordonii. The parameters were calibrated by measurements made from biological experiments in monospecies biofilms which will be used to test different hypotheses for either independence or competition for substrate between species, mutual benefit/syntrophy or production of toxic molecules for dual- and three-species biofilms. Finally, the validation of the model will be done by comparing the simulated results with those of the experimental results obtained for dual and three-species biofilms. Altogether this study will enable a better understanding of the iron driven pathogenesis of the oral microbiota.
... M101 releases O 2 according to a simple gradient without requiring any allosteric effector, providing the right amount of O 2 to hypoxic tissues. This non-glycosylated protein induces no immunogenic or allergenic response upon intravenous injection in mice, nor does it induce significant vasoconstriction or impact on heart rate after intravenous injection in rodents [32]. ...
... In our study, the addition of HEMO 2 life V R induce a smaller PVR after graft reperfusion compared to Group 1 (Perfadex V R alone). It can therefore be assumed that the addition of HEMO 2 life V R during the preservation period decreases the functional consequences of the second phase of reperfusion (between 2 and 4 h) [32,33,41,42]. ...
Article
Full-text available
We describe the results of adding a new biological agent HEMO2life® to a standard preservation solution for hypothermic static lung preservation aiming to improve early functional parameters after lung transplantation. HEMO2life® is a natural oxygen carrier extracted from Arenicola marina with high oxygen affinity developed as an additive to standard organ preservation solutions. Standard preservation solution (Perfadex®) was compared with Perfadex® associated with HEMO2life® and with sham animals after 24 h of hypothermic preservation followed by lung transplantation. During five hours of lung reperfusion, functional parameters and biomarkers expression in serum and in bronchoalveolar lavage fluid (BALF) were measured. After five hours of reperfusion, HEMO2life® group led to significant improvement in functional parameters: reduction of graft vascular resistance (p < .05) and increase in graft oxygenation ratio (p < .05). Several ischemia-reperfusion related biomarkers showed positive trends in the HEMO2life® group: expression of HMG B1 in serum tended to be lower in comparison (2.1 ± 0.8 vs. 4.6 ± 1.5) with Perfadex® group, TNF-α and IL-8 in BALF were significantly higher in the two experimental groups compared to control (p < .05). During cold ischemia, expression of HIF1α and histology remained unchanged and similar to control. Supplementation of the Perfadex® solution by an innovative oxygen carrier HEMO2life® during hypothermic static preservation improves early graft function after prolonged cold ischemia in lung transplantation.
... Attempts to develop large polymerized bovine Hb by increasing the cross-link density (50:1, i.e., molar ratio of glutaraldehyde to Hb) have yielded no vasoconstriction compared to smaller polymeric Hbs (26), and other toxicities have not yet been explored. Natural acellular Hbs of terrestrial (47) and marine (150) worms are acellular and have evolved in this manner. Hb from the marine worm Arenicola marina is being developed as an HBOC (150), and with similar properties, a zero-linked polymeric Hb (63) has been developed by OXYVITA, Inc., New Windsor, NY. ...
... Natural acellular Hbs of terrestrial (47) and marine (150) worms are acellular and have evolved in this manner. Hb from the marine worm Arenicola marina is being developed as an HBOC (150), and with similar properties, a zero-linked polymeric Hb (63) has been developed by OXYVITA, Inc., New Windsor, NY. ...
Article
Significance: There has been a striking advancement in our understanding of red cell substitutes over the past decade. Although regulatory oversight has influenced many aspects of product development in this period, those who have approached the demonstration of efficacy of red cell substitutes have failed to understand their implication at the level of the microcirculation, where blood interacts closely with tissue. Recent advances: The understanding of the adverse effects of acellular hemoglobin (Hb)-based oxygen carriers (HBOCs) has fortunately expanded from Hb-induced renal toxicity to a more complete list of biochemical mechanism. In addition, various unexpected adverse reactions were seen in early clinical studies. The effects of the presence of acellular Hb in plasma are relatively unique because of the convergence of mechanical and biochemical natures. Critical issues: Controlling the variables using genetic engineering and chemical modification to change specific characteristics of the Hb molecule may allow for solving the complex multivariate problems of acellular Hb vasoactivity. HBOCs may never be rendered free of negative effects; however, quantifying the nature and extent of microvascular complications establishes a platform for designing new ameliorative therapies. Future directions: It is time to leave behind the study of vasoactivity and toxicity based on bench-top measurements of biochemical changes and those based solely on systemic parameters in vivo, and move to a more holistic analysis of the mechanisms creating the problems, complemented with meaningful studies of efficacy.
... Il serait d'un grand intérêt pour prévenir l'anémie en cas de situations d'urgence, telles que des pertes sanguines brutales et importantes, accidentelles ou survenues en temps de guerre. Des administrations intraveineuses d'HEMOXYCarrier® (M101, HEMARINA SA) ont montré une oxygénation rapide et efficace dans des modèles animaux, sans occasionner d'effets secondairesTsai et al., 2012)Des tests ont été réalisés chez la souris par injection intraveineuse d'HEMOXYCarrier®. A cet effet M101 a été taguée avec un marqueur fluorescent rouge qui a révélé une rapide diffusion de la molécule dans la totalité du corps de l'animal, avec une rétention de plusieurs heures sans effets secondaires notables. ...
