Fibrinogenolytic Activity Research Articles

The Contrasting Effects of Bothrops lanceolatus and Bothrops atrox Venom on Procoagulant Activity and Thrombus Stability under Blood Flow Conditions.

Consumption coagulopathy and hemorrhagic syndrome are the typical features of Bothrops sp. snake envenoming. In contrast, B. lanceolatus envenoming can induce thrombotic complications. Our aim was to test whether crude B. lanceolatus and B. atrox venoms would display procoagulant activity and induce thrombus formation under flow conditions. Fibrin formation in human plasma was observed for B. lanceolatus venom at 250-1000 ng/mL concentrations, which also induced clot formation in purified human fibrinogen, indicating thrombin-like activity. The degradation of fibrinogen confirmed the fibrinogenolytic activity of B. lanceolatus venom. B. lanceolatus venom displayed consistent thrombin-like and kallikrein-like activity increases in plasma conditions. The well-known procoagulant B. atrox venom activated plasmatic coagulation factors in vitro and induced firm thrombus formation under high shear rate conditions. In contrast, B. lanceolatus venom induced the formation of fragile thrombi that could not resist shear stress. Our results suggest that crude B. lanceolatus venom displays amidolytic activity and can activate the coagulation cascade, leading to prothrombin activation. B. lanceolatus venom induces the formation of an unstable thrombus under flow conditions, which can be prevented by the specific monovalent antivenom Bothrofav®.

Antivenom potential of the latex of Jatropha mutabilis baill. (Euphorbiaceae) against Tityus stigmurus venom: Evaluating its ability to neutralize toxins and local effects in mice

Ethnopharmacological relevanceSpecies of the Jatropha genus (Euphorbiaceae) are used indiscriminately in traditional medicine to treat accidents involving venomous animals. Jatropha mutabilis Baill., popularly known as “pinhão-de-seda,” is found in the semi-arid region of Northeastern Brazil. It is widely used as a vermifuge, depurative, laxative, and antivenom. Aim of the studyObtaining the phytochemical profile of the latex of Jatropha mutabilis (JmLa) and evaluate its acute oral toxicity and inhibitory effects against the venom of the scorpion Tityus stigmurus (TstiV). Materials and methodsThe latex of J. mutabilis (JmLa) was obtained through in situ incisions in the stem and characterized using HPLC-ESI-QToF-MS. Acute oral toxicity was investigated in mice. The protein profile of T. stigmurus venom was obtained by electrophoresis. The ability of latex to interact with venom components (TstiV) was assessed using SDS-PAGE, UV–Vis scanning spectrum, and the neutralization of fibrinogenolytic and hyaluronidase activities. Additionally, the latex was evaluated in vivo for its ability to inhibit local edematogenic and nociceptive effects induced by the venom. ResultsThe phytochemical profile of the latex revealed the presence of 75 compounds, including cyclic peptides, glycosides, phenolic compounds, alkaloids, coumarins, and terpenoids, among others. No signs of acute toxicity were observed at a dose of 2000 mg/kg (p.o.). The latex interacted with the protein profile of TstiV, inhibiting the venom's fibrinogenolytic and hyaluronidase activities by 100%. Additionally, the latex was able to mitigate local envenomation effects, reducing nociception by up to 56.5% and edema by up to 50% compared to the negative control group. ConclusionsThe latex of Jatropha mutabilis exhibits a diverse phytochemical composition, containing numerous classes of metabolites. It does not present acute toxic effects in mice and has the ability to inhibit the enzymatic effects of Tityus stigmurus venom in vitro. Additionally, it reduces nociception and edema in vivo. These findings corroborate popular reports regarding the antivenom activity of this plant and indicate that the latex has potential for treating scorpionism.

Comparative functional characterization and in vitro immunological cross-reactivity studies on Daboia russelii and Craspedocephalus malabaricus venom.

