Objective:EPF3 is a fibrinolysin monomer isolated and purified from Pheretima vulgaris Chen,an earthworm used in traditional Chinese medicine as Dilong for treating blood stasis syndrome.Its composition,anticoagulant ...Objective:EPF3 is a fibrinolysin monomer isolated and purified from Pheretima vulgaris Chen,an earthworm used in traditional Chinese medicine as Dilong for treating blood stasis syndrome.Its composition,anticoagulant and fibrinolytic activities,and relevant mechanisms have been confirmed through in vitro experiments.However,whether it has antithrombotic effects in vivo and can be absorbed by the gastrointestinal tract is unknown.This study evaluates the antithrombotic effect in zebrafish and investigates the gastrointestinal stability and intestinal absorption mechanism of this protein in vitro.Methods:The antithrombotic effect of EPF3 in vivo was verified using the zebrafish thrombus model induced by arachidonic acid and FeCl3.Then,the protein bands of EPF3 incubated with simulated gastric fluid(SGF),simulated intestinal fluid(SIF),and homogenate of Caco-2 cells(HC2C)were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis to evaluate its gastrointestinal stability.Finally,the transport behavior and absorption mechanism of EPF3 were studied using Caco-2 cell monolayer.Results:EPF3 could significantly enhance the returned blood volume and blood flow velocity in zebrafish with platelet aggregation thrombus induced by arachidonic acid.It could also prolong the formation time of tail artery thrombus and increase the blood flow velocity in zebrafish with vessel injury thrombus induced by FeCl3.EPF3 was stable in SIF and HC2C and unstable in SGF.The permeability of EPF3 in Caco-2 monolayer was time-dependent and concentration-dependent.The efflux ratio was less than1.2 during transport,and the transport behavior was not affected by inhibitors.EPF3 could reversibly reduce the expression of tight junction-related proteins,including zonula occludens-1,occludin,and claudin-1 in Caco-2 cells.Conclusion:EPF3 could play a thrombolytic and antithrombotic role in zebrafish.It could be transported and absorbed into the intestine through cellular bypass pathway by opening the intestinal epithelium tight junction.This study provides a scientific explanation for the antithrombotic effect of earthworm and provides a basis for the feasibility of subsequent development of EPF3 as an antithrombotic enteric-soluble preparation.Please cite this article as:Zhong WL,Yang JQ,Liu H,Wu YL,Shen HJ,Li PY,Du SY.Antithrombotic effect in zebrafish of a fibrinolytic protein EPF3 from Dilong(Pheretima vulgaris Chen)and its transport mechanism in Caco-2 monolayer through cell bypass pathway.J Integr Med.2025;23(4):415–428.展开更多
Objective: The purpose of this work was to investigate the distribution pattern of fibrinolytic factors and their inhibitors in rabbit tissues. Methods: The components of the fibrinolytic system in extracts from a var...Objective: The purpose of this work was to investigate the distribution pattern of fibrinolytic factors and their inhibitors in rabbit tissues. Methods: The components of the fibrinolytic system in extracts from a variety of rabbit tissues, including tissue plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1), plasminogen (Plg), plasmin (Pl) and α2 plasmin inhibitor (α2PI), were determined by colorimetric assay. Results: The tissue extracts in renal, small intestine, lung, brain and spleen demonstrated strong fibrinolytic function, in which high activity of tPA, Plg and Pl was manifested; whereas in skeletal muscle, tongue and stomach, higher activity of PAI-1 and α2PI showed obviously. Also excellent linear correlations were found between levels of tPA and PAI-1, Pl and α2PI, Plg and Pl. In related tissues, renal cortex and renal marrow showed distinctly higher activity of tPA and lower activity of PAI-1, with the levels of Plg and Pl in renal cortex being higher than those in renal marrow, where the α2PI level was higher than that in renal cortex. Similarly, the levels of tPA, Plg and Pl in small intestine were higher than those in large intestine, but with respect to PAI-1 and α2PI, the matter was reverse. In addition, the fibrinolytic activity in muscle tissue was lower, however, the levels of tPA, Plg, and Pl in cardiac muscle were obviously higher than those in skeletal muscles, and the levels of PAI-1 and α2PI were significantly lower than those in skeletal muscle. Conclusion: Our data demonstrate that a remarkable difference of the fibrinolytic patterns exists in rabbit tissues, which has probable profound significance in understanding the relationship between the function of haemostasis or thrombosis and the physiologic function in tissues.展开更多
A strong fibrinolytic activity was demonstrated in the Semen Sojae Praeparatum(SSP), which is a famous traditional Chinese medicine. To study the activities and dynamic changes of fibrinolytic enzyme, standard fibrin ...A strong fibrinolytic activity was demonstrated in the Semen Sojae Praeparatum(SSP), which is a famous traditional Chinese medicine. To study the activities and dynamic changes of fibrinolytic enzyme, standard fibrin plate was used to determine the fibrinolytic activity. For the first time fibrinolytic enzyme was found during the fermentation of SSP and the fibrinolytic activities of samples were shown to increase significantly over time. In the "yellow cladding" stage, the fibrinolytic activity was 619.75 IU/g. On day 6, 12 and 15 of the "secondary fermentation" stage, the fibrinolytic activity was 711.49 IU/g, 866.67 IU/g, 1 022.31 IU/g, respectively. The results indicate that fibrinolytic enzyme was generated during the fermentation of SSP and it displayed increasing activity which peaked at the "secondary fermentation" stage. The fibrinolytic enzyme was found to not only degrades fibrin directly, but also activate plasminogen to do so.展开更多