Thesis
Full-text available
Ce travail de thèse avait pour objectif le développement de systèmes de culture cellulaire, en 2D et en 3D, en mettant à profit les propriétés d’un transporteur d’oxygène marin, HEMOXCell®. Notre approche générale était articulée selon deux grands axes : un premier concernant l’évaluation de l’utilisation d’HEMOXCell® dans la culture de deux modèles cellulaires, et un second, utilisant les résultats obtenus à des fins d’ingénierie tissulaire. Dans le premier axe, l’évaluation de l’effet dose-réponse d’HEMOXCell® dans la culture des cellules CHO-S et des cellules souches mésenchymateuses (CSM), a permis de déterminer des concentrations de travail optimales, favorisant la viabilité et la prolifération cellulaire. Le modèle cellulaire CHO-S a contribué à la mise en place d’un test de performance de la molécule, et encouragé son utilisation dans des systèmes de bioproduction. Les essais menés sur les CSM ont quant à eux permis de valider l’innocuité de la molécule à de faibles doses et le maintien de l’état « souche ». L’idée d’associer les CSM à des supports poreux est prometteuse pour des applications d’ingénierie tissulaire, mais est soumise aux problèmes liés à l’oxygénation en profondeur des supports. Dans le second axe de ce projet, nous avons oeuvré à améliorer la colonisation de substituts osseux et méniscaux, en culture statique et dynamique, en présence d’HEMOXCell®. Parallèlement, une étude a été menée pour tenter de caractériser les cellules méniscales. Les analyses de la colonisation des biomatériaux suggèrent un effet bénéfique d’HEMOXCell® lorsqu’il est utilisé en complément des milieux de différenciation cellulaire. Ce travail a contribué à améliorer la compréhension de ce transporteur d’oxygène et à l’élargissement de ses potentiels champs d’utilisation notamment dans un cadre thérapeutique.
... ££ p < 0.01 £££ p < 0.001: in comparison with M101 alone. effects of M101 without its oxygen-carrier properties (Tsai et al., 2012), we showed that M101 lost its intracellular SOD activating effect. Thus, although the precise mechanisms are not elucidated, these results suggest that oxygen supply by M101 is an important feature in its hepatoprotective effect. ...
Article
The amanitins (namely α- and β-amanitin) contained in certain mushrooms are bicyclic octapeptides that, when ingested, are responsible for potentially lethal hepatotoxicity. M101 is an extracellular hemoglobin extracted from the marine worm Arenicola marina. It has intrinsic Cu/Zn-SOD-like activity and is currently used as an oxygen carrier in organ preservation solutions. Our present results suggest that M101 might be effective in reducing amanitin-induced hepatotoxicity and may have potential for therapeutic development.
... Le Gall et al. (2014), Le Meur et al. (2010), Lemaire et al. (2019), Rousselot et al. (2006a), (2006b), Moon-Massat (2017) y Tsai et al. (2012) informan que la hemoglobina extracelular de A. marina es una molécula gigante denominada Hemarina-M101 que ha sido purificada y utilizada para la producción de HEMOXYCarrier (HC, Hemarina S.A., France), producto desarrollado como transportador de oxígeno para fines terapéuticos, que en disolución puede alcanzar y entregar oxígeno a diferentes tejidos inaccesible por los glóbulos rojos. Opera en un simple gradiente de presión parcial de oxígeno, permitiendo que la absorción de oxígeno y su liberación no requiere de un efector alostérico. ...
Article
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The world is facing a pandemic caused by the viral infection of the SARS coronavirus-CoV-2 that triggers the COVID-19 disease. Currently, no specific treatment or vaccine is known to counteract the symptoms and conditions caused by the virus, but antiviral, anti-inflammatory, immunomodulatory, mechanical support treatment, hematic, and others are being administered based on multi-organ dysfunction. When facing a new virus, the treatments used have been combined, which have generated secondary clinical effects in the patient. This study will focus on the hematic mechanical support treatment, where the patient is in a state of oxygen deficiency. During this phase of development of the disease that the extracellular hemoglobin extracted from Arenicola marina (Linnaeus, 1758) can be an alternative treatment through a blood transplant, as it can transport up to 40 times more oxygen when compared to hemoglobin in human.
... During the COVID-19 pandemic, two hospitals in France embarked on a clinical trial using sandworm (Arenicola marina) blood to treat COVID-19 patients suffering from respiratory complications (VOA News, 2020). The sandworm blood was tested because of its ability to increase the oxygen level in the tissues of a patient whose respiratory system has been compromisedthe haemoglobin in the worms' blood binds to oxygen 40 times greater than human haemoglobin when saturated (Rousselot et al., 2006;Tsai et al., 2012). ...
Article
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An antiviral vaccine for COVID-19 is not yet available. Research on polychaete has revealed the ability of its purified haemoglobin in assisting oxygenation to vital organs. Polychaete’s blood and tissue extracts were used as an oxygen-carrier and as a reducing agent in the biosynthesis of silver nanoparticles (Ag-NPs), respectively. Polychaete-based oxygen carrier can be used in organ preservation and as potential treatment for near-death COVID-19 patients. Ag-NPs from polychaete, which are known for possessing excellent antimicrobial properties, may serve as a raw material for various products, including disinfectant, coating materials for paint, handrail, personal care and healthcare products, such as personal protective equipment (PPE). The COVID-19 pandemic may be a catalyst to unveil hidden potentials of polychaete, particularly its therapeutic properties in medicine.