Snake venom is a complex mixture of organic and inorganic constituents, including proteins and peptides. Several studies showed that antivenom efficacy differs due to intra- and inter-species venom variation. In the current study, comparative functional characterization of major enzymatic proteins present in Craspedocephalus malabaricus and Daboia russelii venom was investigated through various in vitro and immunological cross-reactivity assays. The enzymatic assays revealed that hyaluronidase and phospholipase A2 activities were markedly higher in D. russelii. By contrast, fibrinogenolytic, fibrin clotting and L-amino acid oxidase activities were higher in C. malabaricus venom. ELISA results suggested that all the antivenoms had lower binding potential towards C. malabaricus venom. For D. russelii venom, the endpoint titration value was observed at 1:72 900 for all the antivenoms. In the case of C. malabaricus venom, the endpoint titration value was 1:2700, except for Biological E (1:8100). All these results, along with the avidity assays, indicate the strength of venom-antivenom interactions. Similarly, the western blot results suggest that all the antivenoms showed varied efficacies in binding and detecting the venom antigenic epitopes in both species. The results highlight the need for species-specific antivenom to better manage snakebite victims.

Variability in antivenom neutralization of Mexican viperid snake venoms.

Each year, 3,800 cases of snakebite envenomation are reported in Mexico, resulting in 35 fatalities. The only scientifically validated treatment for snakebites in Mexico is the use of antivenoms. Currently, two antivenoms are available in the market, with one in the developmental phase. These antivenoms, produced in horses, consist of F(ab')2 fragments generated using venoms from various species as immunogens. While previous studies primarily focused on neutralizing the venom of the Crotalus species, our study aims to assess the neutralization capacity of different antivenom batches against pit vipers from various genera in Mexico. We conducted various biological and biochemical tests to characterize the venoms. Additionally, we performed neutralization tests using all three antivenoms to evaluate their effectiveness against lethal activity and their ability to neutralize proteolytic and fibrinogenolytic activities. Our results reveal significant differences in protein content and neutralizing capacity among different antivenoms and even between different batches of the same product. Notably, the venom of Crotalus atrox is poorly neutralized by all evaluated batches despite being the primary cause of envenomation in the country's northern region. Furthermore, even at the highest tested concentrations, no antivenom could neutralize the lethality of Metlapilcoatlus nummifer and Porthidium yucatanicum venoms. These findings highlight crucial areas for improving existing antivenoms and developing new products. Our research reveals variations in protein content and neutralizing potency among antivenoms, emphasizing the need for consistency in venom characteristics as immunogens. While Birmex neutralizes more LD50 per vial, Antivipmyn excels in specific neutralization. The inability of antivenoms to neutralize certain venoms, especially M. nummifer and P. yucatanicum, highlights crucial improvement opportunities, given the medical significance of these species.

Скринінг продуцентів протеаз серед представників роду Bacillus, ізольованих з прибережної зони Кінбурнської коси

Protease-producing representatives of Bacillus have been isolated from various environments such as the rhizosphere, soil, sewage, food, waste, and saline environments (sea water, marine sediments, hypersaline lakes, salty food, soda lakes, etc.). However, today, little is known about the characteristics of proteases in representatives of the genus Bacillus, isolated from the coastal and aquatic environments. In this regard, the purpose of this work was to screen for the presence of different types of proteolytic activity in Bacillus cultures isolated from the coastal zone of the Kinburn split. Methods. The objects of the study were 15 cultures (L1-L15), isolated from the dry grass of the coastal zone of the Kinburn spit (Mykolaiv region). The cultures were grown under conditions of deep cultivation at 28 °С, with a mixing speed of the nutrient medium of 230 rpm for 2 days. Methods of determining proteolytic (caseinolytic, elastolytic, fibrinolytic, fibrinogenolytic, and collagenase) activity in the culture liquid supernatant were used. Results. Study of the spectrum of proteolytic activities of 15 strains of Bacillus sp. showed that L9, L1, L2 and L4 exhibited higher levels of proteolytic activity compared to the other strains tested. Bacillus sp. L9 and L2 showed the highest elastase activity (35.80 and 33.80 U/mL, respectively), L1, L2, and L4 − fibrinogenolytic (12.50, 11.20 and 10.33 U/mL, respectively), and L1 and L4 − fibrinolytic (13.00 and 10.90 U/mL, respectively) activity. Conclusions. According to its catalytic properties, a number of representatives of Bacillus sp. isolated from the dry grass of the coastal zone of the Kinburn split may be promising for further research as producers of enzymes with elastotic, fibrinolytic, and fibrinogenolytic activities.

