Fibrin Plate Method Research Articles

Development of a Methodology Based on Optical Interferometry for Measuring Fibrinolytic Activity.

Fibrinolytic activity assay is particularly important for the detection, diagnosis, and treatment of cardiovascular disease and the development of fibrinolytic drugs. A novel efficacious strategy for real-time and label-free dynamic detection of fibrinolytic activity based on ordered porous layer interferometry (OPLI) was developed. Fibrin or a mixture of fibrin and plasminogen (Plg) was loaded into the highly ordered silica colloidal crystal (SCC) film scaffold to construct a fibrinolytic response interference layer to measure fibrinolytic activity with different mechanisms of action. Fibrinolytic enzyme-triggered fibrinolysis led to the migration of interference fringes in the interferogram, which could be represented by optical thickness changes (ΔOT) tracked in real time by the OPLI system. The morphology and optical property of the fibrinolytic response interference layer were characterized, and the Plg content in the fibrinolytic response interference layer and experimental parameters of the system were optimized. The method showed adequate sensitivity for the fibrinolytic activity of lumbrokinase and streptokinase, with wide linear ranges of 12-6000 and 10-2000 U/mL, respectively. Compared with the traditional fibrin plate method, it has a lower detection limit and higher linearity. The whole kinetic process of fibrinolysis by these two fibrinolytic drug models was recorded in real time, and the Michaelis constant and apparent kinetic parameters were calculated. Importantly, some other blood proteins were less interfering with this system, and it showed reliability in fibrin activity detection in real whole blood samples. This study established a better and more targeted research method of in vitro fibrinolysis and provided dynamic monitoring data for the analysis of fibrinolytic activity of whole blood.

Purification and Properties of a Plasmin-like Marine Protease from Clamworm (Perinereis aibuhitensis).

Marine organisms are a rich source of enzymes that exhibit excellent biological activity and a wide range of applications. However, there has been limited research on the proteases found in marine mudflat organisms. Based on this background, the marine fibrinolytic enzyme FELP, which was isolated and purified from clamworm (Perinereis aibuhitensis), has exhibited excellent fibrinolytic activity. We demonstrated the FELP with a purification of 10.61-fold by precipitation with ammonium sulfate, ion-exchange chromatography, and gel-filtration chromatography. SDS-PAGE, fibrin plate method, and LC-MS/MS indicated that the molecular weight of FELP is 28.9 kDa and identified FELP as a fibrinolytic enzyme-like protease. FELP displayed the maximum fibrinolytic activity at pH 9 (407 ± 16 mm2) and 50 °C (724 ± 27 mm2) and had excellent stability at pH 7-11 (50%) or 30-60 °C (60%), respectively. The three-dimensional structure of some amino acid residues of FELP was predicted with the SWISS-MODEL. The fibrinolytic and fibrinogenolytic assays showed that the enzyme possessed direct fibrinolytic activity and indirect fibrinolysis via the activation of plasminogen; it could preferentially degrade Aα-chains of fibrinogen, followed by Bβ- and γ-chains. Overall, the fibrinolytic enzyme was successfully purified from Perinereis aibuhitensis, a marine Annelida (phylum), with favorable stability that has strong fibrinolysis activity in vitro. Therefore, FELP appears to be a potent fibrinolytic enzyme with an application that deserves further investigation.

Screening of a Novel Fibrinolytic Enzyme-Producing Streptomyces from a Hyper-Arid Area and Optimization of Its Fibrinolytic Enzyme Production

Fibrinolytic enzymes are a kind of proteolytic enzymes that can hydrolyze fibrin and dissolve blood clots. They could be used as a therapeutic agent for treating thrombosis. It is important for the treatment of cardiovascular disease to find and develop new thrombolytic drugs. In order to explore new fibrinolytic enzymes, a strain named 214L-11 with protease and fibrinolytic enzyme activity, which was isolated from the Flaming Mountain of Xinjiang Province, was screened using the skimmed milk plate, the blood powder agarose plate and the fibrin plate methods. Phylogenetic analyses showed that strain 214L-11 shared the highest similarity with Streptomyces fumanus NBRC 13042T (98.88%), which indicated that it represented a potential novel species in the Streptomyces genus. The fibrinolytic enzyme produced by 214L-11 displayed thrombolytic and anticoagulant activities, and it could degrade a single specific protein in the thrombus, thereby destroying the thrombus structure. The fermentation medium optimized through response surface methodology was 15 g/L soluble starch, g/L KNO3 0.58, 0.43 g/L peptone, 0.01 g/L FeSO4·7H2O, 0.5 g/L MgSO4·7H2O, 0.2 g/L Mn2+, 0.5 g/L NaCl and 1 L distilled water, pH 8, and the maximum amount of fibrinolytic enzyme produced by strain 214L-11 in the optimal fermentation medium was 1255.3 FU/mL. Overall, the fibrinolytic enzyme-producing strain was screened from the Flaming Mountain of Xinjiang for the first time, which provided a basis for further research and the development of new efficient and safe hemolytic drugs.