The gene encoding fibrinolytic enzyme from Bacillus sp. zlw-2 was cloned and sequenced （accession no. EU734749）, which was 1146 bp, encoded 381 amino acids and had 99% homology with Nattokinase YF308 and NAT. The ge...The gene encoding fibrinolytic enzyme from Bacillus sp. zlw-2 was cloned and sequenced （accession no. EU734749）, which was 1146 bp, encoded 381 amino acids and had 99% homology with Nattokinase YF308 and NAT. The genes encoding pre-pro-fibrinolytic enzyme （including signal peptide, propeptide, and mature peptide） and fibrinolytic enzyme （including mature peptide） were cloned into pET28a vector respectively and then transformed into Escherichia coli BL21 （DE3）. The recombinant ofpre-pro-fibrinolytic enzyme showed enzyme activity of 183 U mL^-1, while no detectable enzyme activity could be found from the recombinant of the mature peptide.展开更多
Thrombus disease, one of the common cardiovascular diseases, has attracted worldwide at- tention for its rising mortality and morbidity. Due to the distinct shortages of current fibrinolytic drugs, new fibrinolytic ag...Thrombus disease, one of the common cardiovascular diseases, has attracted worldwide at- tention for its rising mortality and morbidity. Due to the distinct shortages of current fibrinolytic drugs, new fibrinolytic agents warrant investigation. In this study, 8 fibrinolytic enzyme-producing strains were isolated from Douchi--a traditional Chinese food, and strain XY-1 which produced the largest amount of the enzyme was chosen for the following experiments. The enzyme produced by strain XY-1 was named Douchi fibrinolytic enzyme （DFE）. We optimized the liquid culture medium of strain XY-1 for enzyme production using Plackett-Burman and Box-Behnken design. The predicted maximal DFE yield was 19.78 FU/mL with 11.4 g/L peptone, 0.5 g/L magnesium sulfate and 1 g/L sodium chloride. How- ever, we acquired maximal production of 21.33 FU/mL in actual experiments, equal to 107.84% of the theoretical value, and the yield had been increased by 79.55% as compared to the yield of un-optimized culture. It was demonstrated that the combined use of Plackett-Burman design and response surface methodology in fermentation optimization can effectively and rapidly increase DFE production.展开更多
A novel potent protease, Urechis unicinctus fibrinolytic enzyme （UFE）, was firstly discovered. The enzymatic properties of UFE were further investigated.As a low molecular mass protein,UFE appeared to be very stable...A novel potent protease, Urechis unicinctus fibrinolytic enzyme （UFE）, was firstly discovered. The enzymatic properties of UFE were further investigated.As a low molecular mass protein,UFE appeared to be very stable to heat and pH.When temperature was below 50 ℃ ,the remnant enzyme activity remained almost unchanged, but when temperature was raised to 60 ℃ ,the remnant enzyme activity began to decrease rapidly. UFE was quite stable in the range of pH value from 3 to 12,especially in slightly alkaline pH value.Mn^2＋ ,Cu^2＋ and Fe^2＋ ions were activators of UFE, while Fe^3＋ and Ag^＋ ions were inhibitors of UFE.Fe^2＋ ion along with Fe^3＋ ion might regulate UFE activity in vivo. The optimum pH and temperature of UFE were about 8 and 50 ℃ ,respectively. Other characteristics of this enzyme were also studied. Systematic research results are significant when UFE is applied for medical and industrial purposes.展开更多
By using equilibrium dialysis, atomic absorption spectrometry, fluorescence titration and determination of fluorescence lifetime, it can be determined that each fibrinolytic principle(FP) molecule contains one Ca 2...By using equilibrium dialysis, atomic absorption spectrometry, fluorescence titration and determination of fluorescence lifetime, it can be determined that each fibrinolytic principle(FP) molecule contains one Ca 2+ binding site and one Ca 2+ ion. The energy transfer between Tb 3+ and the Trp residue in FP was studied through fluorescence spectroscopy. Our studies show that the Ca 2+ ion in an FP molecule can be substituted by Tb 3+ ion. In FP molecule, the excition energy can be transfered from the Trp residue as an energy donor to Tb 3+ as an acceptor, substituted into FP and located near the Trp residue. The distance between Tb 3+ and the Trp residue, ～0 375 nm, was worked out with the experimental data and Forster theory.展开更多
In ST-segment elevation myocardial infarction (STEMI), acute reperfusion of the infarct-related artery (IRA)is the main goal in the early minutes after the patient seeks medical attention. Fibrinolytic therapy (FT) an...In ST-segment elevation myocardial infarction (STEMI), acute reperfusion of the infarct-related artery (IRA)is the main goal in the early minutes after the patient seeks medical attention. Fibrinolytic therapy (FT) and/or primary coronary intervention (PCI) were proven to be effective in opening the IRA.展开更多
The fibrinoiytic activity of peritoneal fluid (PF) and the incidence of luteinized unruptured follicle syndrome (LUFS) in 36 infertile women with endometriosis and 27 without endometriosis were studied The results sho...The fibrinoiytic activity of peritoneal fluid (PF) and the incidence of luteinized unruptured follicle syndrome (LUFS) in 36 infertile women with endometriosis and 27 without endometriosis were studied The results showed that tissue plasminapen activity was significantly diminsihed during the early luteal phase in patients with endometriosis (P<0.05), but no significant difference between these two groups during the late follicular phase, and reduction of t -PA activity was associated with the incidence of LUF. It is postulated that decreare in t-PA activity due to LUF maybe resposible for infertility.展开更多