Isolation and Functional Characterization of Erythrofibrase: An Alfa-Fibrinogenase Enzyme from Trimeresurus erythrurus Venom of North-East India.

Green pit viper bites induce mild toxicity with painful local swelling, blistering, cellulitis, necrosis, ecchymosis and consumptive coagulopathy. Several bite cases of green pit vipers have been reported in several south-east Asian countries including the north-eastern region of India. The present study describes isolation and characterization of a haemostatically active protein from Trimeresurus erythrurus venom responsible for coagulopathy. Using a two-step chromatographic method, a snake venom serine protease erythrofibrase was purified to homogeneity. SDS-PAGE of erythrofibrase showed a single band of ~30 kDa in both reducing and non-reducing conditions. The primary structure of erythrofibrase was determined by ESI LC-MS/MS, and the partial sequence obtained showed 77% sequence similarity with other snake venom thrombin-like enzymes (SVTLEs). The partial sequence obtained had the typical 12 conserved cysteine residues, as well as the active site residues (His57, Asp102 and Ser195). Functionally, erythrofibrase showed direct fibrinogenolytic activity by degrading the Aα chain of bovine fibrinogen at a slow rate, which might be responsible for causing hypofibrinogenemia and incoagulable blood for several days in envenomated patients. Moreover, the inability of Indian polyvalent antivenom (manufactured by Premium Serum Pvt. Ltd., Maharashtra, India) to neutralize the thrombin-like and plasmin-like activity of erythrofibrase can be correlated with the clinical inefficacy of antivenom therapy. This is the first study reporting an α-fibrinogenase enzyme erythrofibrase from T. erythrurus venom, which is crucial for the pathophysiological manifestations observed in envenomated victims.

Rapid Separation and Display of Active Fibrinogenolytic Agents in Sipunculus nudus through Fibrinogen-Polyacrylamide Gel Electrophoresis.

Fibrinogenolytic agents that can dissolve fibrinogen directly have been widely used in anti-coagulation treatment. Generally, identifying new fibrinogenolytic agents requires the separation of each component first and then checking their fibrinogenolytic activities. Currently, polyacrylamide gel electrophoresis (PAGE) and chromatography are mostly used in the separating stage. Meanwhile, the fibrinogen plate assay and reaction products based PAGE are usually adopted to display their fibrinogenolytic activities. However, because of the spatiotemporal separation of those two stages, it is impossible to separate and display the active fibrinogenolytic agents with the same gel. To simplify the separating and displaying processes of fibrinogenolytic agent identification, we constructed a new fibrinogen-PAGE method to rapidly separate and display the fibrinogenolytic agents of peanut worms (Sipunculus nudus)in this study. This method includes fibrinogen-PAGE preparation, electrophoresis, renaturation, incubation, staining, and decolorization. The fibrinogenolytic activity and molecular weight of the protein can be detected simultaneously. According to this method, we successfully detected more than one active fibrinogenolytic agent of peanut wormhomogenate within 6 h. Moreover, this fibrinogen-PAGE method is time and cost-friendly. Furthermore, this method could be used to study the fibrinogenolytic agents of the other organisms.

