Effect of Guhong injection combined with atyplase on vascular endothelial function and fibrinolytic system and related factors in patients with acute cerebral infarction

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.

转枯草芽孢杆菌纤溶酶(Bacillus subtilis fibrinolytic enzyme,BSFE)基因对烟草生长发育的影响

将枯草芽孢杆菌纤溶酶(Bacillussubtilisfibrinolyticenzyme,BSFE)基因及其信号肽序列转入烟草,转基因烟草表现出不同于野生型烟草的生长特性,如营养器官生长速率下降、叶片数目减少、叶片狭长、叶片宽度/叶片长度值降低以及植株矮小、节间缩短等。推测其与外源BSFE的蛋白水解活性有关。

Cloning and Expression of One Fibrinolytic Enzyme from Bacillus sp. zlw-2

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.

Enzymatic properties of UFE,a novel marine fibrinolytic enzyme

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.

Optimization of Douchi Fibrinolytic Enzyme Production by Statistical Experimental Methods

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.

Interaction between Tb^(3 + )and Fibrinolytic Principle from Agkistrodon Acutus Venom

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.

Novel distribution pattern of fibrinolytic components in rabbit tissues extract: a preliminary study

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.

Lessons learned from the TIMI trials in rescue interventions for elderly patients after failed fibrinolytic therapy:look beyond the TIMI flow

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.

FIBRINOLYTIC ACTIVITY OF PERITONEAL FLUID AND LUF IN INFERTILE WOMEN WITH ENDOMETRIOSIS

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.

EFFECTS OF EXERCISE ON THE FIBRINOLYTIC ACTIVITY INPATIENTS WITH UNSTABLE ANGINA PECTORIS

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.

Physicochemical Properties and Fibrinolytic Activity of Ginseng Powder Fermented with <i>Bacillus subtilis</i>Isolated from <i>Cheonggukjang</i>

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%.

Screening and Preservation of Douchi Fibrinolytic Enzyme Producing Strain

[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.

Fibrinolytic Activity of Some Nigerian Medicinal Plants

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.

Production and Characterization of New Fibrinolytic Protease from Mucor subtillissimus UCP 1262 in Solid-State Fermentation

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.

Studies on a Novel Fibrinolytic Enzyme(Nattokinase)in the Vegetable Cheese Natto

The culture condition of bacillus strain and the extraction method of Nattokinase were reported. The fibrinolytic activity of nattokinese was appraised.

Effect of reperfusion strategy on QT dispersion in patients with acute myocardial infarction:Impact on in-hospital arrhythmia

BACKGROUND Myocardial ischemia and ST-elevation myocardial infarction(STEMI)increase QT dispersion(QTD)and corrected QT dispersion(QTcD),and are also associated with ventricular arrhythmia.AIM To evaluate the effects of reperfusion strategy[primary percutaneous coronary intervention(PPCI)or fibrinolytic therapy]on QTD and QTcD in STEMI patients and assess the impact of the chosen strategy on the occurrence of in-hospital arrhythmia.METHODS This prospective,observational,multicenter study included 240 patients admitted with STEMI who were treated with either PPCI(group I)or fibrinolytic therapy(group II).QTD and QTcD were measured on admission and 24 hr after reperfusion,and patients were observed to detect in-hospital arrhythmia.RESULTS There were significant reductions in QTD and QTcD from admission to 24 hr in both group I and group II patients.QTD and QTcD were found to be shorter in group I patients at 24 hr than those in group II(53±19 msec vs 60±18 msec,P=0.005 and 60±21 msec vs 69+22 msec,P=0.003,respectively).The occurrence of in-hospital arrhythmia was significantly more frequent in group II than in group I(25 patients,20.8%vs 8 patients,6.7%,P=0.001).Furthermore,QTD and QTcD were higher in patients with in-hospital arrhythmia than those without(P=0.001 and P=0.02,respectively).CONCLUSION In STEMI patients,PPCI and fibrinolytic therapy effectively reduced QTD and QTcD,with a higher observed reduction using PPCI.PPCI was associated with a lower incidence of in-hospital arrhythmia than fibrinolytic therapy.In addition,QTD and QTcD were shorter in patients not experiencing in-hospital arrhythmia than those with arrhythmia.

Targeted thrombolysis strategies for neuroprotective effect

Stroke is usually treated by systemic thrombolytic therapy if the patient presents within an appropriate time window. There is also widespread interest in the development of thrombolytic agents that can be used in cases of delayed presentation. Current agents that can be used in cases of delayed presentation of nerve damage by thrombus. Current systemic thrombolytic therapy is associated with adverse effects such as fibrinogenolysis and bleeding. In an attempt to increase the efficacy, safety, and specificity of thrombolytic therapy, a number of targeted thrombolytic agents have been studied in recent years. This review focuses on the concepts underlying targeted thrombolytic therapy and describes recent drug developments in this field.

