Fibrin Plate Assay Research Articles

Surface Decoration of Selenium Nanoparticles by Proteins from the Culinary-Medicinal Shiitake Mushroom, Lentinus edodes (Agaricomycetes), for Enhanced Fibrinolytic Activity.

Lentinus edodes (shiitake mushroom) has exhibited fibrinolytic activity. We synthesized and characterized selenium nanoparticles (SeNPs) using protein precipitated from the mushroom. We also investigated the fibrinolytic activity of the SeNPs. The proteins from a crude extract of L. edodes were recovered through the use of aqueous 2-phase separation, and these we used as the capping agent in SeNP biosynthesis. We characterized the SeNPs using UV-visible spectrophotometry, field emission scanning electron microscopy (FESEM), energy dispersive X-ray (EDX), transmission electron microscopy (TEM), particle size distribution analysis, and Fourier transform infrared spectroscopy (FT-IR). The fibrinolytic capability of the SeNPs was tested through an in vitro fibrin plate assay. The UV-visible spectra showed maximal absorbance at 220 nm. FESEM images showed that the SeNPs were dispersed and did not clump. The TEM images revealed a spherical shape and average size of the SeNPs. The particle size distribution analysis confirmed the mean size of the SeNPs at 64.53 nm. A strong signal for the presence of selenium was observed in the EDX analysis. The FT-IR spectrum revealed the involvement of protein functional groups in the reduction of sel-enite. Overall, the SeNPs capped with protein from shiitake mushroom were effective as an in vitro fibrinolytic agent.

A Novel Thrombolytic and Anticoagulant Serine Protease from Polychaeta, Diopatra sugokai.

Ischemic stroke can result from blockage of blood vessels, forming fibrin clots in the body and causing irreparable brain damage. Remedial thrombolytic agents or anticoagulants have been studied; however, because the FDA-approved tissue plasminogen activator has low efficacy and side effects, it is necessary to develop safer and more effective treatment candidates. This study aimed at assessing the fibrinolytic and anticoagulation features of a novel serine protease extracted and purified from Diopatra sugokai, a polychaeta that inhabits tidal flats. The purified serine protease was obtained through ammonium sulfate precipitation, affinity chromatography, and ion-exchange chromatography. Its molecular size was identified via SDS-PAGE. To characterize its enzymatic activities, the protease activity at various pH and temperatures, and in the presence of various inhibitors, was measured via azocasein assay. Its fibrinolytic activity and anticoagulant effect were assessed by fibrin zymography, fibrin plate assay, and fibrinogenolytic activity assays. The novel 38 kDa serine protease had strong indirect thrombolytic activity rather than direct activity over broad pH (4-10) and temperature (37°C-70°C) ranges. In addition, the novel serine protease exhibited anticoagulant activity by degrading the α-, β-, and γ-chains of fibrinogen. In addition, it did not produce cytotoxicity in endothelial cells. Therefore, this newly isolated serine protease is worthy of further investigation as a novel alkaline serine protease for thrombolytic therapy against brain ischemia.

Purification of serine protease from polychaeta, Lumbrineris nipponica, and assessment of its fibrinolytic activity

Ischemic stroke and cardiovascular disease can occur from blockage of blood vessels by fibrin clots formed naturally in the body. Therapeutic drugs of anticoagulant or thrombolytic agents have been studied; however, various problems have been reported such as side effects and low efficacy. Thus, development of new candidates that are more effective and safe is necessary. The objective of this study is to evaluate fibrinolytic activity, anti-coagulation, and characterization of serine protease purified from Lumbrineris nipponica, polychaeta, for new thrombolytic agents. In the present study, we isolated and identified a new fibrinolytic serine protease from L. nipponica. The N-terminal sequence of the identified serine protease was EAMMDLADQLEQSLN, which is not homologous with any known serine protease. The size of the purified serine protease was 28kDa, and the protein purification yield was 12.7%. The optimal enzyme activity was observed at 50°C and pH 2.0. A fibrin plate assay confirmed that indirect fibrinolytic activity of the purified serine protease was higher than that of urokinase-PA, whereas direct fibrinolytic activity, which causes bleeding side effects, was relatively low. The serine protease did not induce any cytotoxicity toward the endothelial cell line. In addition, anticoagulant activity was verified by an in vivo DVT animal model system. These results suggest that serine protease purified from L. nipponica has the potential to be an alternative fibrinolytic agent for the treatment of thrombosis and use in various biomedical applications.