Role of gamma radiation as an agent modulator of Mucor subtilissimus UCP1262 Fibrinolytic Enzyme (MsFE)

Thrombosis is a dangerous condition that can result in major damage. Fibrinolytic enzymes are significant therapeutic agents for thrombosis. This study focused on MsFE, a fibrinolytic protease from M. subtilissimus UCP1262, exploring its physicochemical traits and the impact of gamma radiation on its activity. Investigations included: fibrinogenolytic activity, effects of solvents and ions, gamma rays, enzyme shelf life, FTIR, CD, clotted times determination, and enzymatic kinetics. Notably, the major results obtained show the protease retained 90 % activity in organic solvents, FTIR indicated carbonyl functional groups and Circular dichroism spectroscopic studies indicated that the secondary structure (β-sheet) of enzyme remains mostly unaffected over pH 2.0–9.0, whereas considerable changes were observed in the tertiary structure. Kinetic analysis revealed altered parameters post-irradiation. Gamma radiation initially enhanced enzyme activity (10 kGy) but progressively decreased beyond 20 kGy. Metallic ions, especially magnesium, augmented irradiated enzyme activity. This study highlights gamma radiation's potential as a modulator for enhanced fibrinolytic activity, suggesting future biomedical applications.

Biochemical and biological characterization of the venoms of Naja kaouthia and Naja mandalayensis from Myanmar and neutralization effects of BPI cobra antivenom

Snakebite is a neglected public health issue, with many scientific and medical issues to be solved. Cobras are among the most common venomous snakes in Myanmar and are responsible for a considerable number of severe snakebite envenoming. There are three species of cobra (Naja kaouthia, Naja mandalayensis and Ophiophagus hannah) in Myanmar. The study aims to characterize the N. kaouthia and N. mandalayensis venoms and to investigate the efficacy of anti-cobra antivenom (BPI) against the two venoms. Protein components and fibrinogenolytic activity were determined by SDS-PAGE. Enzymatic activities for PLA2, protease and acetylcholinesterase were determined by spectrophotometric method. Anticoagulant activity was determined by recalcification time of citrated human plasma. Myotoxicity, necrotizing activity, median lethal dose (LD50) and median effective dose (ED50) were determined by WHO recommended methods. The SDS-PAGE displayed the proteins and enzymes containing in two venoms were different. N. kaouthia venom exhibited more in PLA2, acetylcholinesterase, anticoagulant, fibrinogenolytic and necrotizing activities than N. mandalayensis venom. N. mandalayensis venom had more protease activity and myotoxicity than N. kaouthia venom. The median lethal dose (LD50) of N. kaouthia and N. mandalayensis venom was 4.33 μg/mouse and 5.04 μg/mouse respectively. Both venoms induced fibrinogen Aα chain degradation in 30 min (N. kaouthia) and in 6 h (N. mandalayensis). The same median effective dose (ED50) (19.56 μg/mouse) showed that anti-NK antivenom can neutralize against lethal effect of N. mandalayensis venom. It can also neutralize the protease activity, anticoagulant activity and fibrinogenolytic activity of both venoms. Immunodiffusion and immunoblotting studies showed that the antivenom recognized its homologous venom (N. kaouthia) and cross-reacted against the heterologous venom (N. mandalayensis). The anti-NK antivenom is suitable to use for N. mandalayensis bite if monospecific antivenom is not available.

Мixed-ligand complexes of germanium – 3d-metal with 1-hydroxyethane-1,1-diphosphonic acid аnd 2,2′-bipyridine as modulators of Bacillus sp. IMV B-7883 elastase and fibrinogenase activity

We have previously shown that Bacillus sp. IMV B-7883 exhibits both elastase and fibrinogenolytic activity. One of the approaches to enhance enzymatic activity is the use of coordination compounds capable to affect enzyme’s activity or synthesis. The purpose of this work was to study the effect of mixed-ligand complexes of Ge(IV) – Co(II) (Ni(II), Cu(II)) with 1-hydroxyethane-1,1-diphosphonic acid аnd 2,2′-bipyridine on the activity of elastase and fibrinogenase purified from Bacillus sp. IMV B-7883. Previously synthesized and characterized mixed-ligand complexes and enzymes purified from the supernatant of the bacterial culture liquid were used in the study. Elastase activity was determined colorimetrically with the use of Congo red, fibrinogenase activity was estimated by fibrinogen hydrolysis measured by absorption at 275 nm. It was shown that complexes 1 (C132H164Co4Ge6N24O68P12) and 2 (C132H148Ge6N24Ni4O60P12) inhibited activity of Bacillus sp. IMV B-7883 elastase by 54 and 71% respectively, while complex 3 (C92H128Cu4Ge6N16O63P12) enhanced it by 30%. Stimulating effect of all three complexes on fibrinogenase activity was revealed. Thus, complex 1 and 2 activated the enzyme by more than 50% and complex 1 – by 19%. The data obtained indicate a complex mechanism of the studied complexes influence on enzymatic activity depending on both their composition and structure. Keywords: 1-hydroxyethane-1‚1-diphosphonic acid, 2‚2′-bipyridine, Bacillus sp. IMV B-7883, elastase, fibrinogenase, germanium – 3d-metal, mixed-ligand complexes