... Arenicola marina erythrocruorin was used to make HEMOXY Carrier (Hemarina S.A., Morlaix, France) [216]. In several studies, HEMOXY Carrier has been shown to have antioxidant properties and not cause vasoconstriction in rodents [217,218]. Thus, the annelid hemoglobins show some promise in functioning as RBC substitutes; however, more studies are needed. HemoTech by HemoBioTech Inc. (Dallas, TX, USA) is a newer hemoglobin-based blood substitute. ...
Article
Blood transfusion is the second most used medical procedures in health care systems worldwide. Over the last few decades, significant changes have been evolved in transfusion medicine practices. These changes were mainly needed to increase safety, efficacy, and availability of blood products as well as reduce recipients' unnecessary exposure to allogeneic blood. Blood products collection, processing, and storage as well as transfusion practices throughout all patient populations were the main stream of these changes. Health care systems across the world have adopted some or most of these changes to reduce transfusion risks, to improve overall patients' outcome, and to reduce health care costs. In this article, we are going to present and discuss some of these recent modifications and their impact on patients' safety. Copyright © 2016 Elsevier Masson SAS. All rights reserved.
... 28 AmEc has also been successfully transfused into mice and hamsters without eliciting an immune response or significant changes in blood pressure. 29,30 However, AmEc was observed to quickly dissociate from the HBL into dodecamers when exposed to the relatively low ionic strength of human plasma in in vitro studies conducted at 378C, pH 7.4. 29 In contrast, the Ec of the terrestrial oligochaete L. terrestris (LtEc) does not dissociate in human plasma. ...
Article
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While many blood substitutes are based on mammalian hemoglobins (e.g. human hemoglobin, HbA), the naturally extracellular hemoglobins of invertebrates (a.k.a. erythrocruorins, Ecs) are intriguing alternative oxygen carriers. Specifically, the erythrocruorin of Lumbricus terrestris has been shown to effectively deliver oxygen in mice and rats without the negative side effects observed with HbA. In this study, the properties of six oligochaete Ecs (Lumbricus terrestris, Eisenia hortensis, Eisenia fetida, Eisenia veneta, Eudrilus eugeniae, and Amynthas gracilis) were compared in vitro to identify the most promising blood substitute candidate(s). Several metrics were used to compare the Ecs, including their oxidation rates, dissociation at physiological pH, thermal stability, and oxygen transport characteristics. Overall, the Ecs of Lumbricus terrestris (LtEc) and Eisenia fetida (EfEc) were identified as the most promising candidates, since they demonstrated high thermal and oligomeric stability, while also exhibiting relatively low oxidation rates. Interestingly, the O2 affinity of LtEc (P50 = 26.25 mm Hg at 37oC) was also observed to be uniquely lower than EfEc and all of the other Ecs (P50 = 9.29-13.62 mm Hg). Subsequent alignment of the primary sequences of LtEc and EfEc revealed several significant amino acid substitutions within the D subunit interfaces that may be responsible for this significant change in O2 affinity. Nonetheless, these results show that LtEc and EfEc are promising potential blood substitutes that are resistant to oxidation and denaturation, but additional experiments will need to be conducted to determine their safety, efficacy, and the effects of their disparate oxygen affinities in vivo. This article is protected by copyright. All rights reserved.
... Another recent exciting development in the area of novel HBOC molecules is the utilization of large molecular weight extracellular Hb isolated from marine invertebrates like polychaete annelid (e.g., the product HEMOXYCarrier from Hemarina, Morlaix, France) (138). Preclinical studies with this unique Hb molecule have shown reduced microvascular vasoconstriction and no significant impact on mean arterial blood pressure, compared with other HBOCs that utilize bovine or human Hb (139). Further investigation of this system is currently ongoing to establish its clinical potential as an oxygen carrier therapeutic. ...
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... Bovine hemoglobin has been used due to ease of production [6], similarity to human Hb and potentially favourable antioxidative properties [7]. Whereas marine worms produce large Hb polymers (erythrocruorins) that have naturally evolved to function outside the cellular environment and hence can be used as an HBOC without any post purification modifications [8]. However, the most common animal source for HBOC is human. ...
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Hemoglobin (Hb)-based oxygen carriers (HBOCs) have been engineered to replace or augment the oxygen carrying capacity of erythrocytes. However, clinical results have generally been disappointing, in part due to the intrinsic oxidative toxicity of Hb. The most common HBOC starting material is adult human or bovine Hb. However, it has been suggested that fetal Hb may offer advantages due to decreased oxidative reactivity. Large-scale manufacturing of HBOC will likely and ultimately require recombinant sources of human proteins. We, therefore, directly compared the functional properties and oxidative reactivity of recombinant fetal (rHbF) and recombinant adult (rHbA) Hb. rHbA and rHbF produced similar yields of purified functional protein. No differences were seen in the two proteins in: autoxidation rate; the rate of hydrogen peroxide reaction; NO scavenging dioxygenase activity; and the NO producing nitrite reductase activity. The rHbF protein was: less damaged by low levels of hydrogen peroxide; less damaging when added to human umbilical vein endothelial cells (HUVEC) in the ferric form; and had a slower rate of intrinsic heme loss. The rHbA protein was: more readily reducible by plasma antioxidants such as ascorbate in both the reactive ferryl and ferric states; less readily damaged by lipid peroxides; and less damaging to phosphatidylcholine liposomes. In conclusion in terms of oxidative reactivity, there are advantages and disadvantages to the use of rHbA or rHbF as the basis for an effective HBOC.
