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

2015 ◽  
Vol 61 (5) ◽  
pp. 157-164 ◽  
Author(s):  
Manoj Kumar Narasimhan ◽  
Muthukumaran Chandrasekaran ◽  
Mathur Rajesh
Keyword(s):  
Bacillus Cereus ◽  
Enzyme Production ◽  
Blood Clot ◽  
Fibrinolytic Enzyme ◽  
Clot Lysis

scholarly journals Statistical Optimization of Fibrinolytic Enzyme Production Using Agroresidues byBacillus cereusIND1 and Its Thrombolytic ActivityIn Vitro

2014 ◽  
Vol 2014 ◽  
pp. 1-11 ◽  
Author(s):  
Ponnuswamy Vijayaraghavan ◽  
Samuel Gnana Prakash Vincent
Keyword(s):  
Enzyme Production ◽  
Blood Clot ◽  
Exchange Chromatography ◽  
Fibrinolytic Enzyme ◽  
Dihydrogen Phosphate ◽  
Sodium Dihydrogen Phosphate ◽  
Beef Extract ◽  
Diethylaminoethyl Cellulose ◽  
Sodium Dihydrogen

A potent fibrinolytic enzyme-producingBacillus cereusIND1 was isolated from the Indian food, rice. Solid-state fermentation was carried out using agroresidues for the production of fibrinolytic enzyme. Among the substrates, wheat bran supported more enzyme production and has been used for the optimized enzyme production by statistical approach. Two-level full-factorial design demonstrated that moisture, supplementation of beef extract, and sodium dihydrogen phosphate have significantly influenced enzyme production (P<0.05). A central composite design resulted in the production of 3699 U/mL of enzyme in the presence of 0.3% (w/w) beef extract and 0.05% (w/w) sodium dihydrogen phosphate, at 100% (v/w) moisture after 72 h of fermentation. The enzyme production increased fourfold compared to the original medium. This enzyme was purified to homogeneity by ammonium sulfate precipitation, diethylaminoethyl-cellulose ion-exchange chromatography, Sephadex G-75 gel filtration chromatography, and casein-agarose affinity chromatography and had an apparent molecular mass of 29.5 kDa. The optimum pH and temperature for the activity of fibrinolytic enzyme were found to be 8.0 and 60°C, respectively. This enzyme was highly stable at wide pH range (7.0–9.0) and showed 27% ± 6% enzyme activity after initial denaturation at 60°C for 1 h.In vitroassays revealed that the enzyme could activate plasminogen and significantly degraded the fibrin net of blood clot, which suggests its potential as an effective thrombolytic agent.

scholarly journals Cow Dung Is a Novel Feedstock for Fibrinolytic Enzyme Production from Newly Isolated Bacillus sp. IND7 and Its Application in In Vitro Clot Lysis

2016 ◽  
Vol 7 ◽  
Author(s):  
Ponnuswamy Vijayaraghavan ◽  
Arumugaperumal Arun ◽  
Samuel Gnana Prakash Vincent ◽  
Mariadhas Valan Arasu ◽  
Naif Abdullah Al-Dhabi
Keyword(s):  
Enzyme Production ◽  
Fibrinolytic Enzyme ◽  
Clot Lysis ◽  
Cow Dung ◽  
Bacillus Sp

Dependence of Blood Clot Lysis on the Mode of Transport of Urokinase into the Clot - A Magnetic Resonance Imaging Study in Vitro

1991 ◽  
Vol 65 (05) ◽  
pp. 549-552 ◽  
Author(s):  
A Blinc ◽  
G Planinšič ◽  
D Keber ◽  
O Jarh ◽  
G Lahajnar ◽  
...  
Keyword(s):  
Magnetic Resonance Imaging ◽  
Magnetic Resonance ◽  
Pressure Difference ◽  
Volume Flow Rate ◽  
Blood Clot ◽  
Linear Regression Analysis ◽  
Imaging Study ◽  
Clot Lysis ◽  
Resonance Imaging