Objective The purpose of this study was to evaluate the fibrinolytic response to exercise in patients with unstable angina pectoris(UAP) compared with an age - matched control group. Methods We measuredtissure- type p...Objective The purpose of this study was to evaluate the fibrinolytic response to exercise in patients with unstable angina pectoris(UAP) compared with an age - matched control group. Methods We measuredtissure- type plasminogen activator (t- PA) activity and plasminogen activator inhibitor 1(PAI- 1) activitybefore and after treadmill exercise test in 20 healthy subjects and 25 patients with UAP. ResultsResting t - PA activities were similar between two groups, but resting PAI- 1 activity was higher in UAP groupthan in control. Although both groups showed significant increase in t- PA activity with exercise, post - exerciset - PA activity was significantly lower in patients with UAP than in control (0.96± 0.45IU/ml vs 1.89± 0.68I U/ml,P<0.01), Post - exercise PAI- 1 activity was still much higher in UAP group than in control (8.20±2.28A U/ml vs4.21± 0.68A U/ml, P<0.01). Conclusion There existed impaired fibrinolysis in patients with UAP not only at restbut also alter exercise loading.展开更多
In order to obtain all the properties of fermented ginseng, we fermented ginseng using Bacillus subtilis (B. subtilis) isolated from Cheonggukjang. A sterilized ginseng medium was made with 4-year-old ginseng powder a...In order to obtain all the properties of fermented ginseng, we fermented ginseng using Bacillus subtilis (B. subtilis) isolated from Cheonggukjang. A sterilized ginseng medium was made with 4-year-old ginseng powder and distilled water (300% ginseng powder [w/w]), and the ginseng was fermented by B. subtilis (1% ginseng medium) followed by incubation at 37°C for 3, 5, 7, and 10 days. The growth of B. subtilis in the ginseng medium significantly increased up to 9 log CFU/g, but no significant difference was observed after 3 days. As the fermentation progressed, the ginsenoside Rd and Rg+Rh1 contents increased by 255.3% - 322.5% and 165.6% - 228.6%, respectively, whereas the Rc, Re, and Rg1 contents decreased by 30.7% - 39.6%, 10.5% - 12.8%, and 16.2% - 16.6%, respectively. After 3 days of fermentation, a 6.25% - 7.12% viscous substance was produced;thereafter, the viscous substance was gradually reduced until it disappeared. The viscosity of the medium significantly decreased with a longer fermentation time. Fibrinolytic activity increased during 3 - 10 days of fermentation, indicating a relative activity of 85.0% - 100.0%.展开更多
[Objective] To isolate and preserve a high-activity Douchi fibrinolytic enzyme producing strain from Douchi products. [Method] The Douchi flbrinolytic enzyme producing strain was screened on the selected medium prepar...[Objective] To isolate and preserve a high-activity Douchi fibrinolytic enzyme producing strain from Douchi products. [Method] The Douchi flbrinolytic enzyme producing strain was screened on the selected medium prepared with self-made pork blood powder, the strain with the highest activity was screened out according to the size of hydrolysis cycle, and then preserved in LB medium. [ Rebait] A Douchi fibrinolytic enzyme producing strain with high thrombolytic activity was successfully screened out from the Douchi produced in Hunan, Guangdong and Jiangxi Prov- inces. [ Ceadusioe] The study lays foundation for the development of new-type thrombolytic drugs.展开更多
Objective:To explore the effect of Guhong Injection combined with atyplase on vascular endothelial function and fibrinolytic system and related factors in patients with acute cerebral infarction.Methods:To select 90 p...Objective:To explore the effect of Guhong Injection combined with atyplase on vascular endothelial function and fibrinolytic system and related factors in patients with acute cerebral infarction.Methods:To select 90 patients with acute cerebral infarction in our hospital,divided into control group(48 cases)and observation group(48 cases)randomly.Patients in the control group were treated with alteplase on the basis of routine symptomatic treatment,and the observation group was treated with Guhong injection on the basis of the control group.Before and after treatment,the serum levels of related factors were detected and compared between the two groups.Results:Before treatment,there were no significant differences in serum related factors(ET-1,PAO,H-FABP,VEGF,S100β,BDNF,CEC,Fibulin-5,vWF,P-selectin,t-PA,PAI-1)between the two groups;After treatment,the serum levels of ET-1,PAO,H-FABP,S100β,CEC,Fibulin-5,vWF,P-selectin,PAI-1 in the observation group were significantly lower than those in the control group,and the serum levels of VEGF,BDNF and t-PA were higher than those in the control group,there were significant differences between the two groups.Conclusion:Guhong injection was added to patients with acute cerebral infarction on the basis of routine symptomatic treatment and ateplase,could significantly improve the level of serum related factors,it was more conducive to the control of symptoms and rehabilitation of patients,the effect was definite,it was worth further study and application in clinic.展开更多
The first line drugs in the management of thromboernbolic diseases in Nigeria include Urokinase and Streptokinase. These drugs come with their attendant shortcomings. The need to source for cheap and accessible altern...The first line drugs in the management of thromboernbolic diseases in Nigeria include Urokinase and Streptokinase. These drugs come with their attendant shortcomings. The need to source for cheap and accessible alternative therapy forms the basis of this study. The fibrinolytic potentials of 10 Nigerian medicinal plants： Anona senegalensis, Buchholzia coriaceae, Citrullus colocynthis, Cnidoscolus aconitifolius, Curculigo pilosa, Nicotiana tabaccum, Parinari curatellifolia, Pepperomia pellucida, Sida acuta and Xylopia aethiopica extracts were evaluated using water and streptokinase as negative and positive controls respectively. Trace metal （Cu, Fe, Ca and Zn） analysis of the plant extracts was carried out using atomic absorption spectrophotometer. Methodology involved in vitro clot lysis method, the crude methanolic extract ofParinari curatellifolia （56.122%）, Cnidoscolus aconitifolius （48.385%）, Anona senegalensis （46.360%）, Xylopia aethiopica （43.205%） and Buchholzia coriaceae （27.062%） were found to have significant （P 〉 0.001） fibrinolytic activity. Maximum effect was exhibited by the positive standard： Streptokinase （60.20 ± 4.30%）, followed by Parinari curatellifolia （56.122 ± 3.86%）, our findings reveal that fibrinolytic activity is independent of elemental Copper or Iron content of the extracts. This study indicated that methanolic extracts of Parinari curatellifolia, Cnidoscolus aconitifolius, Anona senegalensis, Xylopia aethiopica and Buchholzia coriaceae could serve as cheap and accessible alternative.展开更多