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In blood, the primary role of RBCs is to transport oxygen via highly regulated mechanisms involving hemoglobin (Hb). Hb is a tetrameric porphyrin protein comprising of two α- and two β-polypeptide chains, each containing an iron-containing heme group capable of binding one oxygen molecule. In military as well as civilian trauma, exsanguinating hemorrhage can lead to suboptimal tissue oxygenation and subsequent morbidity and mortality. In such cases, transfusion of whole blood or RBCs can significantly improve survival. However, blood products including RBCs have limited availability and portability and present additional challenges related to type matching, pathogenic contamination risks, and short shelf-life. These issues lead to substantial logistical barriers to their pre-hospital use in austere battlefield and remote civilian conditions. While robust efforts are underway to resolve these issues, recent research breakthroughs have led to bioinspired engineering of RBC surrogates, using various cross-linked, polymeric, and encapsulated forms of Hb. These “next-generation” Hb-based oxygen carriers (HBOCs) can potentially provide therapeutic oxygenation when whole blood or RBCs are not available. Several of these HBOCs have undergone rigorous pre-clinical and clinical evaluation, but have not yet received clinical approval in the USA for human use. This chapter will comprehensively review both historical and new HBOC designs, including current state-of-the-art and novel molecules in development, along with a critical discussion of successes and challenges in this field.
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This chapter highlights the diversity of invertebrate hemoglobin structures, and gathers together up of 100 years of research data. Among invertebrates, annelid hemoglobin structures have been the most widely studied, probably due to their typical symmetric shapes and sizes that allow direct observations to be made using transmission electron microscopy. Hemoglobin seems to be present in all phyla, which suggests a very ancient origin. Using the hemoglobin of the marine annelid Arenicola marina, the French biotechnology startup HEMARINA is developing medical products. The first generations of blood substitutes (hemoglobin oxygen carriers) were manufactured using intracellular hemoglobin (human and bovine) in order to function outside the red blood cells. However, HEMARINA technologies are based on a natural extracellular hemoglobin. This circulatory pigment, which is present in the blood vessels of A. marina, has evolved over a million years and is able to function extracellularly. Currently, two main products are under development at HEMARINA, namely HEMO 2 life® (for organ preservation) and HEMOXYCarrier® (as a universal oxygen carrier).
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The growing use of marginal organs for transplantation pushes current preservation methods towards their limits, and the need for improvement is pressing. We previously demonstrated the benefits of M101, a natural extracellular oxygen carrier compatible with hypothermia, for the preservation of healthy renal grafts in a porcine model of auto‐transplantation. Herein, we use a variant of this pre‐clinical model to evaluate M101 potential benefits both in static storage (CS) and in machine perfusion (MP) preservation in the transplantation outcomes for marginal kidneys. In the CS arm, despite the absence of obvious benefits within the first two weeks of follow‐up, M101 dose‐dependently improved long‐term function, normalizing creatininemia after 1 and 3 months. In the MP arm, M101 improved short and long‐term functional outcomes as well as tissue integrity. Importantly, we provide evidence for the additivity of machine perfusion and M101 functional effects, showing that the addition of the compound further improves organ preservation, by reducing short‐term function loss, with no loss of function or tissue integrity recorded throughout the follow‐up. Extending previous observations with healthy kidneys, the present results point at the M101 oxygen carrier as a viable strategy to improve current organ preservation methods in marginal organ transplantation. This article is protected by copyright. All rights reserved.
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Oxygen therapeutics are being developed for a variety of applications in transfusion medicine. In order to reduce the side-effects (vasoconstriction, systemic hypertension, and oxidative tissue injury) associated with previous generations of oxygen therapeutics, new strategies are focused on increasing the molecular diameter of hemoglobin obtained from mammalian sources via polymerization and encapsulation. Another approach towards oxygen therapeutic design has centered on using naturally occurring large molecular diameter hemoglobins (i.e. erythrocruorins) derived from annelid sources. Therefore, the goal of this study was to purify erythrocruorin from the terrestrial worm Lumbricus terrestris for diverse oxygen therapeutic applications. Tangential flow filtration (TFF) was used as a scalable protein purification platform to obtain a >99% pure LtEc product, which was confirmed by size exclusion high performance liquid chromatography and SDS-PAGE analysis. In vitro characterization concluded that the ultra-pure LtEc product had oxygen equilibrium properties similar to human red blood cells, and a lower rate of auto-oxidation compared to human hemoglobin, both of which should enable efficient oxygen transport under physiological conditions. In vivo evaluation concluded that the ultra-pure product had positive effects on the microcirculation sustaining functional capillary density compared to a less pure product (~86% purity). In summary, we purified an LtEc product with favorable biophysical properties that performed well in an animal model using a reliable and scalable purification platform to eliminate undesirable proteins.