Thrombolytic potential in bacteria isolated from fermented durian tempoyak

Abstract. Priskila C, Vidian V, Sanjaya A, Sugata M, Pinontoan R. 2022. Thrombolytic potential in bacteria isolated from fermented durian tempoyak. Biodiversitas 23: 5731-5737. Cardiovascular disease is one of the leading causes of death worldwide and is primarily caused by dysregulation in the blood clotting system leading to the formation of unwanted blood clots (thrombi). Thrombolytic therapy, which aims to break down unwanted blood clots, employs various agents possessing thrombolytic activity. However, the harmful side effects of conventional thrombolytic agents necessitate the exploration of new and safer agents with high thrombolytic activities. Thrombolytic agents can be obtained from various sources, including fermented foods. Tempoyak is a condiment made from fermented durian, which is found in some regions of Indonesia, and is reported to contain beneficial microorganisms. The aim of this study was to isolate and identify thrombolytic agents from tempoyak. After several pure microbial colonies were successfully isolated from tempoyak, they were screened for proteolytic activity on skim milk agar medium. The isolated colonies with proteolytic activity were then subjected to several tests, including the whole blood clot lysis test, euglobulin clot lysis test, fibrin plate method, and zymography. Subsequently, the isolates with thrombolytic and fibrinolytic activity potential were identified in terms of their morphology and biochemistry. In this study, three bacteria with thrombolytic activity potential were successfully isolated. Fibrin zymography indicated that the molecular weights of the proteins that play roles in the fibrinolytic activity ranged from approximately 30 to 153 kDa, which were suspected to correspond to serine protease, cysteine protease, and/or metalloprotease. All three isolates were identified as belonging to the genus Corynebacterium. This is the first report discussing the thrombolytic and fibrinolytic activities of Corynebacterium sp. derived from fermented food.

Cultivation conditions and biochemical characterization of the proteolytic enzymes with fibrinolytic action obtained from mushrooms in the last ten years

Fibrinolytic enzymes are proteases that are capable of degrading the fibrin mesh present in blood clots and can be obtained through fungal extracts. Thus, the objective of this paper was to seek out and compare all the information regarding the production and biochemical characterization of fibrinolytic enzymes of mushroom species published in papers in the last ten years. The research was carried out in eight national and international electronic databases using the terms: “fungal enzymes”, “fibrinolytic Proteases”, “thrombosis” and “mushrooms”. The results obtained were analyzed according to each step. In the first stage, the titles and abstracts of all papers were analyzed independently; in the second stage, all papers were read and those that did not meet the inclusion criteria were excluded. Then, the remaining papers were reviewed and the data were tabulated and compared. As a result, it was found that enzymes with fibrinolytic action were obtained from eight species of mushrooms in the period of this research: P. sajor-cashew, P. ferulae, P. ostreatus, L. shimeji, A. polytricha, H. erinaceum, C. comatus and C. militaris. The enzymes were identified as protease SPPs, metalloproteases, serine proteases and serine metalloproteases. The methods used to evaluate fibrinolytic activity were the fibrin plate method and/or the formation of artificial thrombus through thrombin and fibrinogen. The molecular mass of these enzymes ranged from ~18 to 66 kDa and, for the biochemical characterization of the extracts, the pH ranged from 4.0 to 9.5 and the optimum temperature from 25 to 70 ºC. Via the review, it was observed that few articles were published on the subject in question, which made it necessary to carry out more studies to discover the biotechnological properties of mushrooms and define their applications in the most diverse sectors of industries, not only in commercial species of mushrooms, but also in native species.