Fibrinolytic enzymes have received attention regarding their medicinal potential for thrombolytic diseases, a leading cause of morbidity and mortality worldwide. Various natural enzymes purified from animal, plant and...Fibrinolytic enzymes have received attention regarding their medicinal potential for thrombolytic diseases, a leading cause of morbidity and mortality worldwide. Various natural enzymes purified from animal, plant and microbial sources have been extensively studied. The aim of this work was to produce fibrinolytic protease by solid state fermentation using agro industrial substrates. Rhizopus arrhizus var. arrhizus UCP 1295 and Mucor subtillissimus UCP 1262 filamentous fungi species isolated from soil of Caatinga-PE, Brasil, were used as producer microorganisms. Wheat bran was shown to be the best substrate for the production of the enzyme and by using a 23 full factorial design the main effects and interactions of the quantity of the substrate wheat bran, moisture and temperature on the fibrinolytic enzyme production and protease were evaluated. The best results for fibrinolytic and protease activities, 144.58 U/mL and 48.33 U/mL, respectively, were obtained with Mucor subtillissimus UCP 1262 using as culture medium 3 g wheat bran, 50% moisture at a temperature of 25°C for 72 hours. The optimum temperature for the produced enzyme was 45°C and most of its original activity was retained after being subjected to 80°C for 120 min. The protease activity was enhanced by K+, Ca+ and Mn+;but with Cu+ there was an inhibition. The specificity to chromogenic substrate and the inhibition by PMSF indicates that it is a chymotrypsin-like serine protease. Presented results suggest that this enzyme produced by solid-state fermentation is an interesting alternative as a candidate for thrombolytic therapy.展开更多
The culture condition of bacillus strain and the extraction method of Nattokinase were reported. The fibrinolytic activity of nattokinese was appraised.
Three fibrinolytic proteases, which were designated as BPGFP1, BPGFP2 and BPGFP3 individually, were purified from the gut extract of larvae of Boettcherisca peregrina fed on artificial diet containing fibrin rich...Three fibrinolytic proteases, which were designated as BPGFP1, BPGFP2 and BPGFP3 individually, were purified from the gut extract of larvae of Boettcherisca peregrina fed on artificial diet containing fibrin rich pig blood coagulated block. BPGFP1 consists of two subunits with MW 32 000 and 30 000. Both BPGFP2 and BPGFP3 are monomer with MW 40 000 and 28 000, respectively. These three proteases are similar in substrate and inhibitor specificity. All of them possess high activities against fibrinolytic protease specific substrates such as fibrin, Chromzym P, Chromzym UK and S 2288. They also strongly hydrolyze trypsin specific substrates Bz Phe Val Arg NA, cBz Gly Pro Arg NA, Bz Pro Phe Arg NA and Bz Val Gly Arg NA. PMSF, STI, LBTI and SBBI can inhibit activity of these proteases. Activities of these three fibrinolytic proteases were found to be maximal at alkaline range of pH 9.0～10.0.展开更多
BACKGROUND Pediatric complicated community-acquired pneumonia(CCAP)is on the rise.The three subtypes include para-pneumonic effusion(PPE),necrotizing pneumonia(NP),and empyema.AIM To study different sub-types of pedia...BACKGROUND Pediatric complicated community-acquired pneumonia(CCAP)is on the rise.The three subtypes include para-pneumonic effusion(PPE),necrotizing pneumonia(NP),and empyema.AIM To study different sub-types of pediatric CCAP,and compare their etiology,clinical profile,and outcome in the post-pneumococcal vaccination era.METHODS This prospective observational study was conducted over a 2-year period.All details(demographics,clinical,management,and outcomes)were recorded.Continuous data were presented either as mean and SD,or as median and interquartile range.Categorical data were presented as frequencies and percentages(%).Data were analyzed by using the IBM SPSS Statistics for Windows,version 21(IBM Corp.,Armonk,NY,United States).RESULTS Of the 80 cases included(71%aged 4-8 years),the distribution was as follows:PPE(42%),empyema(39%),and NP(19%).Bacterial etiology was identified in 28%(empyema 63%,P=0.012).Staphylococcus aureus(45%)was most common followed by Escherichia coli(E.coli)(22.7%),and Streptococcus pneumoniae(13.6%).Patients with empyema,compared to PPE and NP,were less likely to receive prior antibiotics(32%vs 56%and 58%,respectively,P=0.03).Duration(days,mean±SD)of hospitalization was longer in children with NP compared to empyema and PPE(17.7±9.8,16.1±7.5,and 13.6±4.2,respectively).All children recovered with the medical management except 2 children requiring decortication.CONCLUSION Staphylococcus aureus and E.coli are the most common bacterial etiology in the post-pneumococcal vaccination era.Empyema might be related to a delay in antibiotics administration.NP is the most severe pediatric CCAP with prolonged hospitalization.展开更多