Recent advances in the diagnosis and treatment of acute myocardial infarction

The Third Universal Definition of Myocardial Infarction(MI) requires cardiac myocyte necrosis with an increase and/or a decrease in a patient's plasma of cardiac troponin(cT n) with at least one cT n measurement greater than the 99 th percentile of the upper normal reference limit during:(1) symptoms of myocardialischemia;(2) new significant electrocardiogram(ECG) ST-segment/T-wave changes or left bundle branch block;(3) the development of pathological ECG Q waves;(4) new loss of viable myocardium or regional wall motion abnormality identified by an imaging procedure; or(5) identification of intracoronary thrombus by angiography or autopsy.Myocardial infarction,when diagnosed,is now classified into five types.Detection of a rise and a fall of troponin are essential to the diagnosis of acute MI.However,high sensitivity troponin assays can increase the sensitivity but decrease the specificity of MI diagnosis.The ECG remains a cornerstone in the diagnosis of MI and should be frequently repeated,especially if the initial ECG is not diagnostic of MI.There have been significant advances in adjunctive pharmacotherapy,procedural techniques and stent technology in the treatment of patients with MIs.The routine use of antiplatelet agents such as clopidogrel,prasugrel or ticagrelor,in addition to aspirin,reduces patient morbidity and mortality.Percutaneous coronary intervention(PCI) in a timely manner is the primary treatment of patients with acute ST segment elevation MI.Drug eluting coronary stents are safe and beneficial with primary coronary intervention.Treatment with direct thrombin inhibitors during PCI is non-inferior to unfractionated heparin and glycoprotein Ⅱb/Ⅲa receptor antagonists and is associated with a significant reduction in bleeding.The intra-coronary use of a glycoprotein Ⅱb/Ⅲa antagonist can reduce infarct size.Pre- and post-conditioning techniques can provide additional cardioprotection.However,the incidence and mortality due to MI continues to be high despite all these recent advances.The initial ten year experience with autologous human bone marrow mononuclear cells(BMCs) in patients with MI showed modest but significant increases in left ventricular(LV) ejection fraction,decreases in LV endsystolic volume and reductions in MI size.These studies established that the intramyocardial or intracoronary administration of stem cells is safe.However,many of these studies consisted of small numbers of patients who were not randomized to BMCs or placebo.The recent LateT ime,Time,and Swiss Multicenter Trials in patientswith MI did not demonstrate significant improvement in patient LV ejection fraction with BMCs in comparison with placebo.Possible explanations include the early use of PCI in these patients,heterogeneous BMC populations which died prematurely from patients with chronic ischemic disease,red blood cell contamination which decreases BMC renewal,and heparin which decreases BMC migration.In contrast,cardiac stem cells from the right atrial appendage and ventricular septum and apex in the SCIPIO and CADUCEUS Trials appear to reduce patient MI size and increase viable myocardium.Additional clinical studies with cardiac stem cells are in progress.

Evaluation of cytotoxic and anti-tumor activity of partially purified serine protease isolate from the Indian earthworm Pheretima posthuma

Objective:To isolate,partially purify and evaluate the cytotoxic and antitumor activity of a serine protease from the chosen Indian earthworm Pheretima posthuma.Methods:Whole animal extract was prepared and purified its protein constituents by size and charge based chromatographic separation techniques using Sephadex G-50 and DEAE-Cellulose resin respectively.Average molecular weight of the protein isolate was determined and analyzed for its cytotoxic property against Vero cells in different dilutions(1:20 and 1:40)and anti-tumor activity by MTT assay(a colorimetric assay)using breast cancer cell line MCF-7,with tamoxifen as standard.Results:One of the protein constituents after purification was characterized as serine protease by Caseinolytic plate diffusion assay.Average molecular weight of this purified isolate was determined,by SDS-PAGE analysis with standard protein ladder,as of 15 kDa.The performed tests suggested that the 15kDa fraction has potent cytotoxic activity and satisfactory antitumor activity as well in vitro.Conclusions:Exact molecular mechanism of the cytotoxic and antitumor activities is yet to be explored and currently we are working on ultra-purification and biophysical characterization of this fraction.Further investigation into the mechanism(s)of cytotoxic and antitumor activities at molecular level would be useful in treatment of various classes of cancer and viral infections in future.

GAP-43 Expression and Pathological Changes of Temporal Infarction in R.ats and Effects of Batroxobin

To study the changes of the expression of growth-associated protein-43 (GAP-43) and pathology in temporal infarction of rats photochemically induced and the effects of batroxobin. METHODS: Immunohistochemical technique and hematoxylin-eosin stain was used to show the changes of the expression of GAP-43 and pathology. RESULTS: In infarction group, GAP-43 expression was markedly increased on the infarction and surrounding tissues at 24 h cerebral infarction. The expression reached peak level at 72 h after cerebral infarction and was decreased at 7 d after cerebral infarction. However, in batroxobin-treated group, GAP-43 expression was increased and the pathological changes were much slight as compared with infarction group. CONCLUSION: The expression of GAP-43 increases in infarction of temporal neocortex and batroxobin promotes the expression of GAP-43 and ameliorates the pathological changes in infarction of temporal neocortex.