<i>Aspergillus tabacinus</i> as a producer of antithrombotic proteases

Microfungi of the genus Aspergillus are well-known as producers of fibrinolytic and plasminogen activating proteases. But for development of new antithrombotics we should use strains which extracellular proteases correspond to these criteria: 1) demonstrate anticoagulant, fibrinolytic and plasminogen activating activities at the same time; 2) have narrow substrate specificity; 3) are able to hydrolase substrates of two following each other proteins of hemostasis system. According to these criteria Aspergillus tabacinus was chosen. Maximal activities of culture liquid of this strain grown in optimal conditions were 87 Е × 10–3 with activated protein C substrate S-2366 and 73 Е × 10–3 with thrombin substrate Chromozym TH. Fibrinogenolytic activity of lyophilized enzyme preparation after ammonium sulfate precipitation and dialysis was 779.1 Е/mg of protein.

Penicillium citrinum CFAM 521 Isolated From the Amazon Region: A Novel Source of a Fibrinolytic Enzyme.

Fibrinolytic agents are essential in treating thrombosis, playing a critical role in improving survival rates in cardiovascular diseases. Microbial fibrinolytic proteases have emerged as promising alternatives due to their affordability, specificity, lower toxicity, and reduced side effects. Consequently, the search for microorganisms capable of producing these enzymes has gained significant economic importance in the pharmaceutical industry. This study reports and characterizes a novel fibrinolytic enzyme produced by Penicillium citrinum CFAM 521, a strain isolated from the Amazon region. The enzyme was purified using a polyethylene glycol (PEG)-phosphate salt aqueous two-phase system (ATPS). The effects of PEG molecular weight, PEG concentration, and phosphate concentration on the protease partition coefficient (K) were evaluated through a 22 full factorial design. The enzyme exhibited both fibrinolytic and fibrinogenolytic activities. After partitioning in a two-phase system with 10% (w/w) PEG and 15% (w/w) sodium phosphate, the fibrinolytic proteases were predominantly retained in the salt-rich bottom phase (K = 0.33). The enzyme has a molecular weight of 34 kDa, with optimal pH and temperature at 9°C and 37°C, respectively. Inhibitory analysis confirmed that it is a serine protease, and its activity was enhanced by the addition of Mn2+. Notably, the enzyme exhibited no hemolytic activity. Therefore, P. citrinum CFAM 521 represents a novel source of fibrinolytic enzymes, highlighting its potential as an alternative for the development of thrombolytic agents.

Influence of Cultivation Temperature on Activity of Proteolitic Enzymes of Bacteria Isolated from the Deep-Water Bottom Sediments of the Black Sea

The cultivation temperature was shown to affect elastase, fibrinogenolytic and collagenase activity of the studied strains of <i>Bacillus subtilis</i> 1, <i>Priestia megaterium</i> 55, <i>Bacillus licheniformis</i> 249, isolated from the deep-water bottom sediments of the Black Sea. The enzymes' synthesis dynamics in different strains at different temperatures was different.