Objective To measure the concentration of D-dimer (DD), tissue plasminogen activator (t-PA), plasminogen activator inhibitor-1 (PAI-1) and plasminogen (PLG) activity in plasma and cerebrospinal fluid in patients with ...Objective To measure the concentration of D-dimer (DD), tissue plasminogen activator (t-PA), plasminogen activator inhibitor-1 (PAI-1) and plasminogen (PLG) activity in plasma and cerebrospinal fluid in patients with acute cerebral infarction and to investigate their clinical significance.Methods The concentrations of D-dimer, t-PA, and PAI-1 in plasma and cerebrospinal fluid in patients were measured by enzyme-linked immunosorbent assay (ELISA). The PLG biological activity was detected using the chromophore method. The results were compared with those of the controls.Results The concentrations of D-dimer, t-PA and PAI-1 in cerebrospinal fluid and plasma in patients with acute cerebral infarction were much higher than those of normal subjects (P<0.01). Conversely, the level of PLG activity was significantly lower in the patients than in the controls (P<0.01).Conclusion Hypercoagulability and secondary hyperfibrinolysis exist in patients with acute cerebral infarction.展开更多
基金supported by grants from the Fundamental Research Funds for the Central Universities(No.2020-JYB-ZDGG-032)National Natural Science Foundation of China(No.82104352)。
文摘Objective:EPF3 is a fibrinolysin monomer isolated and purified from Pheretima vulgaris Chen,an earthworm used in traditional Chinese medicine as Dilong for treating blood stasis syndrome.Its composition,anticoagulant and fibrinolytic activities,and relevant mechanisms have been confirmed through in vitro experiments.However,whether it has antithrombotic effects in vivo and can be absorbed by the gastrointestinal tract is unknown.This study evaluates the antithrombotic effect in zebrafish and investigates the gastrointestinal stability and intestinal absorption mechanism of this protein in vitro.Methods:The antithrombotic effect of EPF3 in vivo was verified using the zebrafish thrombus model induced by arachidonic acid and FeCl3.Then,the protein bands of EPF3 incubated with simulated gastric fluid(SGF),simulated intestinal fluid(SIF),and homogenate of Caco-2 cells(HC2C)were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis to evaluate its gastrointestinal stability.Finally,the transport behavior and absorption mechanism of EPF3 were studied using Caco-2 cell monolayer.Results:EPF3 could significantly enhance the returned blood volume and blood flow velocity in zebrafish with platelet aggregation thrombus induced by arachidonic acid.It could also prolong the formation time of tail artery thrombus and increase the blood flow velocity in zebrafish with vessel injury thrombus induced by FeCl3.EPF3 was stable in SIF and HC2C and unstable in SGF.The permeability of EPF3 in Caco-2 monolayer was time-dependent and concentration-dependent.The efflux ratio was less than1.2 during transport,and the transport behavior was not affected by inhibitors.EPF3 could reversibly reduce the expression of tight junction-related proteins,including zonula occludens-1,occludin,and claudin-1 in Caco-2 cells.Conclusion:EPF3 could play a thrombolytic and antithrombotic role in zebrafish.It could be transported and absorbed into the intestine through cellular bypass pathway by opening the intestinal epithelium tight junction.This study provides a scientific explanation for the antithrombotic effect of earthworm and provides a basis for the feasibility of subsequent development of EPF3 as an antithrombotic enteric-soluble preparation.Please cite this article as:Zhong WL,Yang JQ,Liu H,Wu YL,Shen HJ,Li PY,Du SY.Antithrombotic effect in zebrafish of a fibrinolytic protein EPF3 from Dilong(Pheretima vulgaris Chen)and its transport mechanism in Caco-2 monolayer through cell bypass pathway.J Integr Med.2025;23(4):415–428.
文摘Objective: The purpose of this work was to investigate the distribution pattern of fibrinolytic factors and their inhibitors in rabbit tissues. Methods: The components of the fibrinolytic system in extracts from a variety of rabbit tissues, including tissue plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1), plasminogen (Plg), plasmin (Pl) and α2 plasmin inhibitor (α2PI), were determined by colorimetric assay. Results: The tissue extracts in renal, small intestine, lung, brain and spleen demonstrated strong fibrinolytic function, in which high activity of tPA, Plg and Pl was manifested; whereas in skeletal muscle, tongue and stomach, higher activity of PAI-1 and α2PI showed obviously. Also excellent linear correlations were found between levels of tPA and PAI-1, Pl and α2PI, Plg and Pl. In related tissues, renal cortex and renal marrow showed distinctly higher activity of tPA and lower activity of PAI-1, with the levels of Plg and Pl in renal cortex being higher than those in renal marrow, where the α2PI level was higher than that in renal cortex. Similarly, the levels of tPA, Plg and Pl in small intestine were higher than those in large intestine, but with respect to PAI-1 and α2PI, the matter was reverse. In addition, the fibrinolytic activity in muscle tissue was lower, however, the levels of tPA, Plg, and Pl in cardiac muscle were obviously higher than those in skeletal muscles, and the levels of PAI-1 and α2PI were significantly lower than those in skeletal muscle. Conclusion: Our data demonstrate that a remarkable difference of the fibrinolytic patterns exists in rabbit tissues, which has probable profound significance in understanding the relationship between the function of haemostasis or thrombosis and the physiologic function in tissues.
基金Supported by the National Natural Science Foundation of China(81660664)the Natural Science Foundation of Jiangxi Province(20192ACBL21032,20192BBGL70051,20192BAB205098,20171BAB21 5061,GJJ150844,GJJ160858)+1 种基金Jiangxi Provincial Health and Family Planning Commission(2017Z016)the Natural Science Foundation of Jiangxi University of Traditional Chinese Medicine(2014BS013).