Isolation of Bacillus spp. with High Fibrinolytic Activity and Performance Evaluation in Fermented Douchi

Fibrinolytic enzymes are effective and highly safe in treating cardiovascular and cerebrovascular diseases. Therefore, screening fibrinolytic enzyme-producing microbial strains with excellent fermentation performance is of great value to industrial applications. The fibrin plate method was used in screening strains with high yields of fibrinolytic enzymes from different fermented food products, and the screened strains were preliminarily identified using molecular biology. Then, the strains were used for solid-state fermentation of soybeans. Moreover, the fermentation product douchi was subjected to fibrinolytic activity measurement, sensory evaluation, and biogenic amine content determination. The fermentation performance of each strain was comprehensively evaluated through principal component analysis. Finally, the target strain was identified based on strain morphology, physiological and biochemical characteristics, 16S rDNA sequence, and phylogenetic analysis results. A total of 15 Bacillus species with high fibrinolysin activity were selected. Their fibrinolytic enzyme-producing activity levels were higher than 5,500 IU/g. Through molecular biology analysis, we found 4 strains of Bacillus subtilis, 10 strains of Bacillus amyloliquefaciens, and 1 strain of Bacillus velezensis. The principal component analysis results showed that SN-14 had the best fermentation performance and reduced the accumulation of histamine and total amine, the fibrinolytic activity of fermented douchi reached 5,920.5 ± 107.7 IU/g, and the sensory score was 4.6 ± 0.3 (out of 5 points). Finally, the combined results of physiological and biochemical analyses showed SN-14 was Bacillus velezensis. The high-yield fibrinolytic and excellent fermentation performance strain Bacillus velezensis SN-14 has potential industrial application.

Fibrinolytic characteristics of <i>Bacillus subtilis</i> G8 isolated from natto

Due to the high prevalence of vascular obstructive diseases, discovering potent, safe, and affordable fibrinolytic agents is of importance. There is particular interest concerning the use of functional foods that have a fibrinolytic activity, such as natto, a Japanese fermented soy-based product made with Bacillus subtilis (natto) strain BEST195. We recently isolated another bacterial strain from natto commercialized in Indonesia, B. subtilis G8, which has proven to exert fibrinolytic activity. Herein, a further characterization of B. subtilis G8 was assessed through a comparison with commercialized nattokinase, the major fibrinolytic enzyme of B. subtilis, by utilizing various in vitro fibrinolytic assays, namely whole blood clot lysis, euglobulin clot lysis, the fibrin plate method, and zymography. Both nattokinase and B. subtilis G8 were able to dissolve both whole blood and euglobulin clots. Furthermore, both nattokinase and B. subtilis G8 were able to lyse blood clots, presumably due to their ability to directly lyse fibrin. Finally, a crude extract of B. subtilis G8 displayed six zymogram bands of approximately 42.0, 35.5, 30.8, 26.7, 20.0, and 13.7 kDa, with the strongest activity observed at 20.0 kDa. This indicates that B. subtilis G8 contained several fibrinolytic enzymes, which might have comprised nattokinase and other fibrinolytic enzymes. In summary, we demonstrated that a crude extract of B. subtilis G8 has potent fibrinolytic activity and that the activity was mediated by various fibrinolytic enzymes.

Physicochemical Properties and Bioactivities of Rice Beans Fermented by Bacillus amyloliquefaciens

The purpose of this study was to investigate the physicochemical properties and bioactivities of rice beans (Vigna umbellata) fermented by Bacillus amyloliquefaciens. The fermentation conditions were optimized on the basis of the fibrinolytic activity. Under the optimal fermentation conditions, the fibrinolytic activity reached a maximum of 78.0 FU·g−1 (4890 IU·g−1, fibrin plate method, FU: fibrin degradation unit). The contents of peptides (which increased from 2.1 to 10.9 g per 100 g), total phenolics (from 116.7 to 388.5 mg gallic acid per 100 g), total flavonoids (from 235.5 to 354.3 mg rutin per 100 g), and anthocyanin (from 20.1 to 47.1 mg per 100 g), as well as the superoxide dismutase activity (from 55.3 to 263.6 U·g−1) in rice beans were significantly increased after fermentation. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2-azinobis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS) free radical scavenging activities and ferric reducing antioxidant power (FRAP) of fermented rice beans were 1.9–4.8 times higher than those of unfermented rice beans. Moreover, fermentation induced an increase in the dipeptidyl peptidase IV (DPP-IV) inhibition, α-glucosidase inhibition, and anticoagulant activities of rice beans. Rice beans fermented by Bacillus amyloliquefaciens may serve as a functional food with potential benefits for the prevention of thrombotic diseases.