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We report the development of a mathematical model that quantifies the effects of small changes in systemic hematocrit (Hct) on the transport of nitric oxide (NO) in the microcirculation. The model consists of coupled transport equations for NO and oxygen (O2) and accounts for both shear-induced NO production by the endothelium and the effect of changing systemic Hct on the rate of NO production and the rate of NO scavenging by red blood cells. To incorporate the dependence of the plasma layer width on changes in Hct, the model couples the hemodynamics of blood in arterioles with NO and O2 transport in the plasma layer. A sensitivity analysis was conducted to determine the effects of uncertain model parameters (the thicknesses of endothelial surface layers and diffusion coefficients of NO and O2 in muscle tissues and vascular walls) on the model's predictions. Our analysis reveals that small increases in Hct may raise NO availability in the vascular wall. This finding sheds new light on the experimental data that show that the blood circulation responds to systematic increases of Hct in a manner that is consistent with increasing NO production followed by a plateau.
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Heme oxygenase is a heme-oxidizing enzyme which generates biliverdin and carbon monoxide (CO). The present study was designed to elucidate whether CO endogenously produced by this enzyme serves as an active vasorelaxant in the hepatic microcirculation. Microvasculature of the isolated perfused rat liver was visualized by dual-color digital microfluorography to alternately monitor sinusoidal lining and fat-storing Ito cells. In the control liver, the CO flux in the venous effluent ranged at 0.7 nmol/min per gram of liver. Administration of a heme oxygenase inhibitor zinc protoporphyrin IX (1 microM) eliminated the baseline CO generation, and the vascular resistance exhibited a 30% elevation concurrent with discrete patterns of constriction in sinusoids and reduction of the sinusoidal perfusion velocity. The major sites of the constriction corresponded to local sinusoidal segments colocalized with Ito cell which were identified by imaging their vitamin A autofluorescence. The increase in the vascular resistance and sinusoidal constriction were attenuated significantly by adding CO (1 microM) or a cGMP analogue 8-bromo-cGMP (1 microM) in the perfusate. From these findings, we propose that CO can function as an endogenous modulator of hepatic sinusoidal perfusion through a relaxing mechanism involving Ito cells.
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The effect of molecular dimension of hemoglobin (Hb)-based O(2) carriers on the diameter of resistance arteries (A(0), 158 +/- 21 microm) and arterial blood pressure were studied in the conscious hamster dorsal skinfold model. Cross-linked Hb (XLHb), polyethylene glycol (PEG)-conjugated Hb, hydroxyethylstarch-conjugated XLHb, polymerized XLHb, and PEG-modified Hb vesicles (PEG-HbV) were synthesized. Their molecular diameters were 7, 22, 47, 68, and 224 nm, respectively. The bolus infusion of 7 ml/kg of XLHb (5 g/dl) caused an immediate hypertension (+34 +/- 13 mmHg at 3 h) with a simultaneous decrease in A(0) diameter (79 +/- 8% of basal value) and a blood flow decrease throughout the microvascular network. The diameter of smaller arterioles did not change significantly. Infusion of larger O(2) carriers resulted in lesser vasoconstriction and hypertension, with PEG-HbV showing the smallest changes. Constriction of resistance arteries was found to be correlated with the level of hypertension, and the responses were proportional to the molecular dimensions of the O(2) carriers. The underlying mechanism is not evident from these experiments; however, it is likely that the effects are related to the diffusion properties of the different Hb molecules.
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The hamster window chamber model was used to study systemic and microvascular hemodynamic responses to extreme hemodilution with low- and high-viscosity plasma expanders (LVPE and HVPE, respectively) to determine whether plasma viscosity is a factor in homeostasis during extreme anemic conditions. Moderated hemodilution was induced by two isovolemic steps performed with 6% 70-kDa dextran until systemic hematocrit (Hct) was reduced to 18% (level 2). In a third isovolemic step, hemodilution with LVPE (6% 70-kDa dextran, 2.8 cP) or HVPE (6% 500-kDa dextran, 5.9 cP) reduced Hct to 11%. Systemic parameters, cardiac output (CO), organ flow distribution, microhemodynamics, and functional capillary density, were measured after each exchange dilution. Fluorescent-labeled microspheres were used to measure organ (brain, heart, kidney, liver, lung, and spleen) and window chamber blood flow. Final blood and plasma viscosities after the entire protocol were 2.1 and 1.4 cP, respectively, for LVPE and 2.8 and 2.2 cP, respectively, for HVPE (baseline = 4.2 and 1.2 cP, respectively). HVPE significantly elevated mean arterial pressure and CO compared with LVPE but did not increase vascular resistance. Functional capillary density was significantly higher for HVPE [87% (SD 7) of baseline] than for LVPE [42% (SD 11) of baseline]. Increases in mean arterial blood pressure, CO, and shear stress-mediated factors could be responsible for maintaining organ and microvascular perfusion after exchange with HVPE compared with LVPE. Microhemodynamic data corresponded to microsphere-measured perfusion data in vital organs.