Proteolytic Activity of Bacillus Strains Isolated from Soil of Rice Agrocenosis

Microorganisms are the most common sources of commercial enzymes due to their physiological and biochemical properties, facile culture conditions, and ease of cell manipulation. Among microbial enzymes, proteases are ubiquitous in nature and have been found in all living forms encompassing the eukaryotes like plants, animals, fungi, and protists as well as the prokaryotic domains of bacteria and archaea. Proteases are the most important for the industry and constitute approximately 60% of the total industrial enzyme market. Among the bacteria, the genus Bacillus has a very prominent place in terms of the commercial production of proteases. Earlier from the water and bottom sediments of the Black Sea, we have isolated a number of producers of proteolytic enzymes from Bacillus species. The aim of this work was to investigate the ability of representatives of a number of soil bacilli species to synthesize enzymes that hydrolyze such protein substrates as elastin, fibrin, fibrinogen, and keratin. Methods. The objects of the study were 8 cultures (KS 1 — KS 8) isolated from the soil of the rice agrocenosis. Cultures were grown under conditions of deep cultivation at 28 °С, with a mixing speed of for the nutrient medium of 230 rpm for 4 days. Methods of determining proteolytic (caseinolytic, elastolytic, fibrinolytic, fibrinogenolytic, and keratinase) activity in the culture liquid supernatant were used. Disulfide reductase activity was measured spectrophotometrically at 412 nm by evaluating the yellow sulfide formed during the reduction of 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB). Results. The study of the spectrum of proteolytic activities of 8 freshly isolated strains showed that only KS 6 under experimental conditions did not show the ability to hydrolyze any of the studied substrates (casein, elastin, fibrin, fibrinogen, and keratin). Strains KS 1, KS 2, KS 7, and KS 8 showed higher levels of activity compared to other strains studied. The most interesting for further research are: І) strain KS 1, which showed the highest fibrinolytic activity, ІІ) strain KS 2 as the most effective producer with elastase and fibrinogenolytic activity, III) KS 7 and KS 8, which simultaneously showed the highest rates as keratinase (7 U/mL and 9 U/mL) and sulfate reductase (33 μmol/min and 31 μmol/min) activity, respectively. Conclusions. According to the catalytic properties, a number of representatives of Bacillus, isolated from the soil of the rice agrocenosis may be promising for further research as an enzyme producer with proteolytic activity.

Isolation and characterization of Bacillus sp. IMV B-7883 proteases

The representatives of Bacillus are some of the best protease producers studied so far since they exhibit broad substrate specificity, significant activity, stability, simple downstream purification, short period of fermentation and low cost. Earlier, we showed that Bacillus sp. IMV B-7883 strain synthesizes an extracellular proteases, which exhibit elastolytic and fibrinogenolytic activity. The aim of the work was to isolate and purify these enzymes from the culture liquid of the Bacillus sp. IMV B-7883 strain, as well as to study their properties. Isolation and purification of proteases was carried out by precipitation of the culture liquid with ammonium sulfate, gel permeation and ion exchange chromatography and rechromatography on Sepharose 6B. As a result, proteases with elastolytic and fibrinogenolytic activity with a molecular weight of 23 and 20 kDa respectively were isolated with elastase activity increased by 63.6 and fibrinogenolytic activity by 44.1 times. The enzyme with elastase activity had a pH-optimum of 7.0 and hydrolyzed only elastin, while the enzyme with fibrinogenolytic activity was an alkaline protease with a pH-optimum of 8.0 and in addition to fibrinogen, showed specificity for fibrin and, in trace amounts, for collagen. Keywords: Bacillus sp. IMV B-7883, elastase, fibrinogenase, pH optimum, substrate specificity

