Maximum Fibrinolytic Activity Research Articles

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.

Produksi Enzim Fibrinolitik Tempe oleh Rhizopus oryzae FNCC 6078

ackground Rhizopus oryzae FNCC 6078 had been evaluated producing fibrinolytic enzyme under solid state fermentation. Soybean had been used to produce fibrinolytic enzyme through fermentation in tempeh. The main purpose of this study was to reveal optimum condition for fermentation. The parameters of the condition were inoculum volume, incubation period and temperature. Optimum condition was defined by maximum fibrinolytic activity. Methode Fibrinolytic activity was measured using spectrophotometry at 274 nm. Result optimum condition for producing fibrinolytic enzyme was 1,5 mL volume of inoculum of Rhizopus oryzae suspension in 25%T, 42 hours for incubation period and 35oC temperature incubation.

피브린 Zymographic Gel에서 Bacillus amyloliquefaciens G-13으로부터 혈전용해효소의 생성에 영향을 미치는 물리화학적 요인들의 비교

Our previous research demonstrated the characteristics and fibrinolytic activities of NaCl- and capsaicin-resistant bacterium Bacillus amyloliquefaciens G-13, isolated from mustard leaf kimchi [1]. In this study, we extended the work to the comparison of various physicochemical factors (e.g., temperature, pH, NaCl, capsaicin, metal ions, and inhibitors) influencing the production of fibrinolytic enzyme by strain G-13. Initially, a time course of the fibrinolytic activity of strain G-13 was measured by the fibrin plate assay, and after 84 hours of incubation, the maximum fibrinolytic activity was about 3.42 times that of plasmin used as the standard. Through a fibrin zymographic assay, four bands (23, 34, 45, and 67 kDa) of fibrinolytic enzyme from strain G-13 were compared in fibrin zymographic gels. The strain could grow and produce the fibrinolytic enzyme in the presence of NaCl (1-10%) or capsaicin (0-300 ㎍/mL), respectively. The optimum pH and temperature for the enzymatic activity were 8 and 35℃. In the study of the effects of metal ions on fibrinolytic activity, K⁺, CaSUP2+/SUP, and MgSUP2+/SUP ions increased the activity and maintained it overall, but FeSUP3+/SUP, ZnSUP2+/SUP, BaSUP2+/SUP, and HgSUP2+/SUP ions rapidly decreased the activity of the fibrinolytic enzyme. The enzyme was completely inhibited by phenylmethanesulfonyl fluroide (PMSF), suggesting that it was a serine protease. These results provide important clues for understanding the characteristics of fibrinolytic bacteria by confirming the effects of B. amyloliquefaciens G-13 on various physicochemical factors influencing the production of fibrinolytic enzymes through fibrin zymography.

Purification and characterization of a novel fibrinolytic enzyme from Whitmania pigra Whitman

Developing an effective fibrinolytic drug for treating thrombolysis with minimal undesirable side effects is of great importance. In the current study, an optimum solvent was selected for the extraction of fibrinolytic active components. Furthermore, a strong fibrinolytic enzyme named WPI01 was purified from Whitmania pigra Whitman through various chromatographic steps. WPI01 has a molecular mass of 27044.297 Da, and the N-terminal 8 amino acid sequence was determined as VVGGVEAR. WPI01 was stable within the pH range of 6.0–10.0 and with maximum fibrinolytic activity at 40 °C and a pH of 8.0. At 500 U/mL, WPI01 induced 50.59% blood clot reduction in vitro within 6 h, which was higher than that induced by urokinase at 1000 U/mL. In an analysis of the plasminogen activator activity, WPI01 produced obvious halos on heated and unheated fibrin plates, suggesting that WPI01 may not only act as a plasminogen activator but also degrade fibrin clots directly, and more study is needed to support this. In conclusion, WPI01 is obviously different from known fibrinolytic enzymes in terms of substrate specificity and fibrinolytic mode of action, suggesting that it is a novel fibrinolytic enzyme with potential applications in the treatment and prevention of thrombosis.