Purification and antithrombotic activity of wulfase, a fibrinolytic enzyme from the fruit bodies of the edible and medicinal mushroom Sparassis crispa Wulf. ex. Fr.

A fibrinolytic protease, wulfase, exhibiting antithrombotic potency was purified from the fruit bodies of Sparassis crispa Wulf. ex. Fr. Wulfase showed a single band of approximately 90 kDa by SDS-PAGE and fibrin zymography. The fibrinolytic activity of wulfase determined by fibrin plate assay revealed that it could directly degrade fibrin clot. The enzyme could inhibit activities of factor Xa and thrombin. Turbidity and electron-microscopy analysis using fluorescent conjugate demonstrated that wulfase also inhibited fibrin polymer formation. The anticoagulant effect of the enzyme was further confirmed in human plasma. These results suggest that wulfase may be useful for reducing or preventing thrombotic challenge.

Effects on fibrinogen, fibrin, and blood coagulation of proteolytic extracts from fruits of Pseudananas macrodontes, Bromelia balansae, and B. hieronymi (Bromeliaceae) in comparison with bromelain.

Extracts rich in cysteine proteases obtained from fruits of Pseudananas macrodontes (Pm), Bromelia balansae (Bb), and B. hieronymi (Bh) have previously shown an anti-inflammatory effect on animal models. Given the close relationship between hemostasis and inflammation, it is attractive to investigate therapeutic agents capable of modulating both systems. The aim of this work was to study the effect of Pm, Bb, and Bh on fibrin(ogen) and blood coagulation compared with stem bromelain (Bro). Action on fibrinogen was electrophoretically and spectrophotometrically evaluated, fibrinolytic activity was measured both electrophoretically and by the fibrin plate assay, and the effect on blood coagulation was studied by conventional coagulation tests (PT and APPT). All extracts showed the same proteolytic preference for fibrinogen subunits, that is Aα > Bβ, whereas γ was partially hydrolyzed by 100-fold concentration increase. Unlike Bro, cysteine proteases of Pm, Bb, and Bh increased absorbance at 540 nm of fibrinogen solution, suggesting thrombin-like activity, which was time-dependent and reached maximum values at lower concentration. All extracts showed the same proteolytic preference for fibrin subunits; however Pm, Bb, and Bh showed lower fibrinolytic activity than Bro at the assayed concentrations. Although Bb acted only as anticoagulant, Pm, Bh, and unexpectedly Bro showed dual action on blood coagulation: at low concentration showed procoagulant effect and at high concentration anticoagulant effect. Results reveal new plant species as potential sources of pharmacological agents for the treatment of a wide range of hemostatic disorders as well as to wound healing.

SCREENING AND BIOSYTNTHESIS OF FIBRINOLYTIC ENZYME FROM ASPERGILLUS JAPONICUM

Fibrin is a protein that forms in the blood clots after trauma or injury. This is essential to stop blood loss. There are more than twenty enzymes in the body that assist in clotting of the blood, while only one that can break the clot down. It is an endogenously produced fibrinolytic enzyme called plasmin. Streptokinase is an extracellular metallo-enzyme produced by beta-haemolytic streptococcus and is used as an effective and cheap clot-dissolving medication in some cases of myocardial infarction (heart attack) and pulmonary embolism. It belongs to a group of medications known as fibrinolytics. Fibrinolytic enzymes can be found in a variety of foods, such as Japanese Natto, Tofuyo, Korean Chungkook-Jang soy sauce, and edible honey mushroom. Fibrinolytic enzymes producing Aspergillus japonicum KSS 05 strain were screened for the production by fibrin plate assay method. The maximum zone of fibrin hydrolysis were found 6 mm diameter. Further the Aspergillus japonicum KSS 05 were employed for the production by submerged fermentation and it showed 235 IU by pH 6, temperature 30 0 C and 1 ml inoculums size. Key words: Fermentation kinetics, effect of pH, fibrinolytic enzymes and fibrin