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Annelid erythrocruorins are extracellular respiratory complexes assembled from 180 subunits into hexagonal bilayers. Cryo-electron microscopic experiments have identified two different architectural classes. In one, designated type I, the vertices of the two hexagonal layers are partially staggered, with one hexagonal layer rotated by about 16 degrees relative to the other layer, whereas in the other class, termed type II, the vertices are essentially eclipsed. We report here the first crystal structure of a type II erythrocruorin, that from Arenicola marina, at 6.2 A resolution. The structure reveals the presence of long continuous triple-stranded coiled-coil "spokes" projecting towards the molecular center from each one-twelfth unit; interdigitation of these spokes provides the only contacts between the two hexagonal layers of the complex. This arrangement contrasts with that of a type I erythrocruorin from Lumbricus terrestris in which the spokes are broken into two triple-stranded coiled coils with a disjointed connection. The disjointed connection allows formation of a more compact structure in the type I architecture, with the two hexagonal layers closer together and additional extensive contacts between the layers. Comparison of sequences of the coiled-coil regions of various linker subunits shows that the linker subunits from type II erythrocruorins possess continuous heptad repeats, whereas a sequence gap places these repeats out of register in the type I linker subunits, consistent with a disjointed coiled-coil arrangement.
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Many researchers have tested a hemoglobin (Hb) solution as a possible oxygen carrier after discovering that one Hb molecule contains four hemes that bind and release oxygen reversibly, and that blood type antigens are expressed on the surface of red blood cells (RBCs). However, various side effects emerged during the long development of Hb-based oxygen carriers (HBOCs). The physiological significance of the RBC structure is undergoing reconsideration. Fundamentally, excessive native Hb molecules are toxic, but encapsulation can shield this toxic effect. So-called liposomeencapsulated Hb or Hb-vesicles that mimic the cellular structure of RBCs have been developed for clinical applications.
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To compare survival after exchange transfusion followed by hemorrhage using: 1) the vasoactive, oxygen-carrying, bovine hemoglobin-based blood substitute Oxyglobin (Biopure, 12.9 g hemoglobin/dL); and 2) the hydroxyethyl starch plasma expander Hextend (high molecular weight and low degree of substitution, 6%). Comparison between treatments. Laboratory. Awake hamster chamber window model. Fifty percent blood volume exchange transfusion followed by a 60% hemorrhage over 1 hr, followed by 1 hr of observation. Measurement of blood gases, mean arterial blood pressure, functional capillary density, arteriolar and venular diameter, and Po2 tension distribution. Survival with Oxyglobin was 100% and only 50% for the Hextend group. Vasoconstriction was evident in the microcirculation. Mean arterial pressure was higher in the Oxyglobin group. Functional capillary density was significantly reduced, although to a lesser extent by Oxyglobin. There was no difference in microvascular Po2 distribution after 1 hr of shock between groups. Higher mean arterial pressure during the initial stages of hemorrhage could be due to vasoconstriction in the Oxyglobin group as compared to the Hextend group. It is concluded that the pressor effect due to a vasoactive oxygen carrier may be beneficial in maintaining perfusion in conditions of severe hemodilution followed by hypovolemia.
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Publisher Summary The primary difficulty in assaying Superoxide dismutase (SOD) for its enzymatic activity consists in the free radical nature of its substrate O 2 · - which can only be supplied by generation within the assay medium. The substrate O 2 · - cannot easily be detected directly by conventional analytical tools. Routine testing of SOD, therefore, is performed according to a general principle, which is explained in the chapter. If an absolute measure of physiological levels of SOD is intended, a direct immunochemical method is suggested in addition to activity measurements. This chapter focuses on measuring SOD in biological media. It presents a qualitative test for SOD activity based on the reduction of nitro blue tetrazolium (NBT) by O 2 · - , a simple immunological determination of the SOD molecule of sufficient sensitivity and avoiding labeled reagents, and an indirect measure of SOD activity to be used in purified samples.
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An experimental model was designed for direct, quantitative studies of hemodynamic and morphologic parameters in the microcirculation. It consists of implanting a modified Algire chamber in the dorsal skin flap of hamsters and the implementation of two permanent catheters in jugular vein and carotid artery. The microcirculation was studied using intravital microscopy and television techniques for in situ measurements of blood cell velocity and vascular diameters. Due to the poor contrast between blood cells, blood capillaries and surrounding s.c. tissue, microvascular beds were visualized using fluorescent microscopy after i.v. injection of 0.2 ml of 5% FITC-Dextran 150. The combination of optical elements and low amounts of FITC-Dextran improved the contrast of the televised image without changing macro- and microhemodynamic parameters, and blood plasma was delineated as bright structure against the substantially darker background of red blood cells and surrounding tissue. This permitted the quantitative study of practically all blood vessels within a given field of s.c. tissue in unanesthetized animals. Blood cell velocity in arterioles was 0.7–1.1 mm/s, 0.2–0.7 mm/s in midcapillaries and reached 0.6 mm/s in collecting venules. Since i.v. injection of drugs and systemic pressure measurements are possible in this model, it provides a unique means for studying the reactivity of the microcirculation over a prolonged period.