Screening, production and biochemical characterization of a new fibrinolytic enzyme produced by Streptomyces sp. (Streptomycetaceae) isolated from Amazonian lichens

ABSTRACT Thrombosis is a pathophysiological disorder caused by accumulation of fibrin in the blood. Fibrinolytic proteases with potent thrombolytic activity have been produced by diverse microbial sources. Considering the microbial biodiversity of the Amazon region, this study aimed at the screening, production and biochemical characterization of a fibrinolytic enzyme produced by Streptomyces sp. isolated from Amazonian lichens. The strain Streptomyces DPUA1576 showed the highest fibrinolytic activity, which was 283 mm2. Three variables at two levels were used to assess their effects on the fibrinolytic production. The parameters studied were agitation (0.28 - 1.12 g), temperature (28 - 36 ºC) and pH (6.0 - 8.0); all of them had significant effects on the fibrinolytic production. The maximum fibrinolytic activity (304 mm2) was observed at 1.12 g, 28 ºC, and pH of 8.0. The crude extract of the fermentation broth was used to assess the biochemical properties of the enzyme. Protease and fibrinolytic activities were stable during 6 h, at a pH ranging from 6.8 to 8.4 and 5.8 to 9.2, respectively. Optimum temperature for protease activity ranged between 35 and 55 °C, while the highest fibrinolytic activity was observed at 45 ºC. Proteolytic activity was inhibited by Cu2+ and Co2+ ions, phenylmethylsulfonyl fluoride (PMSF) and pepstatin A, which suggests that the enzyme is a serine protease. Enzymatic extract cleaved fibrinogen at the subunits Aα-chain, Aβ-chain, and γ-chain. The results indicated that Streptomyces sp. DPUA 1576 produces enzymes with fibrinolytic and fibrinogenolytic activity, enzymes with an important application in the pharmaceutical industry.

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.

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.

Transient expression of optimized and synthesized nattokinase gene in melon (<i>Cucumis melo</i> L.) fruit by agroinfiltration

A synthetic gene (sNK) encoding Nattokinase (NK) was constructed by modifying its sequence based on the codon usage in plants. A version (sNKi) incorporating the first intron from the tomato E8 gene was also constructed. The synthetic sNK and sNKi genes were transiently expressed in melon (Cucumis melo L.) fruit. Quantitative real-time reverse transcription PCR (qRT-PCR) analysis and fibrinolytic activity assays showed that the expression level of recombinant NK controlled by E8 promoter was higher than that controlled by 35S promoter, with the maximum fibrinolytic activity of 79.30 U ml−1. The intron could enhance the expression of sNK to an extent of 27.42%. Orthogonal tests determined the optimal agroinfiltration as: an acetosyringone concentration of 0.25 mM, an OD600=0.6 and harvesting 5 days after infiltration.

Purification and characterization of a fibrinolytic protease from Aspergillus oryzae KSK-3

An enzyme from Aspergillus oryzae KSK-3, isolated from commercial rice-koji for miso brewing, showed fibrinolytic activity in liquefied rice culture and was analyzed. A culture filtrate of A. oryzae KSK-3 was concentrated by ultrafiltration and subsequently purified to electrophoretic homogeneity by ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration. The molecular weight of the purified enzyme was estimated to be approximately 30kDa by SDS-PAGE and high-performance liquid chromatography–size exclusion chromatography. Its maximum fibrinolytic activity was observed at pH 6 and 50°C. The purified protease was stable between pH 4 and 9, at temperatures of up to 50°C. The activity of the enzyme was highest with S-2238 and was considerably inhibited by phenylmethylsufonyl fluoride and pefabloc SC. These results indicate that the enzyme is a serine protease. Moreover, the enzyme is edible and exhibited very high productivity (2,960 U urokinase per milliliter of culture broth). Taken together, the findings of this study indicate that the A. oryzae KSK-3 enzyme may be used as a natural agent for oral fibrinolytic therapy and nutraceutical applications.

Purification and characterization of a fibrinolytic protease from Aspergillus <i>oryzae</i> KSK-3

An enzyme from Aspergillus oryzae KSK-3, isolated from commercial rice-koji for miso brewing, showed fibrinolytic activity in liquefied rice culture and was analyzed. A culture filtrate of A. oryzae KSK-3 was concentrated by ultrafiltration and subsequently purified to electrophoretic homogeneity by ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration. The molecular weight of the purified enzyme was estimated to be approximately 30 kDa by SDS-PAGE and high-performance liquid chromatography–size exclusion chromatography. Its maximum fibrinolytic activity was observed at pH 6 and 50°C. The purified protease was stable between pH 4 and 9, at temperatures of up to 50°C. The activity of the enzyme was highest with S-2238 and was considerably inhibited by phenylmethylsufonyl fluoride and pefabloc SC. These results indicate that the enzyme is a serine protease. Moreover, the enzyme is edible and exhibited very high productivity (2,960 U urokinase per milliliter of culture broth). Taken together, the findings of this study indicate that the A. oryzae KSK-3 enzyme may be used as a natural agent for oral fibrinolytic therapy and nutraceutical applications.