Optimasi Produksi Fibrinolitik oleh Bacillus megaterium BM 9.1 menggunakan media Limbah Pertanian sebagai sumber karbon

The study of effect agro recidues carbon sources on fibrinolytic enzyme production by Bacillus megaterium BM 9.1 was done by using fibrin plate method. Bacillus megaterium BM 9.1 grown on agro residues medium with solid state fermentation. The fibrinolytic enzymes activity is declared by fibrinolytic index. Fibrinolytics index is the comparisson between clear zones and wall diameters. From various carbon sources tested, Bacillus megaterium BM 9.1 which is grown in banana peel, pineaple skin, corn husk, rice bran and corn cob could produce fibrinolytic enzymes. Optimum fibrinolytic activities found in a nutrient agar and banana peel medium at fibrinolytic index 4,95 ± 0,08 (p<0,05). The comparisson fibrinolytic index between Bacillus megaterium BM 9.1 which grown in combination nutrient agar, banana peel and nutrien agar standart is 117,%. Banana peel concentration optimation was done at concentration of 0,5 %, 1 %, 2 % and 4 % and the fibrinolytic index was found respectively according to 4,26 ± 0,11 ; 4,32 ± 0,03 ; 4,61 ± 0,03; and 4,35 ± 0,05. The optimum fibrinolytic activity was found in banana peel on consentration 2 %.

Cost-effective fibrinolytic enzyme production by Bacillus subtilis WR350 using medium supplemented with corn steep powder and sucrose

The goal of this study was to develop a cheap and simple medium and to optimize fermentation parameters for fibrinolytic enzyme production by Bacillus subtilis WR350. A low-cost medium containing 35 g/L sucrose, 20 g/L corn steep powder and 2 g/L MgSO4·7H2O was developed via single-factor and orthogonal experiments. A cheap nitrogen source, corn steep powder, was used to replace the soy peptone present in the initial medium. The highest fibrinolytic activity of 5865 U/mL was achieved using the optimized medium in a 100-L fermenter with an aeration rate of 1.0 vvm and an agitation speed of 200 rpm. The resulting enzyme yield was among the highest described in the literature with respect to fibrinolytic activity, as determined by the fibrin plate method. Techno-economic evaluation indicated that the cost of the optimized medium was only 8.5% of the cost of the initial medium, and the total fermentation cost of fibrinolytic enzyme production using the optimized medium was 23.35% of the cost of using the initial medium.

Novel Fibrinolytic Protease Producing Streptomyces radiopugnans VITSD8 from Marine Sponges.

Fibrinolytic enzymes have received more attention due to their medicinal potential for thrombolytic diseases. The aim of this study is to characterize the in vitro fibrinolytic nature of purified protease producing Streptomyces radiopugnans VITSD8 from marine brown tube sponges Agelas conifera. Three varieties of sponge were collected from the Rameshwaram Sea coast, Tamil Nadu, India. The fibrinolytic activity of Streptomyces sp. was screened and determined by casein plasminogen plate and fibrin plate methods respectively. The crude caseinolytic protease was purified using ammonium sulfate fractionation, affinity and ion-exchange chromatography. Based on the morphological, biochemical, and molecular characterization, the isolate VITSD8 was confirmed as Streptomyces radiopugnans. Maltose and peptone were found to be the best carbon and nitrogen sources for the production of fibrinolytic protease. The carbon and nitrogen source peptone showed (781 U/mL) enzyme activity. The optimum pH and temperature for fibrinolytic protease production was found to be 7.0 and 33 °C respectively. The purified enzyme showed a maximum specific activity of 3891 U. The blood clot lysis activity was compared with the standard, and it was concluded that a minimum of 0.18 U (10 µL) of purified protease was required to dissolve the blood clot. This is the first report which exploits the fibrinolytic protease activity of Streptomyces radiopugnans VITSD8 extracted from a marine sponge. Hence the investigation suggests a potential benefit of purified fibrinolytic protease which will serve as an excellent clot buster alternative.