SummaryMagnetic resonance imaging was employed to study the dependence of clot lysing patterns on two different modes of transport of urokinase into whole blood clots. In one group of clots (nonperfused clots, n1 = 10), access of urokinase to the fibrin network was possible by diffusion only, whereas in the other group (perfused clots, n2 = 10) bulk flow of plasma containing urokinase was instituted through occlusive clots by a pressure difference of 3 .7 kPa (37 cm H2O) across 3 cm long clots with a diameter of 4 mm. It was determined separately that this pressure difference resulted in a volume flow rate of 5.05 ± 2.4 × 10−2 ml/min through occlusive clots. Perfused clots diminished in size significantly in comparison to nonperfused ones already after 20 min (p <0.005). Linear regression analysis of two-dimensional clot sizes measured by MRI showed that the rate of lysis was more than 50-times faster in the perfused group in comparison to the nonperfused group. It was concluded that penetration of the thrombolytic agent into clots by perfusion is much more effective than by diffusion. Our results might have some implications for understanding the differences in lysis of arterial and venous thrombi.

Effect of Heparin on TAFI-Dependent Inhibition of Fibrinolysis: Relative Importance of TAFIa Generated by Clot-Bound and Fluid Phase Thrombin

2002 ◽  
Vol 88 (08) ◽  
pp. 282-287 ◽  
Author(s):  
Anna Pentimone ◽  
Bianca Binetti ◽  
Marialisa Cramarossa ◽  
Donatella Piro ◽  
Nicola Semeraro ◽  
...  
Keyword(s):  
Thrombin Generation ◽  
Blood Clot ◽  
Fluid Phase ◽  
Clot Lysis ◽  
Relative Importance ◽  
Thrombin Activatable Fibrinolysis Inhibitor ◽  
Dependent Inhibition ◽  
Fibrinolysis Inhibitor ◽  
Dependent Mechanism

SummaryHeparin has been proposed to enhance thrombolysis by inhibiting thrombin-dependent generation of activated TAFI (thrombin activatable fibrinolysis inhibitor), a carboxypeptidase that inhibits fibrinolysis. We evaluated the effect of heparin in an in vitro thrombolysis model consisting of a radiolabelled blood clot submerged in defibrinated plasma. Fibrinolysis was induced by adding t-PA (250 ng/ml) and calcium to the plasma bath. Control experiments indicated that thrombin generation induced by recalcification caused significant TAFI activation and inhibited clot lysis. Heparin (up to 1 U/ml), added to the plasma bath, failed to enhance clot lysis. Thrombin generation in the fluid phase was totally inhibited by heparin at concentrations > 0.5 U/ml. In contrast, thrombin generation on the clot surface was not inhibited by heparin (1 U/ml). TAFIa generation did occur in heparin-containing samples (1 U/ml) and amounted to about 10% of TAFIa formed in control samples. This low amount of TAFIa did exert antifibrinolytic activity as indicated by the observation that the addition of a specific TAFIa inhibitor (PTI) along with heparin enhanced clot lysis. Hirudin (10 µg/ml), at variance with heparin, inhibited clot-bound thrombin and enhanced clot lysis. These data show that heparin is unable to stimulate fibrinolysis through a TAFI-dependent mechanism, most likely because of its inefficiency in inhibiting thrombin generation on the clot surface. Moreover, they suggest that clot-bound thrombin plays a major role in TAFI-mediated inhibition of fibrinolysis through “localized” TAFIa generation.

scholarly journals PRODUCTION AND PARTIAL CHARACTERIZATION OF FIBRINOLYTIC ENZYME FROM A SOIL ISOLATE ASPERGILLUS CARBONARIUS S-CSR-0007

2016 ◽  
Vol 8 (12) ◽  
pp. 142 ◽  
Author(s):  
Afini A.v. M. ◽  
Sooraj S. Nath ◽  
Smitha K. V. ◽  
Kunhi A.a. M.
Keyword(s):  
Ammonium Sulfate ◽  
Substrate Concentration ◽  
Fibrinolytic Activity ◽  
Blood Clot ◽  
Maximum Activity ◽  
Fibrinolytic Enzyme ◽  
Clot Lysis ◽  
Aspergillus Carbonarius ◽  
Lysis Activity ◽  
Wide Range