Production of a Fibrinolytic Enzyme from <i>Coprinus comatus</i> YY-20

A strong fibrin-specific fibrinolytic enzyme was produced by liquid culture ofCoprinus comatusYY-20. Soybean cake powder and sucrose as substrates resulted in the highest fibrinolytic activity. The optimal temperature for the production of the enzyme was found to be 21-25°C in the optimal medium (Soybean cake powder 50 g/L, sucrose 20 g/L). The optimal initial pH of the medium was found to be at the range of 5-8. The maximum fibrinolytic activity was observed at the stationary phase of cell growth. These results suggest that the culture liquid ofCoprinus comatusYY-20 has potential as a source of physiologically active fibrinolytic enzymes.

민자주방망이버섯으로부터 혈전용해효소의 정제 및 특성 연구

민자주방망이버섯으로부터 분리한 혈전용해효소의 비활성은 22.78 U/mg 이었으며, fibrin를 직접 용해하는 fibrinolytic enzyme 이었다. 15번째까지 N-terminal amino acid 서열 분석 결과, Tyr-Pro-Ser-Pro-Ser-His-Gln-Thr-Ala-Val-Asn-Ala-Ile-Ile-X로 지금까지 발표되지 않은 새로운 효소였다. 분자량은 34 KDa 이고 pH 7.0 부터 pH 9.5의 넓은 영역에서 높은 활성을 나타내는 alkaline protease 였으며, <TEX>$55^{\circ}C$</TEX>에서 가장 큰 활성을 보이는 이 효소는 serine protease 저해제인 phenylmethylsulfonyl fluoride를 첨가한 경우 효소의 활성이 전혀 나타나지 않는 것으로 미루어 높은 혈전용해 활성을 갖는 새로운 serine protease로 생각된다. 또한 <TEX>$Hg^{2+}$</TEX>의 금속이온을 첨가한 경우에도 효소의 활성은 완전히 사라졌다. Fibrinolytic enzyme has been isolated and purified from the edible mushroom, Lepista nuda. The apparent molecular mass of purified enzyme was estimated to be 34 KDa by SDS-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of the enzyme was Tyr-Pro-Ser-Pro-Ser-His-Gln-Thr-Ala-Val-Asn-Ala-Ile-Ile-X. It has a pH optimum at <TEX>$7.0.{\sim}9.5$</TEX>, suggesting that the purified enzyme is an alkaline protease. It shows the maximum fibrinolytic activity at <TEX>$55^{\circ}C$</TEX>. The fibrinolytic activity was inhibited by phenylmethylsulfonyl fluoride, indicating that the purified enzyme is a serine protease. The activity of the purified enzyme was totally inhibited by <TEX>$Hg^{2+}$</TEX>.

Fibrinolytic Enzyme Production by Bacillus subtilis KH-4 Isolated from Deonjang

A strong fibrin-specific fibrinolytic enzyme was produced from Bacillus subtilis KH-4 isolated from Deonjang, a Korean fermented soybean paste similar to Japanese miso. The addition of glucose as a carbon source resulted in the highest levels of caseinolytic and fibrinolytic activities. Likewise, the addition of yeast extract as the nitrogen source resulted in the highest caseinolytic and fibrinolytic activities (3473.2 unit and 47.4 munit, respectively). It was observed that out of all metal ion sources only calcium (chloride) enhanced caseinolytic and fibrinolytic activities, with increases of 4949.3 unit and 58.2 unit/mg, respectively. The optimal temperature for the production of the enzyme was found to be 40℃ in the optimal medium (glucose 20 g, yeast extract 5 g, CaCl₂ 1 g, and NaCl 2 g). The maximum fibrinolytic activity was observed at the late stationary phase. B. subtilis KH-4 produced a fibrinolytic enzyme at 40℃, after 30 h growth, which increased up to 54 h and then remained constant. These results suggest that Deonjang has potential as a source of physiologically active anti-thromotic enzymes.

Characterization of a metalloenzyme from a wild mushroom, Tricholoma saponaceum.