文摘A strong fibrinolytic activity was demonstrated in the Semen Sojae Praeparatum(SSP), which is a famous traditional Chinese medicine. To study the activities and dynamic changes of fibrinolytic enzyme, standard fibrin plate was used to determine the fibrinolytic activity. For the first time fibrinolytic enzyme was found during the fermentation of SSP and the fibrinolytic activities of samples were shown to increase significantly over time. In the "yellow cladding" stage, the fibrinolytic activity was 619.75 IU/g. On day 6, 12 and 15 of the "secondary fermentation" stage, the fibrinolytic activity was 711.49 IU/g, 866.67 IU/g, 1 022.31 IU/g, respectively. The results indicate that fibrinolytic enzyme was generated during the fermentation of SSP and it displayed increasing activity which peaked at the "secondary fermentation" stage. The fibrinolytic enzyme was found to not only degrades fibrin directly, but also activate plasminogen to do so.
基金funded by the Science and Technology Research Project during the 11th Five-Year Plan period of Hebei Province, China (06220106D)
文摘The gene encoding fibrinolytic enzyme from Bacillus sp. zlw-2 was cloned and sequenced （accession no. EU734749）, which was 1146 bp, encoded 381 amino acids and had 99% homology with Nattokinase YF308 and NAT. The genes encoding pre-pro-fibrinolytic enzyme （including signal peptide, propeptide, and mature peptide） and fibrinolytic enzyme （including mature peptide） were cloned into pET28a vector respectively and then transformed into Escherichia coli BL21 （DE3）. The recombinant ofpre-pro-fibrinolytic enzyme showed enzyme activity of 183 U mL^-1, while no detectable enzyme activity could be found from the recombinant of the mature peptide.
基金supported by a grant from the Ministry of Environmental Protection (No. 200909036)
文摘Thrombus disease, one of the common cardiovascular diseases, has attracted worldwide at- tention for its rising mortality and morbidity. Due to the distinct shortages of current fibrinolytic drugs, new fibrinolytic agents warrant investigation. In this study, 8 fibrinolytic enzyme-producing strains were isolated from Douchi--a traditional Chinese food, and strain XY-1 which produced the largest amount of the enzyme was chosen for the following experiments. The enzyme produced by strain XY-1 was named Douchi fibrinolytic enzyme （DFE）. We optimized the liquid culture medium of strain XY-1 for enzyme production using Plackett-Burman and Box-Behnken design. The predicted maximal DFE yield was 19.78 FU/mL with 11.4 g/L peptone, 0.5 g/L magnesium sulfate and 1 g/L sodium chloride. How- ever, we acquired maximal production of 21.33 FU/mL in actual experiments, equal to 107.84% of the theoretical value, and the yield had been increased by 79.55% as compared to the yield of un-optimized culture. It was demonstrated that the combined use of Plackett-Burman design and response surface methodology in fermentation optimization can effectively and rapidly increase DFE production.
文摘A novel potent protease, Urechis unicinctus fibrinolytic enzyme （UFE）, was firstly discovered. The enzymatic properties of UFE were further investigated.As a low molecular mass protein,UFE appeared to be very stable to heat and pH.When temperature was below 50 ℃ ,the remnant enzyme activity remained almost unchanged, but when temperature was raised to 60 ℃ ,the remnant enzyme activity began to decrease rapidly. UFE was quite stable in the range of pH value from 3 to 12,especially in slightly alkaline pH value.Mn^2＋ ,Cu^2＋ and Fe^2＋ ions were activators of UFE, while Fe^3＋ and Ag^＋ ions were inhibitors of UFE.Fe^2＋ ion along with Fe^3＋ ion might regulate UFE activity in vivo. The optimum pH and temperature of UFE were about 8 and 50 ℃ ,respectively. Other characteristics of this enzyme were also studied. Systematic research results are significant when UFE is applied for medical and industrial purposes.
文摘By using equilibrium dialysis, atomic absorption spectrometry, fluorescence titration and determination of fluorescence lifetime, it can be determined that each fibrinolytic principle(FP) molecule contains one Ca 2+ binding site and one Ca 2+ ion. The energy transfer between Tb 3+ and the Trp residue in FP was studied through fluorescence spectroscopy. Our studies show that the Ca 2+ ion in an FP molecule can be substituted by Tb 3+ ion. In FP molecule, the excition energy can be transfered from the Trp residue as an energy donor to Tb 3+ as an acceptor, substituted into FP and located near the Trp residue. The distance between Tb 3+ and the Trp residue, ～0 375 nm, was worked out with the experimental data and Forster theory.
文摘In ST-segment elevation myocardial infarction (STEMI), acute reperfusion of the infarct-related artery (IRA)is the main goal in the early minutes after the patient seeks medical attention. Fibrinolytic therapy (FT) and/or primary coronary intervention (PCI) were proven to be effective in opening the IRA.
文摘The fibrinoiytic activity of peritoneal fluid (PF) and the incidence of luteinized unruptured follicle syndrome (LUFS) in 36 infertile women with endometriosis and 27 without endometriosis were studied The results showed that tissue plasminapen activity was significantly diminsihed during the early luteal phase in patients with endometriosis (P<0.05), but no significant difference between these two groups during the late follicular phase, and reduction of t -PA activity was associated with the incidence of LUF. It is postulated that decreare in t-PA activity due to LUF maybe resposible for infertility.