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Ischemia-reperfusion injury was investigated in terms of functional capillary density (FCD), capillary red blood cell velocity (cRBCv), and arteriolar and venular diameter after 4-h ischemia in the unanesthetized hamster skin-fold preparation. Animals in group 1 were studied by transillumination. Group 2 received a bolus injection of fluorescein isothiocyanate (FITC)-dextran (mol wt 150,000) and was studied by transillumination (zone 1) and epi-illumination (zone 2). In group 1, FCD decreased after ischemia (92% of baseline, 30 min), returning to control up to 24 h. cRBCv increased after reperfusion, being 175% of baseline at 24 h. Arterioles and venules dilated for 24 h after reperfusion. In group 2/zone 2, FCD progressively decreased to 11% of control, arteriolar dilation was inhibited, and cRBCv increased 30 min and 2 h after reperfusion. Tissue perfusion index (FCD x cRBCv) increased 158% in group 1 at 24 h, did not change in group 2/zone 1, and was 9% of control at 24 h in group 2/zone 2 (P < or = 0.05). We conclude that increased perfusion is a normal reaction to ischemia-reperfusion injury in this model, and previously observed capillary no reflow is due to FITC-dextran phototoxicity.
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To elucidate the quaternary structure of the extracellular haemoglobin (Hb) of the marine polychaete Arenicola marina (lugworm) it was subjected to multi-angle laser-light scattering (MALLS) and to electrospray-ionisation mass spectrometry (ESI-MS). It was also subjected to SDS/PAGE analysis for comparative purposes. MALLS analysis gave a molecular mass of 3648 +/- 24 kDa and a gyration radius of 11.3 +/- 1.7 nm. Maximum entropy analysis of the multiply charged electrospray spectra of the native, dehaemed, reduced and carbamidomethylated Hb forms, provided its complete polypeptide chain and subunit composition. We found, in the reduced condition, eight globin chains of molecular masses 15952.5 Da (a1), 15974.8 Da (a2), 15920.9 Da (b1), 16020.1 Da (b2), 16036.2 Da (b3), 16664.8 Da (c), 16983.2 Da (d1), 17033.1 Da (d2) and two linker chains L1, 25174.1 Da, and L2, 26829.7 Da. In the native Hb, chains b, c, d occur as five disulphide-bonded trimer subunits T with masses of 49560.4 Da (T1), 49613.9 Da (T2), 49658.6 Da (T3), 49706.8 Da (T4), 49724.5 Da (T5). Linker chains L1 and L2 occur as one disulphide-bonded homodimer 2L1 (D1) of 50323.1 Da and one disulphide-bonded heterodimer L1-L2 (D2) of 51 981.5 Da. Polypeptide chains a and d possess one free cysteine residue and chains d possess an unusual total of five cysteine residues. Semi-quantitative analysis of ESI-MS data allowed us to propose the following model for the one-twelfth protomer: [(3a1)(3a2)2T] (T corresponding to either T3, T4 or T5). From electron micrograph data T1 and T2 are probably located at the centre of the molecule as mentioned in previous studies. The Hb would thus be composed of 198 polypeptide chains with 156 globin chains and 42 linker chains, each twelfth being in contact with 3.5 linker subunits, providing a total mass of 3682 kDa including haems in agreement with the experimental molecular mass determined by MALLS. From ESI-MS relative intensities and the model proposed above, the globin/linker ratio gave 0.71:0.29 and 0.73:0.27, respectively. The estimation of haem content by pyridine haemochromogen and by cyanmethaemoglobin (HiCN) methods also support the globin chain number provided by ESI-MS.
Article
The clinical utility of cross-linked tetrameric hemoglobin solutions is limited by peripheral vasoconstriction thought to be due to scavenging of nitric oxide. In addition, transfusion of crude preparations of hemoglobin polymers can cause arterial hypertension. We tested the hypothesis that eliminating low-molecular-weight components from the polymer solution would prevent extravasation and its associated pressor response. A zero-link polymer of bovine hemoglobin was developed without chemical linkers left between the tetramers. Transfusion of unprocessed preparations of these polymers in rats resulted in appearance of the polymer in the renal hilar lymph. However, eliminating the low-molecular-weight components with a 300-kDa diafiltration resulted in an average hydrodynamic radius of 250 A and in undetectable levels of polymer in hilar lymph. Exchange transfusion in anesthetized rats and cats and in awake cats produced no increase in arterial pressure. In anesthetized cats, exchange transfusion with an albumin solution reduced hematocrit from 30 to 18%, increased cerebral blood flow, and dilated pial arterioles. In contrast, reducing hematocrit by transfusing the diafiltered polymer did not increase cerebral blood flow as pial arterioles constricted. These results are consistent with the hypothesis that the increase in arterial pressure associated with cell-free hemoglobin transfusion depends on hemoglobin extravasation. Constriction observed in the cerebrovascular bed with a nonextravasating hemoglobin polymer at low hematocrit is presumably a regulatory response to prevent overoxygenation at low blood viscosity.