Two kinds of metalloendopeptidases from the fruiting bodies of Tricholoma saponaceum (TSMEP1 and TSMEP2) have been purified, and TSMEP1 has been characterized based on their fibrinolytic activity. The enzymes have the same N-terminal amino acid sequence, Ala-Leu-Tyr-Val-Gly-X-Ser-Pro-X-Gln-Gln-Ser-Leu-Leu-Val, but slightly different molecular weights of 18,147 and 17,947, as measured by matrix assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry. The N-terminal sequence do not match with any known protein or open reading frame. TSMEP1 hydrolyzes fibrinogen as well as fibrin, but does not show any proteolytic activity for other blood proteins such as thrombin, human albumin, human IgG, hemoglobin, or urokinase. The enzyme hydrolyzes both A alpha and B beta subunits of human fibrinogen with equal efficiency but didn't show any reactivity for the gamma form of human fibrinogen. The enzymatic activity is strongly inhibited by EDTA and 1,10-phenanthroline, indicating that the enzymes are metalloproteases. No inhibition was found with phenylmethylsulfonyl fluoride (PMSF), L-trans-epoxysuccinyl leucylamido-(4-guanidino)-butane (E-64), pepstatin and 2-mercaptoethanol. The activity of the purified enzyme was increased by Mg2+, Fe2+, Zn2+, and Co2+, and slightly decreased by Ca2+, but the enzyme activity was dramatically decreased by Cu2+, and totally inhibited by Hg2+. It has broad substrate specificity for synthetic peptides, and keep the high activity from pH 7.5 to 9, suggesting that the purified enzyme was a basic protease. The enzyme was stable up to 30 degrees C and the maximum fibrinolytic activity was at 55 degrees C.

A fibrinolytic metalloprotease from the fruiting bodies of an edible mushroom, Armillariella mellea.

A fibrinolytic metalloprotease has been purified from the fruiting bodies of the edible honey mushroom (Armillariella mellea). The enzyme has a molecular weight of 18538.1508, as measured by MALDI-TOF mass spectrometry and includes Zn2+ ion as found by ICP/MS. The N-terminal amino acid sequence, XXYNGXTXSRQTTLV, do not match any known protein or open reading frame. It hydrolyzes fibrinogen as well as fibrin, but does not show any proteolytic activity for other blood proteins such as thrombin, human albumin, bovine albumin, human IgG, hemoglobin, or urokinase. This protease hydrolyzes both A alpha and B beta subunits of human fibrinogen with equal efficiency. The enzyme activity was strongly inhibited by EDTA and 1,10-phenanthroline, indicating that the enzyme is a metalloprotease. No inhibition was found with PMSF, E-64, pepstatin, and 2-mercaptoethanol. The activity of the purified enzyme was slightly increased by Mg2+, Zn2+, and Co2+, but the enzyme was totally inhibited by Hg2+. It has broad substrate specificity for synthetic peptides, and a pH optimum at 7, suggested that the purified enzyme was a neutral protease. It was thermally stable up to 60 degrees C and the maximum fibrinolytic activity was at 55 degrees C.

Human protein c induces anticoagulation and increased fibrinolytic activity in the cat

The effect of activated human Protein C (PCa) infusion on the coagulation and fibrinolytic systems of the cat was examined. Cats received bolus i.v. injections of PCa of 3 μg/mL (n=7), 10 μg/mL (n = 2) or 16 μg/mL (n = 1) of calculated total blood volume. Control cats(n=7) received either unactivated Protein C (10 μg/mL) or the activation vehicle containing heparin and antithrombin in tris-saline buffer. Citrated blood samples were drawn prior to infusion and 5, 10, 20, 40, 60, 120 and 180 minutes post infusion. PCa caused an immediate, dose dependent increase in anticoagulation as measured by APTT which began to normalize within 20 minutes. PCa also increased fibrinolytic activity measured by euglobulin clot lysis time and an 1251 whole blood clot lysis assay. However, the profibrinolytic effect of PCa did not parallel the anticoagulant effect. In all treated cats, maximum fibrinolytic activity did not occur until 40–120 minutes after infusion. Control cats demonstrated no significant change in coagulation or fibrinolytic activity. Our results demonstrate that infusion of activated human Protein C can induce anticoagulation and fibrinolytic activity in the cat.

Changes in coagulation and fibrinolysis occurring in dogs during hypothermia

Changes in fibrinolysis, platelets and coagulation during hypothermia were investigated in anesthetized dogs. Strong activation of plasma fibrinolysis was observed in the hypothermia group. The maximum fibrinolytic activity determined with standard fibrin plates was markedly higher than that of the control sample (mean values: 67 and 15 mm 2/0.03 ml plasma, respectively). On the other hand, no fibrinolysis was observed by the plasminogen free plate method. Activation was not observed in the control group or the hypothermia group premedicated with chlorpromazine. The platelet count was decreased from 100% to 14.7% and the collagen induced platelet aggregability from 61% to 2.3% during hypothermia. Recovery from both reductions occurred on rewarming. In the control and premedicated groups, the recalcification time was reduced progressively (from 93.6 to 60.9 sec and from 101.1 to 71.8 sec as mean times, respectively). The tendencey towards shortening of the recalcification time was not related to the core temperature.