Purification and biochemical characterization of a novel fibrinolytic enzyme, PSLTro01, from a medicinal animal Porcellio scaber Latreille

A novel protease, named PSLTro01, with fibrinolytic and anticoagulant activity was isolated from Porcellio scaber Latreille and was purified by a combination of hollow fibre membrane molecular weight cut-off (MWCO), ammonium sulfate fractionation, gel filtration and ion-exchange chromatography. PSLTro01 is a single-chain protein with a molecular mass of 38,497Da as estimated by non-reduced SDS-PAGE and MALDI-TOF MS spectrometry, and its N-terminal 15 amino acid sequence was determined as DINGGGATLPQPLYQ. PSLTro01 is stable in the range of 20–40°C and pH 6.0–10.0, with a maximum fibrinolytic activity at 40°C and pH 7.0. The PSLTro01-induced fibrinolytic activity was not influenced by K+ or Na+ but was slightly increased by Mg2+ and completely inhibited by aprotinin and pepstatin A. Fibrin plate assays revealed that PSLTro01 could not directly degrade fibrin but was a plasminogen activator. PSLTro01 exhibited high specificity for the substrate S-2251 for plasmin, followed by S-2238 for thrombin and S-2444 for urokinase. Moreover, the fibrinogenolysis pattern of PSLTro01 was Aα-chains>Bβ-chains>γ-chain. Tail-thrombus of the enzyme treated group was significantly shorter than the physiological saline treated group and the thrombus decrement was correlated with the enzyme dose. PSLTro01 prolongs both thrombin time (TT) and activated partial thromboplastin time (APTT). These results indicate that PSLTro01 may have potential applications in the prevention and treatment of thrombosis.

Synthesis of fibrinolytic active silver nanoparticle using wheat bran xylan as a reducing and stabilizing agent

A facile synthesis of highly stable silver nanoparticles (AgNPs) was reported using a biopolymer, xylan as both a reducing and stabilizing agent. Xylan was isolated from waste biomass, wheat bran (WB) by alkaline treatment and was characterized by Fehling's test, dinitrosalicylic acid assay, FTIR, 1H NMR and 13C NMR. The synthesized nanoparticles were characterized by UV–Vis spectroscopy and transmission electron microscopy. The nanoparticles were polydispersed with the size ranging from 20 to 45nm. The synthesized WB-xylan AgNPs showed excellent free radical scavenging activity. In addition, WB-xylan AgNPs showed fibrinolytic activity as evidenced by the zone of clearance in fibrin plate assay. The biomedical potential of the WB-xylan AgNPs was demonstrated by dissolution of preformed blood clots. These results suggest that the development of xylan-metal nanoparticle composite would be feasible to treat thrombus related diseases.

Lack of TAFI increases brain damage and microparticle generation after thrombolytic therapy in ischemic stroke

BackgroundThrombin-activatable fibrinolysis inhibitor (TAFI) plays an important role in coagulation and fibrinolysis. Whereas TAFI deficiency may lead to a haemorrhagic tendency, data from TAFI knockout mice (TAFI−/−) are controversial and no differences have been reported in these animals after ischemic stroke. There are also no data regarding the role of circulating microparticles (MPs) in TAFI−/−. Objectivesto examine the effect of tPA on the rate of intracranial haemorrhage (ICH) and on MPs generated in a model of ischemic stroke in TAFI−/− mice. MethodsThrombin was injected into the middle cerebral artery (MCA) to analyse the effect of tPA (10mg/Kg) on the infarct size and haemorrhage in the absence of TAFI. Immunofluorescence for Fluoro-Jade C was performed on frozen brain slides to analyse neuronal degeneration after ischemia. MPs were isolated from mouse blood and their concentrations calculated by flow cytometry. ResultsCompared with saline, tPA significantly increased the infarct size in TAFI−/− mice (p<0.05). Although plasma fibrinolytic activity (fibrin plate assay) was higher in these animals, no macroscopic or microscopic ICH was detected. A positive signal for apoptosis and degenerating neurons was observed in the infarct area, being significantly higher in tPA treated TAFI−/− mice (p<0.05). Interestingly, higher numbers of MPs were found in TAFI−/− plasma as compared to wild type, after stroke (p<0.05). ConclusionsTAFI deficiency results in increased brain damage in a model of thrombolysis after ischemic stroke, which was not associated with bleeding but with neuronal degeneration and MP production.