<p><strong>Objective: </strong>This work was undertaken with the aim of isolating and screening fungal soil isolates with fibrinolytic activity.</p><p><strong>Methods: </strong>Soil sample near slaughter house was collected and screened for fibrinolytic activity by using fibrin-agar. Enzyme production was optimized under various parameters like pH, temperature, substrate concentration and purified partially by ammonium sulphate precipitation. The stability of the partially purified enzyme was analyzed under the influence of a wide range of pH, temperature, and substrate concentrations.<strong></strong></p><p><strong>Results: </strong>Among the seven isolates screened, <em>Aspergillus carbonarius</em> S-CSR-0007 exhibited largest clear zone and was selected for further studies. Among the various substrates tested casein was found to support the highest caseinolytic activity of 816 U/ml and fibrinolytic activity of 510 U/ml. The culture supernatant of <em>A. carbonarius</em> S-CSR-0007 was fractionated by ammonium sulfate precipitation followed by dialysis, and maximum activity was obtained in the fraction with 80% ammonium sulfate, with an enzyme activity of 1200 U/ml using tyrosine as standard. The partially purified fibrinolytic enzyme showed optimal activity at 45 °C and pH 7.0. The enzyme was stable up to a temperature of 50 °C and pH 8.0, and the optimum substrate concentration was 4%.</p><p><strong>Conclusion: </strong>The crude enzyme showed high blood clot lysis activity, which may be a good candidate in the pharmaceutical industry. However, more studies need to be carried out to establish its clinical use.</p>

In vitro whole blood clot lysis by rt-PA is inversely correlated with baseline plasma levels of t-PA antigen

1994 ◽  
Vol 8 ◽  
pp. 43
Author(s):  
M. Colucci ◽  
S. Scopece ◽  
A. Gelato ◽  
L.G. Cavallo ◽  
N. Semeraro
Keyword(s):  
Whole Blood ◽  
Plasma Levels ◽  
Blood Clot ◽  
Clot Lysis

scholarly journals Novel Fibrinolytic Protease Producing Streptomyces radiopugnans VITSD8 from Marine Sponges

Marine Drugs ◽  
2019 ◽  
Vol 17 (3) ◽  
pp. 164 ◽  
Author(s):  
Dhamodharan D ◽  
Jemimah S ◽  
Merlyn S ◽  
Subathra C
Keyword(s):  
Tamil Nadu ◽  
Specific Activity ◽  
Blood Clot ◽  
Exchange Chromatography ◽  
Marine Sponges ◽  
Protease Production ◽  
Clot Lysis ◽  
Carbon And Nitrogen ◽  
Lysis Activity

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

scholarly journals Novel Sequential Screening and Enhanced Production of Fibrinolytic Enzyme byBacillussp. IND12 Using Response Surface Methodology in Solid-State Fermentation

2017 ◽  
Vol 2017 ◽  
pp. 1-13 ◽  
Author(s):  
Ponnuswamy Vijayaraghavan ◽  
P. Rajendran ◽  
Samuel Gnana Prakash Vincent ◽  
Arumugaperumal Arun ◽  
Naif Abdullah Al-Dhabi ◽  
...  
Keyword(s):  
Response Surface Methodology ◽  
Solid State ◽  
Response Surface ◽  
Solid State Fermentation ◽  
Enzyme Production ◽  
Blood Clot ◽  
Fibrinolytic Enzyme ◽  
Surface Model ◽  
Cow Dung ◽  
Enhanced Production

Fibrinolytic enzymes have wide applications in clinical and waste treatment. Bacterial isolates were screened for fibrinolytic enzyme producing ability by skimmed milk agar plate using bromocresol green dye, fibrin plate method, zymography analysis, and goat blood clot lysis. After these sequential screenings,Bacillussp. IND12 was selected for fibrinolytic enzyme production.Bacillussp. IND12 effectively used cow dung for its growth and enzyme production (687±6.5 U/g substrate). Further, the optimum bioprocess parameters were found out for maximum fibrinolytic enzyme production using cow dung as a low cost substrate under solid-state fermentation. Two-level full-factorial experiments revealed that moisture, pH, sucrose, peptone, and MgSO4were the vital parameters with statistical significance (p<0.001). Three factors (moisture, sucrose, and MgSO4) were further studied through experiments of central composite rotational design and response surface methodology. Enzyme production of optimized medium showed4143±12.31 U/g material, which was more than fourfold the initial enzyme production (978±36.4 U/g). The analysis of variance showed that the developed response surface model was highly significant (p<0.001). The fibrinolytic enzyme digested goat blood clot (100%), chicken skin (83±3.6%), egg white (100%), and bovine serum albumin (29±4.9%).

scholarly journals A preliminary investigation of in vitro anti-thrombotic and anti-platelet activity of Pinus Gerardiana