Proteolitic Activity of Marine Strain Bacillus sp. 051

The main interest in the study of marine microorganisms is due to their ability to produce a wide range of unique enzymes, including peptidases with different specificities. In recent years, interest has increased in peptidases that are able to cleave elastin as a specific substrate. Streptomyces fradiae and Bacillus thermoproteolyticus elastases are among the most potent elastolytic proteinases discovered to date because they are 4-8-fold more effective than pancreatic elastases. The disadvantages of these producers include the fact that most of them are pathogenic for humans, and the elastase enzyme secreted from them is directly involved in the initiation of the pathogenetic process. All this significantly limits the scope of their practical application. Therefore, the search for new, more effective, safe for humans’ producers continues to be an urgent question, taking into account the fact that there are no highly active elastase producers in Ukraine. Previously we found elastase activity in only 4 of the 10 studied isolates of bacteria from the Black Sea. Since among them, the elastase activity of the Bacillus sp. 051 was the highest, the aim of this work was to study the physicochemical properties and substrate specificity of the enzyme. Methods. We used methods of determining proteolytic (caseinolytic, elastolytic, fibrinolytic, fibrinogenolytic) activity. Protein concentration was determined by the Lowry method. The study of the effect of temperature on the enzymatic activity was carried out in the range from 4 to 70 °C and pH values from 2.0 to 12.0, created using 0.01 M phosphate-citrate buffer. Results. It has been shown that the growing temperature of 12°C is the most optimal for biosynthesis of enzyme by the culture of Bacillus sp. 051. The complex enzyme preparation capable of hydrolyzing elastin, casein and fibrinogen. The enzyme showed maximum activity in relation to elastin (3.65 U/mg). The optimum pH of the enzyme action is 8.0, the thermal optimum is 40°C. The rate of casein hydrolysis compared to elastin was 2.7 times lower and amounted to 1.35 U/mg. The complex enzyme preparation also hydrolyzed fibrinogen (1.16 U/mg). Conclusions. According to its physicochemical and catalytic properties, the representative of the Black Sea, Bacillus sp. 051 is promising for further research as an enzyme producer with elastolytic activity.

Cobra venom P-III class metalloproteinase atrase A induces inflammatory response and cell apoptosis in endothelial cells via its metalloproteinase domain

Snake venom metalloproteinases (SVMPs), which are a critical component of viperid and crotalid venoms, play various important roles in the pathogenesis of snakebite envenomation. The SVMPs from elapid venoms are not well elucidated, as compared with those from viperid and crotalid venoms. Atrase A is a nonhemorrhagic P-III SVMP purified from Naja atra venom that possesses only weak fibrinogenolytic activity. In our prior study, we found that atrase A detached adherent cells from the substrate. In this work, we investigated further the effect and mechanism of atrase A on endothelial cells. Oxidative damage, inflammatory mediators, apoptosis, and activation of the NF-κB and MAPK signaling pathways were measured after HMEC-1 cells were exposed to atrase A. The results showed that HMEC-1 cells released inflammatory mediators, exihibited oxidative damage and apoptosis after exposure to atrase A. The Western blot analysis results revealed that atrase A increased Bax/Bcl-2 and caspase-3 levels and activated the NF-κB and MAPK signaling pathways in endothelial cells. The effects on endothelial cells were nearly completely abolished after atrase A was treated with ethylenediamine tetraacetic acid. These results showed that atrase A led to an inflammatory response, cellular injury and apoptosis in endothelial cells, and this effect was due to its metalloproteinase domain. The study contributes to a better understanding of the structures and functions of cobra venom P-III class metalloproteinases.

Serine metalloprotease with plasmin-like fibrinolytic activity of Serratia marcescens isolated from the Amazon basin

Research on new fibrinolytic agents has been oriented towards the discovery of microbial proteases with plasmin-like activity, as alternative thrombolytic agents for thrombosis treatment. This study reports the characterization of an extracellular serine metalloprotease of approximately 56 kDa, with fibrinolytic and fibrinogenolytic activity, isolated from a S. marcescens strain obtained from the Amazon (CBAM 519). The enzyme showed optimum activity at pH 9 and temperature of 37 °C. Activity was reduced in the presence of Na+, Cu2+ and Fe2+ ions, and increased with Mn2⁺. The enzyme did not show hemolytic activity, nor did it activate plasminogen, but it effectively hydrolyzed fibrin and fibrinogen, rapidly degrading all fibrinogen chains Aα, Bβ and γ. Therefore, fibrinolysis was promoted only by the direct route, with a fast acting, potent fibrinolytic activity. Based on this, we expect the enzyme to be a protease of the plasmin type that directly degrades fibrin. These characteristics demonstrate great potential for its application in the treatment of thrombosis.