文摘Objective The purpose of this study was to evaluate the fibrinolytic response to exercise in patients with unstable angina pectoris(UAP) compared with an age - matched control group. Methods We measuredtissure- type plasminogen activator (t- PA) activity and plasminogen activator inhibitor 1(PAI- 1) activitybefore and after treadmill exercise test in 20 healthy subjects and 25 patients with UAP. ResultsResting t - PA activities were similar between two groups, but resting PAI- 1 activity was higher in UAP groupthan in control. Although both groups showed significant increase in t- PA activity with exercise, post - exerciset - PA activity was significantly lower in patients with UAP than in control (0.96± 0.45IU/ml vs 1.89± 0.68I U/ml,P<0.01), Post - exercise PAI- 1 activity was still much higher in UAP group than in control (8.20±2.28A U/ml vs4.21± 0.68A U/ml, P<0.01). Conclusion There existed impaired fibrinolysis in patients with UAP not only at restbut also alter exercise loading.
文摘In order to obtain all the properties of fermented ginseng, we fermented ginseng using Bacillus subtilis (B. subtilis) isolated from Cheonggukjang. A sterilized ginseng medium was made with 4-year-old ginseng powder and distilled water (300% ginseng powder [w/w]), and the ginseng was fermented by B. subtilis (1% ginseng medium) followed by incubation at 37°C for 3, 5, 7, and 10 days. The growth of B. subtilis in the ginseng medium significantly increased up to 9 log CFU/g, but no significant difference was observed after 3 days. As the fermentation progressed, the ginsenoside Rd and Rg+Rh1 contents increased by 255.3% - 322.5% and 165.6% - 228.6%, respectively, whereas the Rc, Re, and Rg1 contents decreased by 30.7% - 39.6%, 10.5% - 12.8%, and 16.2% - 16.6%, respectively. After 3 days of fermentation, a 6.25% - 7.12% viscous substance was produced;thereafter, the viscous substance was gradually reduced until it disappeared. The viscosity of the medium significantly decreased with a longer fermentation time. Fibrinolytic activity increased during 3 - 10 days of fermentation, indicating a relative activity of 85.0% - 100.0%.
文摘[Objective] To isolate and preserve a high-activity Douchi fibrinolytic enzyme producing strain from Douchi products. [Method] The Douchi flbrinolytic enzyme producing strain was screened on the selected medium prepared with self-made pork blood powder, the strain with the highest activity was screened out according to the size of hydrolysis cycle, and then preserved in LB medium. [ Rebait] A Douchi fibrinolytic enzyme producing strain with high thrombolytic activity was successfully screened out from the Douchi produced in Hunan, Guangdong and Jiangxi Prov- inces. [ Ceadusioe] The study lays foundation for the development of new-type thrombolytic drugs.
文摘Objective:To explore the effect of Guhong Injection combined with atyplase on vascular endothelial function and fibrinolytic system and related factors in patients with acute cerebral infarction.Methods:To select 90 patients with acute cerebral infarction in our hospital,divided into control group(48 cases)and observation group(48 cases)randomly.Patients in the control group were treated with alteplase on the basis of routine symptomatic treatment,and the observation group was treated with Guhong injection on the basis of the control group.Before and after treatment,the serum levels of related factors were detected and compared between the two groups.Results:Before treatment,there were no significant differences in serum related factors(ET-1,PAO,H-FABP,VEGF,S100β,BDNF,CEC,Fibulin-5,vWF,P-selectin,t-PA,PAI-1)between the two groups;After treatment,the serum levels of ET-1,PAO,H-FABP,S100β,CEC,Fibulin-5,vWF,P-selectin,PAI-1 in the observation group were significantly lower than those in the control group,and the serum levels of VEGF,BDNF and t-PA were higher than those in the control group,there were significant differences between the two groups.Conclusion:Guhong injection was added to patients with acute cerebral infarction on the basis of routine symptomatic treatment and ateplase,could significantly improve the level of serum related factors,it was more conducive to the control of symptoms and rehabilitation of patients,the effect was definite,it was worth further study and application in clinic.
文摘The first line drugs in the management of thromboernbolic diseases in Nigeria include Urokinase and Streptokinase. These drugs come with their attendant shortcomings. The need to source for cheap and accessible alternative therapy forms the basis of this study. The fibrinolytic potentials of 10 Nigerian medicinal plants： Anona senegalensis, Buchholzia coriaceae, Citrullus colocynthis, Cnidoscolus aconitifolius, Curculigo pilosa, Nicotiana tabaccum, Parinari curatellifolia, Pepperomia pellucida, Sida acuta and Xylopia aethiopica extracts were evaluated using water and streptokinase as negative and positive controls respectively. Trace metal （Cu, Fe, Ca and Zn） analysis of the plant extracts was carried out using atomic absorption spectrophotometer. Methodology involved in vitro clot lysis method, the crude methanolic extract ofParinari curatellifolia （56.122%）, Cnidoscolus aconitifolius （48.385%）, Anona senegalensis （46.360%）, Xylopia aethiopica （43.205%） and Buchholzia coriaceae （27.062%） were found to have significant （P 〉 0.001） fibrinolytic activity. Maximum effect was exhibited by the positive standard： Streptokinase （60.20 ± 4.30%）, followed by Parinari curatellifolia （56.122 ± 3.86%）, our findings reveal that fibrinolytic activity is independent of elemental Copper or Iron content of the extracts. This study indicated that methanolic extracts of Parinari curatellifolia, Cnidoscolus aconitifolius, Anona senegalensis, Xylopia aethiopica and Buchholzia coriaceae could serve as cheap and accessible alternative.