Optimization of Fibrinolytic Enzyme from Streptomyces althioticus BN22

Fibrinolytic enzyme plays an important place in the medical field for cardiovascular disease. Only few studies have reported the fibrinolytic enzyme production from marine actinomycetes, and Streptomyces sp. In this study 114 actinomycetes isolates from different marine soil samples were isolated by using starch casein agar media. These 114 actinomycetes strains were tested for fibrinolytic activity by fibrin plate method. 14 isolates showed fibrinolytic activity amongst the 114 actinomycetes strains, and this was identified based on the clear zone of formation around the colonies. The potent strain of Streptomyces althioticus BN22 was isolated and this was confirmed by 16SrRNA sequencing. The optimization of nutritional and physical parameters for fibrinolytic enzyme production from marine soil was studied for the isolate Streptomyces althioticus. The effects of nutritional and physical parameters were studied for the optimum production of a fibrinolytic enzyme from Streptomyces althioticus BN22 by submerged fermentation. The effects of initial pH, temperature and incubation time were studied. The maximum enzymatic activity was obtained with an initial pH7.5 (45,765 Eu/ml), incubation temperature 50°C (50,827 Eu/ml) and incubation time for (41,175 Eu/ml). The effects of various carbon and nitrogen sources were studied. The results indicated that glucose (51,570 Eu/ml) as carbon source and yeast extract (54,625 Eu/ml) as nitrogen source showed the highest fibrinolytic enzyme activity among them. Thus this study showed its originality by focus, that marine Streptomyces althioticus is an important source for fibrinolytic enzyme production.

Anti-thrombosis Activity of Drone Apis mellifera Pupae Extracts

Drones of honeybee (Apis mellifera) have been regarded as a useful value only when mating with queen bee. However, the drone pupae have been reported to be nutritionally valuable, and a potential beekeeping product. In this study, drone pupae extracted with 5% acetic acid were used to measure anti-thrombosis related fibrinolytic activity using Strup and Mullertz fibrin plate method. As a result, the drone pupae extract showed higher effect of fibrinolytic activity(clear zone diameter 20.83mm) compared to the human plasmin (clear zone diameter 12.93mm) used as a positive control. It was suggested that the extract of drone pupae can be developed as a functional material helping prevention or treatment of various vascular diseases.

Strain improvement, optimization and purification studies for enhanced production of streptokinase from Streptococcus uberis TNA-M1

Screening of isolates for their potency to produce streptokinase was an important criterion of this research. The current study emphasizes the strain improvement, optimization and purification studies for enhanced production of streptokinase from Streptococcus uberis TNA-M1 isolated from bovine milk. The study was carried out on samples collected from milk sample. Primary screening and characterization is used as an excellent source for the isolation of β-hemolytic organisms. Strain improvement was done by both physical & chemical mutagenesis. The enzyme activity was checked by clot lysis assay and confirmed by fibrin plate method. The partially purified and crude enzyme were analysed by high-performance liquid chromatography. Molecular weight & enzyme purity was checked by SDS–PAGE, further confirmed by fibrin zymography. Out of the 3 isolated strains, only one isolate expressed β-haemolysis with streptokinase (SK) activity. Based on the results of radial caseinolytic assay and blood clot dissolving assay, isolate TNA-M1 demonstrated the highest streptokinase activity. Based on morphological, biochemical and molecular characterization, it was identified as Streptococcus uberis and the strain was named as Streptococcus uberis TNA-M1. The results indicated that ultra-violet (UV) and ethyl methane sulfonate (EMS) were effective mutagenic agents for strain improvement of Streptococcus uberis TNA-M1 and enhanced SK productivity. HPLC analysis was performed in order to confirm the presence of streptokinase with the similar retention time (0.875 min) with its standard (0.854) min. SDS-PAGE of the enzyme showed protein band of approximately 47 kDa and confirmed by fibrin zymography. It exhibited fibrinolytic activity, which was more potent than other fibrinolytic enzymes. Glucose and peptone were recorded to be the optimum carbon and nitrogen sources respectively. Thus this study presents its novelty by highlighting the potential of Streptococcus uberis TNA-M1 as a significant source for the production of fibrinolytic enzymes.