Purification and Biochemical Characterization of a Novel Fibrinolytic Enzyme from Streptomyces sp. P3.

A novel proteolytic enzyme with fibrinolytic activity, FSP3, was purified from the recently isolated Streptomyces sp. P3, which is a novel bacterial strain isolated from soil. FSP3 was purified to electrophoretic homogeneity by ammonium sulfate precipitation, anion exchange, and gel filtration. FSP3 is considered to be a single peptide chain with a molecular mass of 44 kDa. The maximum activity of the enzyme was observed at 50°C and pH 6.5, and the enzyme was stable between pH 6 and 8 and below 40°C. In a fibrin plate assay, FSP3 showed more potent fibrinolytic activity than urokinase, which is a clinical thrombolytic agent acting as a plasminogen activitor. The activity was strongly inhibited by the serine protease inhibitor PMSF, indicating that it is a serine protease. Additionally, metal ions showed different effects on the activity. It was significantly suppressed by Mg(2+) and Ca(2+) and completely inhibited by Cu(2+), but slightly enhanced by Fe(2+). According to LC-MS/MS results, its partial amino acid sequences are significantly dissimilar from those of previously reported fibrinolytic enzymes. The sequence of a DNA fragment encoding FSP3 contained an open reading frame of 1287 base pairs encoding 428 amino acids. FSP3 is a bifunctional enzyme in nature. It hydrolyzes the fibrin directly and activates plasminogen, which may reduce the occurrence of side effects. These results suggest that FSP3 is a novel serine protease with potential applications in thrombolytic therapy.

A bifunctional protease from green alga Ulva pertusa with anticoagulant properties: partial purification and characterization

A bifunctional thrombolytic serine protease (ulvease) having both direct-acting fibrinolytic and plasminogen-activating properties was purified from the green alga, Ulva pertusa. Ulvease had maximum fibrinolytic activity at 40 °C and pH 7.0, and the activity was inhibited by Cu2+ and Zn2+. Fibrinolytic activity of ulvease was higher than that of plasmin and u-PA by fibrin plate assay and displayed both plasmin and plasminogen activator-like activities. Ulvease preferentially hydrolyzed the Aα- and Bβ-chains of fibrinogen, but the γ-chain was appeared unaffected. Ulvease prolonged APTT and PT. PFA-100 assay showed that it delayed the closure times of citrated whole human blood. Ulvease showed a strong thrombolytic ability and safety in vivo, which is better than that of u-PA. It protected mice from collagen- and epinephrine-induced pulmonary thromboembolism. These results suggest that ulvease may have a potential clinical application for the treatment and prevention of thrombosis.

Soluble expression, purification, and characterization of active recombinant human tissue plasminogen activator by auto-induction in E. coli.

BackgroundHuman tissue plasminogen activator (tPA) belongs to the serine protease family. It converts plasminogen into plasmin and is used clinically to treat thrombosis. Human tPA is composed of 527 amino acids residues and contains 17 disulfide bonds. Escherichia coli has been used only rarely for the efficient production of recombinant tPA. However, the functional expression of full-length tPA that contains multiple disulfide bonds on an industrial scale remains challenging. Here, we describe the soluble expression and characterization of full-length tPA by auto-induction in E. coli.ResultsWe achieved optimal levels of gene expression, minimized negative effects related to the production of heterologous proteins, and optimized cytoplasmic yields. Three different E. coli strains, BL21 (DE3), Rosetta, and Origami 2, could express tPA using an auto-induction mechanism. In addition, similar yields of recombinant protein were produced at temperatures of 33, 35, and 37°C. The E. coli strain origami 2 could increase disulfide bond formation in cytoplasmic tPA and produce purified soluble recombinant protein (~0.9 mg/l medium). The full-length tPA was monomeric in solution, and fibrin plate assays confirmed that the recombinant tPA displayed serine protease activity.ConclusionsThis is the first report that describes the heterologous expression of correctly folded active full-length tPA. This could provide valuable information for using prokaryotic auto-induction expression systems to produce tPA at industrial and pharmaceutical levels without in vitro refolding during the production step.