Purification and proteomic analysis of potent fibrinolytic enzymes extracted from Lumbricus rubellus

BackgroundLumbrokinase derived from earthworms, Lumbricus rubellus is known to have fibrinolytic enzymes that have potential as therapeutic drugs due to its ability to dissolve fibrin. The current study is aimed to purify the Lumbrokinase from L. rubellus and identify its protein component.MethodsWater extract of local earthworm Lumbricus rubellus revealed several proteins. Therefore, to identify its protein component, purification through HiPrep DEAE fast flow and proteomic analysis were conducted prior to identifications. A combination of two-dimension gel electrophoresis (2DE) and electrospray ionization mass spectrometry analysis was used to identify the purified fractions.ResultsThe purified fractions contain five protein bands, namely F25-1, F25-2, F85-1, F85-2, and F85-3, which displayed strong fibrinogenolytic activity. F25 fractions showed fibrinogenolytic activity of 974.85 U/mg, while F85 fractions showed higher activity of 1,484.11 U/mg. Fractions F85-1, F85-2, and F85-3 showed molecular weights of 42.6 kDa, 27.03 kDa, and 14 kDa, respectively and were identified as Lumbrokinase iso-enzymes.ConclusionThis preliminary study indicates that the F25 and F85 fractions are similar to published fibrinolytic protease-1 and lumbrokinase, respectively, in terms of their amino acid sequence.

ACTION OF VENOM OF VIPERA LEBETINA ON BLOOD COAGULATION in vitro

Aim. In this study we focused on the search of fibrinogen-targeted proteases in the venom of Vipera lebetina. Methods. Fractionation of the venom was performed using FPLC chromatographic system Acta Prime on Q Sepharose. Analysis of protein mixtures was performed using SDS-PAGE. Аction on blood coagulation system was analyzed using APTT assay [2]. Proteolytic action on fibrinogen and identification of protein components with fibrinolytic activity was performed using electrophoresis of mixture of fibrinogen solution (2 mg/ml) with venom`s fractions. For a comprehensive evaluation of the effect of the obtained fractions on hemostasis, an original approach with modified aggregatometry was used [3]. This approach made it possible to take into account the ability of fractions to activate platelets, initiate blood coagulation, or inhibit platelet aggregation. Hemolytic action of fractions was estimated using fresh human red cells. Amount of released hemoglobin was estimated by spectrophotometry on Optizen POP. Results. Crude venom of V. lebetina was fractionated using ion-exchange chromatography on Q Sepharose. Elution was performed using a stepwise gradient of NaCl (0.1, 0.2, 0.3, 0.5, and 0.7 M NaCl) in 0.05 M Tris-HCl buffer at pH 8.3. Fractions eluted at 0.1 and 0.2 M of NaCl contained several proteins with different molecular weights ranging from 75 kDa to low molecular weight fractions according to the SDS-PAGE. Proteins that cleave α- and β-chains of fibrinogen were found in fractions 0.1 and 0.2, indicating the presence of an enzyme with fibrinogenolytic activity in the venom of V. lebetina. The fractions 0.3, 0.5, and 0.7 did not show any significant fibrinogenolytic activity. After platelet aggregation study we concluded that fraction 0.1 contained a protein with fibrinogenolytic activity. An increase in platelet aggregation was observed for the fraction 0.2 after the addition of ADP. This may indicate the presence of an active compound that promotes platelet aggregation. Further research is necessary to determine its nature. Fractions 0.3, 0.5, and 0.7 had no effect on platelet aggregation. A decrease in blood plasma clotting time in APTT to 5 s and 7 s, compared to a control value of 70 s, was shown for fractions eluted at NaCl concentrations of 0.1 M and 0.2 M, respectively. The fractions 0.3, 0.5 had only a slight effect on reducing blood plasma clotting. A slightly increased level of hemolysis was shown in the presence of the unbound fraction and the whole venom. It can be suggested that proteins with phospholipase activity are present in the non-binded fraction. Conclusions. Thus, fibrinogen-specific proteases, hemolytic agents, activators of blood clotting were found in the venom of Vipera lebetina. Most of these compounds must to be purified and can be used for basic biochemical research.