2017 ◽  
Vol 4 (1) ◽  
pp. 1098 ◽  
Author(s):  
Atta-ur Rehman ◽  
Sara Naz ◽  
Muhammad Zaman ◽  
Syed Saeed-ul-Hassan ◽  
Javed Iqbal ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Blood Clot ◽  
Preliminary Investigation ◽  
Circulatory System ◽  
Clot Lysis ◽  
Platelet Activity ◽  
In Vivo Models ◽  
The Stability

Introduction: Hemostasis is a process which preserves the stability of a closed and high-pressure circulatory system after any vascular injury. Circulating platelets are recruited to the site of injury, where they develop a major component of the developing thrombus, blood clotting, started by tissue factor, concludes in the generation of thrombin and fibrin. Thrombosis is a serious event in the arterial diseases and a major cause in the development of myocardial infarction, stroke and venous thrombo-embolism which justify prominent morbidity and mortality rate. The knowledge of molecular and cellular mechanism of the formation of thrombus has developed considerably in the recent studies by using different in-vitro and in-vivo models of diseases. P. gerardiana nut oil has been reported to possess anti-bacterial, anti-fungal, anti-viral, anti-septic, anti-neuralgic, diuretic, expectorant, hypertensive properties. However, hardly, any data is available regarding effects of nut oil on platelet function. In this study, fibrinolytic activity and effect on platelet aggregation were investigated. Method: P. gerardiana nut oil was extracted by using n-Hexane and then concentrated by rotary evaporator. Anti-thrombotic and fibrinolytic activities were evaluated on blood clot formation. Effects on platelet aggregation of the oil were determined based on collagen or epinephrine induced platelet aggregation. Results: P. gerardiana caused blood clot lysis in-vitro. P. gerardiana nut oil inhibited collagen dependent platelet aggregation while accelerated the epinephrine dependent platelet aggregation. In vitro whole blood coagulation was also reduced. In vivo P. gerardiana nut oil has no significant effect on blood cell indices. Conclusion: P. gerardiana nuts oil can be an effective therapy for the treatment of cardiovascular disorders and thromboembolism.

scholarly journals Thrombolytic Potential of Novel Thiol-Dependent Fibrinolytic Protease from Bacillus cereus RSA1

Biomolecules ◽  
2019 ◽  
Vol 10 (1) ◽  
pp. 3 ◽  
Author(s):  
Chhavi Sharma ◽  
Gad Elsayed Mohamed Salem ◽  
Neha Sharma ◽  
Prerna Gautam ◽  
Rajni Singh
Keyword(s):  
Bacillus Cereus ◽  
Fibrinolytic Activity ◽  
Thrombolytic Agent ◽  
Divalent Cations ◽  
Blood Clot ◽  
Fold Increase ◽  
Protease Production ◽  
Stability Range ◽  
Stain Removal

The present study demonstrates the production and thrombolytic potential of a novel thermostable thiol-dependent fibrinolytic protease by Bacillus cereus RSA1. Statistical optimization of different parameters was accomplished with Plackett–Burman design and validated further by central composite design with 30.75 U/mL protease production. Precipitation and chromatographic approaches resulted in 33.11% recovery with 2.32-fold purification. The molecular weight of fibrinolytic protease was 40 KDa and it exhibited a broad temperature and pH stability range of 20–80 °C and pH 5–10 with utmost activity at 50 °C and pH 8, respectively. The protease retained its fibrinolytic activity in organic solvents and enhanced the activity in solutions with divalent cations (Mn2+, Zn2+, and Cu2+). The enzyme kinetics revealed Km and Vmax values of 1.093 mg/mL and 52.39 µg/mL/min, respectively, indicating higher affinity of fibrinolytic activity towards fibrin. Also, complete inhibition of fibrinolytic activity with DFP and a 2-fold increase with DTT and β-mercaptoethanol indicates its thiol-dependent serine protease nature. MALDI–TOF analysis showed 56% amino acid sequence homology with Subtilisin NAT OS = Bacillus subtilis subsp. natto. The fibrinolysis activity was compared with a commercial thrombolytic agent for its therapeutic applicability, and fibrinolytic protease was found highly significant with absolute blood clot dissolution within 4 h in in vitro conditions. The isolated fibrinolytic protease of Bacillus cereus RSA1 is novel and different from other known fibrinolytic proteases with high stability and efficacy, which might have wide medicinal and industrial application as a thrombolytic agent and in blood stain removal, respectively.

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