Fibrinolytic enzyme production by newly isolated Bacillus cereus SRM-001 with enhanced in-vitro blood clot lysis potential.

The discovery of plasmin-like microbial fibrinolytic enzymes having high specificity and negligible side effects is crucial for thrombolytic therapy. Herein, we report one such extra-cellular fibrinolytic enzyme producing Bacillus cereus SRM-001 isolated from the blood-laden soil of a chicken dump yard. The potency of the enzyme was established with fibrin plate assay and in-vitro blood clot lysis assay. The shake-flask operating parameters and media composition were optimized for maximizing the productivity of the enzyme. The operating parameters, pH 7, 37°C, 1% inoculum volume and 24 h inoculum age, were found to be the optimum. The levels of media components, corn flour (0.3% w/v), soyabean powder (1.9% w/v) and MnSO4 (11.5 mM) were optimized by statistical analysis using Box-Behnken design derived RSM. This resulted in an almost 1.8 fold increase in fibrinolytic enzyme productivity. The 3D response surface plots showed soyabean powder and MnSO4 to be the key ingredients for enhancing the enzyme productivity, whereas corn flour had a marginal effect. The in-vitro blood clot lysis assay conducted at near physiological pH 7 at 37°C showed the enzyme to be a potential therapeutic thrombolytic agent.

A Fibrinolytic Enzyme Produced by &lt;em&gt;Bacillus subtilis&lt;/em&gt; Using Chickpea (&lt;em&gt;Cicer arietinum&lt;/em&gt; L.) as Substrate

A Fibrinolytic Enzyme (BSFE) was isolated from fermented chickpeas using Bacillus subtilis. BSFE was purified with ammonium sulfate precipitation, ion exchange and gel filtration chromatography. The fibrin (ogen) olytic activity of BSFE was investigated by means of fibrinolysis plate and hydrolysis of fibrinogen. Through these steps, the purity of the enzyme increased with 74.60-fold with 6.88% recovery activity. The molecular weight of the BSFE was estimated to be 30 kDa by SDS-PAGE. The optimum pH, optimum temperature, pH stability and thermal stability of BSFE were measured, respectively as 8.0, 55°C, 6.0-8.0 and less than 45°C. The activity was inhibited by serine protease inhibitor PMSF as well as metalloprotease inhibitor EDTA, indicating that the BSFE is a serine metalloprotease. In fibrin plate assay, BSFE showed more stronger fibrinolytic activity than that of nattokinase and it specifically hydrolyzed Aα and Bβ chains followed by γ chain of fibrinogen. Therefore, this study provided a method and it for the preparation of multifunctional food of chickpeas which has strong fibrinolytic activity.

Detecting of fibrino(geno)lytic activity of plant latex proteases by a specific fluorogenic substrate and fibrin(ogen)-zymography

Plant latex is repeatedly examined in terms of blood clotting and fibrinolytic activity, due to the proteases often contained. Some examples are the latex of Synadenium grantii Hook (Euphorbiaceae) [1], Pergularia extensa (Jacq.) N.E. Br. (Apocynaceae) [2] or Artocarpus heterophyllus Lam. (Moraceae) [3]. Previously we described the in vitro influence of a protease from the latex of Euphorbia mauritanica L. (Euphorbiaceae) on some steps of the coagulation and fibrinolysis process, by using different methods such as a fibrin plate assay, turbidimetry or in-gel-digestion [4]. We examined over hundred species of the family Euphorbiaceae for fibrinolytic activity with the specific fluorogenic substrate Boc-Val-Leu-Lys-AMC. We have combined the results for the most active plant latices with fibrin(ogen) zymography to confirm the results of the screening and to get information whether the active proteases are the same in the different latices. We also wanted to confirm the results of the above methods for the mentioned isolated protease. The zymograms confirm the activity for all latices tested. However, different proteases seem to be responsible for the activity in each latex.