基金the financial support from CAPES(Coordenacao de Aperfeicoamento de Pessoal de Nível Superior,Brasília,Brasil),FACEPE(Fundacao de Amparo a Ciencia e Tecnologia do Estado de Pernambuco,Recife,Brasil)and the CNPq(Conselho Nacional de Desenvolvimento Científico e Tecnológico,Brasília,Brasil)the project was approved in network RENNORFUN Notice MCT/CNPq/MMA/MEC/CAPES/FNDCTAcao Transversal/FAPs n.47/2010,Sistema Nacional de Pesquisa em Biodiversidade-SISBIOTA/Brasil.
文摘Fibrinolytic enzymes have received attention regarding their medicinal potential for thrombolytic diseases, a leading cause of morbidity and mortality worldwide. Various natural enzymes purified from animal, plant and microbial sources have been extensively studied. The aim of this work was to produce fibrinolytic protease by solid state fermentation using agro industrial substrates. Rhizopus arrhizus var. arrhizus UCP 1295 and Mucor subtillissimus UCP 1262 filamentous fungi species isolated from soil of Caatinga-PE, Brasil, were used as producer microorganisms. Wheat bran was shown to be the best substrate for the production of the enzyme and by using a 23 full factorial design the main effects and interactions of the quantity of the substrate wheat bran, moisture and temperature on the fibrinolytic enzyme production and protease were evaluated. The best results for fibrinolytic and protease activities, 144.58 U/mL and 48.33 U/mL, respectively, were obtained with Mucor subtillissimus UCP 1262 using as culture medium 3 g wheat bran, 50% moisture at a temperature of 25°C for 72 hours. The optimum temperature for the produced enzyme was 45°C and most of its original activity was retained after being subjected to 80°C for 120 min. The protease activity was enhanced by K+, Ca+ and Mn+;but with Cu+ there was an inhibition. The specificity to chromogenic substrate and the inhibition by PMSF indicates that it is a chymotrypsin-like serine protease. Presented results suggest that this enzyme produced by solid-state fermentation is an interesting alternative as a candidate for thrombolytic therapy.
文摘The culture condition of bacillus strain and the extraction method of Nattokinase were reported. The fibrinolytic activity of nattokinese was appraised.
文摘Three fibrinolytic proteases, which were designated as BPGFP1, BPGFP2 and BPGFP3 individually, were purified from the gut extract of larvae of Boettcherisca peregrina fed on artificial diet containing fibrin rich pig blood coagulated block. BPGFP1 consists of two subunits with MW 32 000 and 30 000. Both BPGFP2 and BPGFP3 are monomer with MW 40 000 and 28 000, respectively. These three proteases are similar in substrate and inhibitor specificity. All of them possess high activities against fibrinolytic protease specific substrates such as fibrin, Chromzym P, Chromzym UK and S 2288. They also strongly hydrolyze trypsin specific substrates Bz Phe Val Arg NA, cBz Gly Pro Arg NA, Bz Pro Phe Arg NA and Bz Val Gly Arg NA. PMSF, STI, LBTI and SBBI can inhibit activity of these proteases. Activities of these three fibrinolytic proteases were found to be maximal at alkaline range of pH 9.0～10.0.
文摘BACKGROUND Pediatric complicated community-acquired pneumonia(CCAP)is on the rise.The three subtypes include para-pneumonic effusion(PPE),necrotizing pneumonia(NP),and empyema.AIM To study different sub-types of pediatric CCAP,and compare their etiology,clinical profile,and outcome in the post-pneumococcal vaccination era.METHODS This prospective observational study was conducted over a 2-year period.All details(demographics,clinical,management,and outcomes)were recorded.Continuous data were presented either as mean and SD,or as median and interquartile range.Categorical data were presented as frequencies and percentages(%).Data were analyzed by using the IBM SPSS Statistics for Windows,version 21(IBM Corp.,Armonk,NY,United States).RESULTS Of the 80 cases included(71%aged 4-8 years),the distribution was as follows:PPE(42%),empyema(39%),and NP(19%).Bacterial etiology was identified in 28%(empyema 63%,P=0.012).Staphylococcus aureus(45%)was most common followed by Escherichia coli(E.coli)(22.7%),and Streptococcus pneumoniae(13.6%).Patients with empyema,compared to PPE and NP,were less likely to receive prior antibiotics(32%vs 56%and 58%,respectively,P=0.03).Duration(days,mean±SD)of hospitalization was longer in children with NP compared to empyema and PPE(17.7±9.8,16.1±7.5,and 13.6±4.2,respectively).All children recovered with the medical management except 2 children requiring decortication.CONCLUSION Staphylococcus aureus and E.coli are the most common bacterial etiology in the post-pneumococcal vaccination era.Empyema might be related to a delay in antibiotics administration.NP is the most severe pediatric CCAP with prolonged hospitalization.
文摘Objective To measure the concentration of D-dimer (DD), tissue plasminogen activator (t-PA), plasminogen activator inhibitor-1 (PAI-1) and plasminogen (PLG) activity in plasma and cerebrospinal fluid in patients with acute cerebral infarction and to investigate their clinical significance.Methods The concentrations of D-dimer, t-PA, and PAI-1 in plasma and cerebrospinal fluid in patients were measured by enzyme-linked immunosorbent assay (ELISA). The PLG biological activity was detected using the chromophore method. The results were compared with those of the controls.Results The concentrations of D-dimer, t-PA and PAI-1 in cerebrospinal fluid and plasma in patients with acute cerebral infarction were much higher than those of normal subjects (P<0.01). Conversely, the level of PLG activity was significantly lower in the patients than in the controls (P<0.01).Conclusion Hypercoagulability and secondary hyperfibrinolysis exist in patients with acute cerebral infarction.