Food Origin Fibrinolytic Enzyme With Multiple Actions

Many health related problems such as cardiovascular diseases are associated with the formation of excessive clot in the blood (thrombus). Approaches in cardiovascular disease treatment are preventing the formation or removing the thrombus. The present thrombolytic agents can be classified as plasminogen activators, fibrinolytic enzyme which directly degrades fibrinogen or fibrin and heparin type which act as thrombin inhibitor. Recently, microbial fibrinolytic enzymes of food origin receive more attention that leads to escalating efforts to explore traditional fermented foods as the natural sources. We have successfully isolated microorganism from Indonesian fermented soybean tofu dregs “Oncom” that secretes fibrinolytic enzyme. The microorganism identified as Stenotrophomonas sp. is unique because most of the reported fibrinolytic microorganism belongs to Bacillus sp. This isolate was found to produce extracellular fibrinolytic enzyme which could degrade fibrinogen and fibrin directly as determined by fibrinogen zymography and fibrin plate methods. More importantly, the 30-kD purified enzymes was found to demonstrate not only fibrin and fibrinogen degradation capabilities, but also acted as thrombin inhibitor as determined using specific substrates for thrombin. This is the first report of a fibrinolytic enzyme that demonstrates additional synergistic activities. This finding accentuates the importance of further development of the enzyme into a powerful agent to treat the thrombus-related disease effectively.

Global assays of fibrinolysis.

Fibrinolysis is an important and integral part of the hemostatic system. Acting as a balance to blood coagulation, the fibrinolytic system protects the body from unwanted thrombus formation and occlusion of blood vessels. As long as blood coagulation and fibrinolysis remain in equilibrium, response to injury, such as vessel damage, is appropriately regulated. However, alterations in this balance may lead to thrombosis or bleeding. A variety of methods have been proposed to assess fibrinolytic activity in blood or its components, but due to the complexity of the system, the design of a "gold standard" assay that reflects overall fibrinolysis has remained an elusive goal. In this review, we describe the most commonly used methods that have been described, such as thromboelastography (TEG and ROTEM), global fibrinolytic capacity in plasma and whole blood, plasma turbidity methods, simultaneous thrombin and plasmin generation assays, euglobulin clot lysis time and fibrin plate methods. All of these assays have strengths and limitations. We suggest that some methods may be preferable for detecting hypofibrinolytic conditions, whereas others may be better for detecting hyperfibrinolytic states.

Browplasminin, a condensed tannin with anti-plasmin activity isolated from an aqueous extract of Brownea grandiceps Jacq. flowers

Ethnopharmacological relevanceFollowing Venezuelan traditional medicine, females with heavy menstrual blood loss (menorrhagia) drink Brownea grandiceps Jacq. flowers (BG) decoctions to reduce the bleeding. In a previous study, we demonstrated that BG aqueous extract (E) possesses a potent anti-fibrinolytic activity capable of inhibiting plasmin, the main serine-protease that degrades fibrin. It is widely known that plasmin inhibitors are often used as anti-fibrinolytics to reduce bleeding during surgeries with high risk of blood loss such as cardiac, liver, vascular, tooth extraction and large orthopedic procedures, as well as for menorrhagia treatments. The aim of this work was to isolate and characterize from BGE the compound responsible for the reported anti-fibrinolytic activity. Materials and methodsA decoction of BG was prepared; then it was homogenized, centrifuged and lyophilized to obtain BGE. Subsequently the extract was fractionated by gel filtration and reverse phase using HPLC and the active compound was characterized by MALDI-ToF MS. The kinetic parameters of anti-plasmin activity were evaluated by an amidolytic assay using a chromogenic substrate; also the anti-plasmin activity was estimated by fibrin plate method. Data were analyzed by nonparametric statistics. ResultsThe active compound was a condensed tannin denominated Browplasminin, which is capable of inhibiting the plasmin activity in a dose-dependent manner when measured in fibrin plates or by the amidolytic activity method; it also has a minor effect on the FXa activity. However, it does not affect the activity of other serine-proteases such as trypsin, t-PA or u-PA. Browplasminin consists predominately of heteroflavan-3-ols of catechin with B-type linkages, and extents up to heptadecamers (~ 5000Da), with hexose residues attached to the polymer that presents a high degree of galloylation. Its IC50 for plasmin was 47.80μg/mL and for FXa was 237.08μg/mL, while the Ki were 0.76 and 61.61μg/mL for plasmin and FXa, respectively. ConclusionsThe overall outcome of this study suggests that Browplasminin could be responsible for reducing heavy menstrual bleeding in women because its kinetic parameters showed that is a good plasmin inhibitor.