Expression of cholera toxin B subunit-lumbrokinase in edible sunflower seeds-the use of transmucosal carrier to enhance its fusion protein's effect on protection of rats and mice against thrombosis.

Lumbrokinase (LK) is a group of serine proteases with strong fibrinolytic and thrombolytic activities and is useful for treating diseases caused by thrombus. Cholera toxin B subunit (CTB) has been widely used to facilitate antigen delivery by serving as an effective mucosal carrier molecule for the induction of oral tolerance. We investigate here the application of CTB as a transmucosal carrier in enhancing its fusion protein-LKs effect to protect rats against thrombosis. Thus, in this study, CTB-LK fusion gene separated by a furin cleavage site was expressed in seeds of Helianthus annuus L. The activity of recombinant protein in seeds of transgenic sunflower was confirmed by Western blot analysis, fibrin plate assays and GM1 -ganglioside ELISA. The thrombosis model of rats and mice revealed that the oral administration of peeled seeds of sunflower expressing CTB-LK had a more significant anti-thrombotic effect on animals compared with that administration of peeled seeds of sunflower expressing LK. It is possible to conclude that CTB can successfully enhance its fusion protein to be absorbed in rats or mice thrombosis model. The use of CTB as a transmucosal carrier in the delivery of transgenic plant-derived oral therapeutic proteins was supported. In addition, for the purpose of that recombinant CTB-LK was designed for oral administration, thus the expression of CTB-LK in edible sunflower seeds eliminated the need for downstream processing of proteins.

Starase: A bi-functional fibrinolytic protease from hepatic caeca of Asterina pectinifera displays antithrombotic potential

A bi-functional fibrinolytic serine protease, Starase exhibiting thrombolytic potency was purified from hepatic caeca of Asterina pectinifera. Starase showed a single band of approximately 48kDa by SDS-PAGE and fibrin zymography. The N-terminal sequence of Starase was AIPTEFDARTKKHNN, which does not match with any known fibrinolytic enzyme. Starase had optimum amidolytic activity at 50°C and pH 8.0 and the activity was inhibited by PMSF and APMSF. Starase showed the highest specificity toward the substrate H-D-Val-Leu-Lys-pNA for plasmin followed by pyroGlu-Gly-Arg-pNA for urokinase. The apparent Km and Vmax values of Starase toward a chromogenic substrate for plasmin H-D-Val-Leu-Lys-pNA were determined as 1.37mM and 6.8mM/min/mg respectively. The fibrinolytic activity of Starase by fibrin plate assay displayed that it could not only directly degrade fibrin clot but also activate plasminogen. Starase showed a strong fibrinogenolytic activity, cleaving all three major chains of fibrinogen rapidly. In addition, Starase with more than 1μg could cleave extracellular matrix component type VII collagen, and plasma proteins such as bovine albumin and bovine gamma globulin. It could also inhibit factor Xa and thrombin activity. Starase at a dose of 0.8mg/kg was devoid of hemorrhagic activity and it demonstrated antithrombotic effect in three animal models; FeCl2-induced carotid arterial thrombus model, carrageenan-induced tail thrombosis model and collagen and epinephrine induced pulmonary thromboembolism mice model. These results suggest that Starase has the potential to be a potent thrombolytic agent due to its bi-functional properties (containing both direct-acting and plasminogen-activating activities) and lack of hemorrhagic activity. Although Starase might interfere with the normal composition of the plasma proteins, it may be used not only for the treatment and prevention of thrombosis, but also in a number of biomedical applications.

Isolation and Characterization of a 32-kDa Fibrinolytic Enzyme (FE-32kDa) from Gloydius blomhoffii siniticus Venom: Fibrinolytic Enzyme from Gloydius blomhoffii siniticus Venom.

