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      Characterization of L-amino Acid Oxidase Derived from Crotalus adamanteus Venom: Procoagulant and Anticoagulant Activities

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          Abstract

          Snake venom enzymes of the L-amino acid oxidase (LAAO) class are responsible for tissue hemorrhage, edema, and derangement of platelet function. However, what role, if any, these flavoenzymes play in altering plasmatic coagulation have not been well defined. Using coagulation kinetomic analyses (thrombelastograph-based), it was determined that the LAAO derived from Crotalus adamanteus venom displayed a procoagulant activity associated with weak clot strength (no factor XIII activation) similar to thrombin-like enzymes. The procoagulant activity was not modified in the presence of reduced glutathione, demonstrating that the procoagulant activity was likely due to deamination, and not hydrogen peroxide generation by the LAAO. Further, unlike the raw venom of the same species, the purified LAAO was not inhibited by carbon monoxide releasing molecule-2 (CORM-2). Lastly, exposure of the enzyme to phenylmethylsulfonyl fluoride (PMSF) resulted in the LAAO expressing anticoagulant activity, preventing contact activation generated thrombin from forming a clot. In sum, this investigation for the first time characterized the LAAO of a snake venom as both a fibrinogen polymerizing and an anticoagulant enzyme acting via oxidative deamination and not proteolysis as is the case with thrombin-like enzymes (e.g., serine proteases). Using this thrombelastographic approach, future investigation of purified enzymes can define their biochemical nature.

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          Most cited references26

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          A high-throughput venom-gland transcriptome for the Eastern Diamondback Rattlesnake (Crotalus adamanteus) and evidence for pervasive positive selection across toxin classes.

          Despite causing considerable human mortality and morbidity, animal toxins represent a valuable source of pharmacologically active macromolecules, a unique system for studying molecular adaptation, and a powerful framework for examining structure-function relationships in proteins. Snake venoms are particularly useful in the latter regard as they consist primarily of a moderate number of proteins and peptides that have been found to belong to just a handful of protein families. As these proteins and peptides are produced in dedicated glands, transcriptome sequencing has proven to be an effective approach to identifying the expressed toxin genes. We generated a venom-gland transcriptome for the Eastern Diamondback Rattlesnake (Crotalus adamanteus) using Roche 454 sequencing technology. In the current work, we focus on transcripts encoding toxins. We identified 40 unique toxin transcripts, 30 of which have full-length coding sequences, and 10 have only partial coding sequences. These toxins account for 24% of the total sequencing reads. We found toxins from 11 previously described families of snake-venom toxins and have discovered two putative, previously undescribed toxin classes. The most diverse and highly expressed toxin classes in the C. adamanteus venom-gland transcriptome are the serine proteinases, metalloproteinases, and C-type lectins. The serine proteinases are the most abundant class, accounting for 35% of the toxin sequencing reads. Metalloproteinases are the most diverse; 11 different forms have been identified. Using our sequences and those available in public databases, we detected positive selection in seven of the eight toxin families for which sufficient sequences were available for the analysis. We find that the vast majority of the genes that contribute directly to this vertebrate trait show evidence for a role for positive selection in their evolutionary history. Copyright © 2011 Elsevier Ltd. All rights reserved.
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            Plasma glutathione peroxidase in healthy young adults: influence of gender and physical activity.

            Oxidative stress is implicated in the pathophysiology of many cardiovascular diseases. Plasma glutathione peroxidase (pl x GPx, GPx3) is an antioxidant enzyme found in the extracellular fluid. This study aimed to determine reference values for serum GPx3 concentration and GPx activity in young healthy males and females of similar aerobic fitness and to determine the impact of acute physical activity on serum GPx. 52 young, healthy but not physically trained subjects (24 male, 28 female; age = 20.4 +/- 0.7 yr, cycling VO(2 max) = 39.2 +/- 1.6 mL/kg/min; mean +/- SE) participated in this study. An independent group of 18 subjects participated in an acute, 90 min bout of 50% VO(2 max) cycling exercise. Serum GPx activity and GPx3 protein levels, as well as estradiol and 8-iso- prostaglandin F(2alpha) (8-iso-PGF(2alpha;) an index of lipid peroxidation) were determined. Females had significantly higher serum GPx3 concentration (29.1 +/- 1.6 vs. 24.2 +/- 1.3 mg/L, p < 0.01) and serum GPx activity (256.4 +/- 10.4 vs. 222.8 +/- 15.6 U/L, p < 0.05) than males; specific activity (U/mg) was not different between genders. There was no significant gender difference in 8-iso-PGF(2alpha). No significant correlation was found between either GPx activity or GPx3 concentration and serum estradiol or VO(2)(max). The acute, prolonged, mild intensity exercise did not affect serum GPx activity or 8-iso-PGF(2a) levels in males or females. The results of this study suggest that in a young, healthy but not physically well-trained population females are endowed with slightly higher serum GPx3 concentrations and GPx activities than males, but the functional significance of this has not been established. Furthermore, the results indicate that serum GPx levels are not associated with aerobic fitness level, or serum estradiol concentration and that acute, prolonged, mild exercise does not affect the activity of serum GPx in this population.
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              Toxins in thrombosis and haemostasis: potential beyond imagination.

              R Kini (2011)
              Exogenous factors isolated from venoms of snakes and saliva of haematophagous animals that affect thrombosis and haemostasis have contributed significantly to the development of diagnostic agents, research tools and life-saving drugs. Here, I discuss recent advances in the discovery, structural and functional characterisation, and mechanism of action of new procoagulant and anti-haemostatic proteins. In nature, these factors have evolved to target crucial 'bottlenecks' in the coagulation cascade and platelet aggregation. Several simple protein scaffolds are used to target a wide variety of target proteins and receptors exhibiting functional divergence. Different protein scaffolds have also evolved to target identical, physiologically relevant key enzymes or receptors exhibiting functional convergence. At times, exogenous factors bind to the same target protein, but at distinct sites, to differentially attenuate their functions exhibiting mechanistic divergence within the same family of proteins. The structure-function relationships of these factors are subtle and complicated but represent an exciting challenge. These studies provide ample opportunities to design highly specific and precise ligands to achieve desired biological target function. Although only a small number of them have been characterised to date, the molecular and mechanical diversities of these exogenous factors and their contributions to understanding molecular and cellular events in thrombosis and haemostasis as well as developing diagnostic and research tools and therapeutic agents, is outstanding. Based on the current status, I have attempted to identify future potential and prospects in this area of research.
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                Author and article information

                Journal
                Int J Mol Sci
                Int J Mol Sci
                ijms
                International Journal of Molecular Sciences
                MDPI
                1422-0067
                30 September 2019
                October 2019
                : 20
                : 19
                : 4853
                Affiliations
                Department of Anesthesiology, University of Arizona College of Medicine, Tucson, AZ 85719, USA; vgnielsen333@ 123456gmail.com
                Article
                ijms-20-04853
                10.3390/ijms20194853
                6801523
                31574907
                2931ba46-30d1-4bb4-84f9-a6cf6a26bc2c
                © 2019 by the author.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

                History
                : 12 September 2019
                : 27 September 2019
                Categories
                Article

                Molecular biology
                procoagulant,anticoagulant,l-amino acid oxidase,thrombelastography,fibrinogen polymerizing enzyme,carbon monoxide releasing molecule

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