Antitumor Studies of Earthworm Fibrinolytic Enzyme Component A from Eisenia foetida on Breast Cancer Cell Line MCF-7

Earthworm fibrinolytic enzyme from Chinese earthworm Eisenia foetida was isolated to investigate its antitumor activity in breast cancer cells. The protein isolated was characterised as earthworm fibrinolytic enzyme using the fibrin plate method. The molecular weight of earthworm fibrinolytic enzyme component was determined to be 25 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis assay using a standard protein ladder. Earthworm fibrinolytic enzyme can markedly inhibit the growth and migration of MCF-7 cells in a dose and time dependent manner. In addition, MCF-7 cells treated with earthworm fibrinolytic enzyme (40 μg/ml) began to undergo apoptosis after 24 h. Expression of focal adhesion kinase and CD44v6 measured using reverse transcription polymerase chain reaction and Western blot was down-regulated in a concentration dependent manner (20-80 μg/ml), resulting in the suppression of MCF-7 cells adhesion. The obtained earthworm fibrinolytic enzyme displayed an antitumor effect on MCF-7 cells in vitro, revealing the therapeutic potential of E. foetida.

Novel Sequential Screening and Enhanced Production of Fibrinolytic Enzyme by Bacillus sp. IND12 Using Response Surface Methodology in Solid-State Fermentation.

Fibrinolytic enzymes have wide applications in clinical and waste treatment. Bacterial isolates were screened for fibrinolytic enzyme producing ability by skimmed milk agar plate using bromocresol green dye, fibrin plate method, zymography analysis, and goat blood clot lysis. After these sequential screenings, Bacillus sp. IND12 was selected for fibrinolytic enzyme production. Bacillus sp. IND12 effectively used cow dung for its growth and enzyme production (687 ± 6.5 U/g substrate). Further, the optimum bioprocess parameters were found out for maximum fibrinolytic enzyme production using cow dung as a low cost substrate under solid-state fermentation. Two-level full-factorial experiments revealed that moisture, pH, sucrose, peptone, and MgSO4 were the vital parameters with statistical significance (p < 0.001). Three factors (moisture, sucrose, and MgSO4) were further studied through experiments of central composite rotational design and response surface methodology. Enzyme production of optimized medium showed 4143 ± 12.31 U/g material, which was more than fourfold the initial enzyme production (978 ± 36.4 U/g). The analysis of variance showed that the developed response surface model was highly significant (p < 0.001). The fibrinolytic enzyme digested goat blood clot (100%), chicken skin (83 ± 3.6%), egg white (100%), and bovine serum albumin (29 ± 4.9%).

In vitro fibrinolysis and antithrombosis characterizations of novel recombinant microplasminogen with RGD and GPRP peptides.

Microplasminogen (μPlg), a truncated form of human plasminogen, has considerable potential as a direct-acting thrombolytic agent. To further develop μPlg into a thrombolytic agent with anti-thrombus properties, we constructed two μPlg variants containing tripeptide Arg-Gly-Asp (RGD) and tetrapeptide Gly-Pro-Arg-Pro (GPRP) by site-directed mutagenesis. The recombinant cDNAs were expressed in yeast (Pichia pastoris) and purified to high homogeneity by Ni-NTA affinity chromatography. The specific activities of RGD-μPlg and GPRP-μPlg were 7.7 and 13.3 U/mg, respectively, as determined using the fibrin-plate method. RGD-μPlg significantly inhibited ADP-induced platelet aggregation, which was 33.6- and 14.1-fold higher than the native μPlg and GPRP-μPlg, respectively. On the other hand, GPRP-μPlg prolonged thrombin-initialized fibrinogen polymerization in a concentration-dependent manner, which was 9.2- and 5.7-fold stronger than μPlg and RGD-μPlg, respectively. Under activation by urokinase, μPlg, RGD-μPlg, and GPRP-μPlg all showed over 80% conversions to their active enzyme in 24h. The structure models that docked RGD-μPlg and μPlg activation loops into the enzymatic active site of urokinase showed that Pro559 to Asp559 mutation of RGD-μPlg led to an alteration in the interaction, which possibly explains the slowed activation of RGD-μPlg by urokinase over an 80-min period. In conclusion, this study has presented two recombinant μPlg variants with anti-platelet aggregation and anti-fibrinogen clotting activity, thus suggesting the anti-thrombosis properties of these two μPlg derivatives.