Objectives:This study was undertaken to isolate a fibrinolytic enzyme from the snake venom of Gloydius blomhoffii siniticus and to investigate its enzymatic characteristics and hemorrhagic activity as a potential pharmacopuncture agent.Methods:The fibrinolytic enzyme was isolated by using chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and fibrin plate assay. The characteristics of the enzyme were investigated using fibrin plate assay, protein hydrolysis analysis, and hemorrhage assay. Its amino acid composition was determined.Results:The fibrinolytic enzyme with the molecular weight of 32kDa (FE-32kDa) from Gloydius blomhoffii siniticus showed a fibrin hydrolysis zone at the concentration of 0.2 mg/mL in the fibrin plate assay. The fibrin hydrolysis activity of the enzyme was inhibited completely by ethylenediaminetetraacetic acid (EDTA), ethyleneglycoltetraacetic acid (EGTA), and 1, 10-phenanthroline, thiothreitol and cysteine, and partially by phenylmethanesulfonylfluoride (PMSF). Metal ions such as Fe2+ and Hg2+ inhibited the fibrin hydrolysis completely, but Zn2+ enhanced it. FE-32kDa hydrolyzed α-chain but did not hydrolyze β-chain and γ-chain of fibrinogen. High-molecular-weight polypeptides of gelatin were hydrolyzed partially into low-molecular-weight polypeptides, but the extent of hydrolysis was limited. FE-32kDa induced hemorrhage beneath back skin of mice at the dose of 2 μg.Conclusions:FE-32kDa is a α-fibrin(ogen)olytic metalloprotease that requires Zn2+ for fibrinolytic activity and causes hemorrhage, suggesting that the enzyme is not appropriate for use as a clinical pharmacopuncture.

Effect of Morinda citrifolia fruit extract and its iridoid glycosides on blood fluidity

The aim of this study was to investigate the effect of Morinda citrifolia fruit on blood fluidity. M. citrifolia fruit extract (MCF-ext) was investigated for its influence on blood aggregation and fibrinolysis. MCF-ext inhibited polybrene-induced erythrocyte aggregation and thrombin activity. The fibrinolytic activity of MCF-ext, in the euglobulin lysis time test and fibrin plate assay, is reported here for the first time. One of the active compounds was an iridoid glycoside, asperulosidic acid. The results indicated that MCF-ext is a potentially useful health food which is capable of improving blood flow and preventing lifestyle-related diseases.

New Thrombolytic Agent from Endophytic Fungi and Lignosus rhinocerus

Thrombosis and cardiovascular-related diseases can be caused by fibrin aggregation. Thrombolytic agents have been used to treat thrombolytic problems and cardiovascular-related diseases. There had been searches for new thrombolytic agents from bacteria, fungi, insects and fermented foods. To name a few, recently fungi such as Aspergillus oryzae KSK-3, Fusarium sp. CPCC 480097 and Fusarium sp. BLB were found able in producing protease fibrinolytic enzymes. Potential protease fibrinolytic enzymes were observed from Lignosus rhinocerus bought from Orang Asli of Sungei Perak. Out of the six (6) sclerotia used, one of them, LR-1, produced the biggest clear zone on Skim Milk Agar with a diameter of 1.31 cm. Fibrin plate assay of LR-1 showed that it produced a clear zone of 0.97 cm when the plate was incubated for 18 hours at 37°C Leaves of Hibiscus sp. were sterilised with 70% alcohol and 70% sodium hypochlorite. The leaves were then cut into pieces of 4 cm and placed on Potato Dextrose Agar plate. Fungi grown from the leave pieces were sub cultured onto new Potato Dextrose Agar. This then showed that Lignosus rhinocerus had protease and fibrinolytic activities. Sixteen (16) endophytic fungi were isolated from Hibiscus sp. leaves from six locations in Shah Alam, Selangor. Two endophytic fungi, FH-3 and S-13 showed protease activities; clear zones of 2.89 cm and 2.42 were seen.after 4 days incubation at 27°C. FH-3 had fibrinolytic activity which was observed by the 1.98 cm diameter clear zone on fibrin plate.