Article
Alternative oxygen therapeutics are oxygen carriers that can potentially replace red blood cell transfusion in clinical instances in which banked blood is unavailable or unsafe. Most of the candidate solutions have been based on hemoglobin, derived from animals, outdated banked human blood, or recombinant systems. Other solutions are based on perfluorocarbons. In contrast to hemoglobin, which chemically binds oxygen, perfluorocarbons carry oxygen as a dissolved gas. Most of the products of either category developed over the past two decades have failed attempts at regulatory approval for clinical use, but their development has led to a large body of research that has defined the way oxygen is normally delivered to tissues. From this research, a new generation of products is emerging, designed specifically to target oxygen to the tissues in most need. It is possible that one or more of the earlier products may be approved by regulatory agencies in 1 or 2 years. However, a universally compatible and safe product most likely will be unavailable for several more years.
Article
Chemically modified or genetically engineered haemoglobins (Hbs) developed as oxygen therapeutics (often termed 'blood substitutes') are designed to correct oxygen deficit due to ischaemia in a variety of clinical settings. These modifications are intended to stabilize Hb outside its natural environment--red blood cells--in a functional tetrameric and/or polymeric form. Uncontrolled haem-mediated oxidative reactions of cell-free Hb and its reactions with various oxidant/antioxidant and cell signalling systems have emerged as an important pathway of toxicity. Current protective strategies designed to produce safe Hb-based products are focused on controlling or suppressing the 'radical' nature of Hb while retaining its oxygen-carrying function.
Article
The past 20 years have witnessed the publication of numerous studies on hemoglobins (Hbs) from deep-sea animals. Most of the animals studied were collected at deep-sea hydrothermal vents and cold seeps, both being environments where the physical-chemical conditions may be severely challenging for metazoans. These environments may be characterized by deep, chronic hypoxia and high concentrations of toxic compounds such as sulfide and heavy metals. Many species from these environments express Hbs, even though they belong to taxa that otherwise were characterised by the absence of respiratory pigments. Hbs from vent and seep invertebrates commonly exhibit high affinities for oxygen when compared to related species from normoxic, shallow-water environments, and marked pH-dependence. These high affinities permit uptake of oxygen from hypoxic waters and the strong Bohr effects favor its release in the metabolizing acidic organs.
Article
Nitric oxide (NO) plays a fundamental role in maintaining normal vasomotor tone. Recent data implicate a critical function for hemoglobin and the erythrocyte in regulating the activity of NO in the vascular compartment. Intravascular hemolysis releases hemoglobin from the red blood cell into plasma (cell-free plasma hemoglobin), which is then able to scavenge endothelium-derived NO 600-fold faster than erythrocytic hemoglobin, thereby disrupting NO homeostasis. This may lead to vasoconstriction, decreased blood flow, platelet activation, increased endothelin-1 expression (ET-1), and end-organ injury, thus suggesting a novel mechanism of disease for hereditary and acquired hemolytic conditions such as sickle cell disease and cardiopulmonary bypass. Furthermore, therapy with NO gas inhalation or infusion of sodium nitrite during hemolysis may attenuate this disruption in vasomotor balance by oxidizing plasma cell-free hemoglobin, thereby preventing the consumption of endogenous NO and the associated pathophysiological changes. In addition to providing an NO scavenging role in the physiological regulation of NO-dependent vasodilation, hemoglobin and the erythrocyte may deliver NO as the hemoglobin deoxygenates. While this process has previously been ascribed to S-nitrosated hemoglobin, recent data from our laboratories suggest that deoxygenated hemoglobin reduces nitrite to NO and vasodilates the human circulation along the physiological oxygen gradient. This newly described role of hemoglobin as a nitrite reductase is discussed in the context of blood flow regulation, oxygen sensing, and nitrite-based therapeutics.
Article
The need to develop a blood substitute is now urgent because of the increasing concern over Europe's BSE outbreak and the worldwide HIV/AIDS epidemic, which have cut blood supplies. Extracellular soluble hemoglobin has long been studied for its possible use as a safe and effective alternative to blood transfusion, but this has met with little success. Clinical trials have revealed undesirable side effects-oxidative damage and vasoconstriction-that hamper the application of cell-free hemoglobin as a blood substitute. We have addressed these problems and have found a new promising extracellular blood substitute: the natural giant extracellular polymeric hemoglobin of the polychaete annelid Arenicola marina. Here we show that it is less likely to cause immunogenic response; its functional and structural properties should prevent the side effects often associated with the administration of extracellular hemoglobin. Moreover, its intrinsic properties are of interest for other therapeutic applications often associated with hemorrhagic shock (ischemia reperfusion, treatment of septic shock and for organ preservation prior to transplantation). Moreover, using natural hemoglobin is particularly useful since recombinant DNA techniques could be used to express the protein in large quantities.
Article
An alternative to blood transfusion, based on oxygen-carrying solutions, has been sought for over a century. The present 'first-generation' haemoglobin-products were based on observations that crosslinking with, for example, glutaraldehyde, overcame subunit dissociation and renal toxicity. Experience with these solutions has shown that they can be vasoactive, sometimes increasing blood pressure, sometimes decreasing tissue perfusion and sometimes both. Clinical trials have been disappointing because of unexpected toxicity. The 'second-generation' products are based on a better understanding of the mechanisms of this vasoconstriction. Such products may seem counterintuitive by traditional standards, but it is hoped that they will be less toxic, more beneficial to patients, and more economical to produce.