Prospective <i>In Vivo</i> Assays on the Antithrombotic Potential of Protease Extracted from <i>Bacillus</i> sp. HSFI-12

Dewi, Okta Yosiana; Ethica, Stalis Norma; Sukeksi, Andri; Rakhmawatie, Maya Dian; Darmawati, Sri

doi:10.2991/absr.k.220406.056

Cited by 2 publications

(4 citation statements)

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“…Further tests to con rm the potential of puri ed protease as an antithrombotic agent are prospective to be done in vivo. This is also as stated by Dewi et al (2022) . Further puri cation can be done by subjecting protease dialysate obtained in this study to an ion exchange or gel ltration chromatography (Zilda et al, 2014) .…”

Section: Determination Of Molecular Weight and Speci City Ofsupporting

confidence: 82%

“…Among bacterial strains producing thrombolytic proteases previously isolated from the intestine of sea cucumber Holothuria scabra is Bacillus thuringiensis HSFI-12. The in vitro thrombolytic activity of Bacillus thuringiensis HSFI-12 was found to be competitive when compared to commercial protease Nattokinase, known as a standard antithrombosis agent (Zafrida et al, 2022, Dewi et al, 2022. However, its potential to be an antithrombotic agent needs con rmation and retesting.…”

Section: Introductionmentioning

confidence: 99%

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Characteristics and Substrate Specificity of Semi-Purified Bacterial Protease ofBacillusthuringiensisHSFI-12 with Potential as Antithrombotic Agent

Ferdiani

Zilda²,

Afriansyah

et al. 2023

Sci. Technol. Indones

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Commercial proteases, such as Nattokinase (NK), Staphylokinase (SAK), and Streptokinase (SK) play an important role in the destruction of thrombus, the main cause of death in cardiovascular disease. The latest technology combining enzymes with certain drugs is the target of new research in the thrombolytic area. The first step is to develop protease from Bacillus thuringiensis HSFI-12 bacteria as an antithrombotic agent, characterization of the bacterial enzyme is necessary. This study aims to determine the specificity of protease from Bacillus thuringiensis HSFI-12 to explore its potential as an antithrombotic agent in terms of anticoagulant and fibrinolytic activities. The molecular weight and specificity of bacterial protease were determined with a zymographic method with casein as substrate. Bacillus thuringiensis HSFI-12 was first cultured on Nutrient Agar (NA) media and then on Skim Milk Agar (SMA) media. The obtained crude protease from Skim Milk Broth (SMB) was then concentrated as dialysate. Both crude and dialysate proteases were tested for their specific activity, as well as anticoagulant and fibrinolytic activities. Next, the dialysate’s molecular weight and specificity on the casein substrate were investigated using the zymographic method. As result, protease activity in crude form is lower than that in dialysate, which was 0.5570 ± 0,004 U/mL and 2.1767 ± 0,005 U/mL, respectively. The molecular weight of the obtained bacterial protease was between 117 – 133 kDa and the enzyme is capable of degrading casein as shown on the zymogram. Overall, both crude and dialysate proteases of Bacillus thuringiensis HSFI-12 show potential as an antithrombotic agent for exhibiting anticoagulant and antiplatelet activities. Yet, it could not exhibit direct fibrinolytic activity implying the possibility that the enzyme plays a role as a plasminogen activator, which can dissolve fibrin by activating plasmin.

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Section: Determination Of Molecular Weight and Speci City Ofsupporting

confidence: 82%

Section: Introductionmentioning

confidence: 99%

Characteristics and Substrate Specificity of Semi-Purified Bacterial Protease ofBacillusthuringiensisHSFI-12 with Potential as Antithrombotic Agent

Ferdiani

Zilda²,

Afriansyah

et al. 2023

Sci. Technol. Indones

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“…14 In vivo activity and the antithrombotic mechanism of action of the Bacillus thuringiensis HSFI-12 protease was yet to be known. 11,15 In vivo antithrombotic assays of protease enzymes in experimental animals can be done on the thrombosis model, for example thrombosis model carried out by carrageenan induction in rat's tails. 16,17 This study aimed to evaluate the thrombolytic in vivo activity of protease concentrates from the crude enzyme B thuringiensis HSFI-12.…”

Section: Enzyme Productionmentioning

confidence: 99%

“…Other haematology assays that also evaluated were leukocytes as white blood cells (WBC) count, erythrocytes as red blood cells (RBC) count and platelets (PLT) count. 15 Figure 1: Schematic of the injection sites in the rat tail for in vivo thrombosis evaluation after bacterial protease treatment 22 isolates were grown in Luria Bertani Agar (LBA, Oxoid, UK) media containing 2 % skim milk. The clear zone formed around the colony was the parameter of the isolate's ability to produce proteases.…”

Section: Enzyme Productionmentioning

confidence: 99%

In vivo antithrombotic potential of protease from Bacillus thuringiensis HSFI-12

Dewi,

Zilda,

Rakhmawatie

et al. 2023

Scripta Medica

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Background/Aim: Cardiovascular diseases (CVDs) are the primary noncommunicable disease at the global level due to abnormal platelet aggregation by fibrin forming clots in blood vessels called thrombus. The search for thrombolytic drugs is largely carried out to treat thrombosis. Crude extract and dialysate protease of Bacillus thuringiensis HSFI-12 is known to have thrombolytic activity in vitro. The in vivo thrombolytic activity evaluation of concentrated protease of the bacterium is yet to be done. This study aimed to evaluate in vivo thrombolytic activity of concentrated protease produced by ultrafiltration of crude B thuringiensis HSFI-12 protease using Rattus norvegicus as animal model. Methods: Carrageenan was used as thrombosis induction agent in rats. Intravenous injection of B thuringiensis HSFI-12 concentrated protease doses of 75, 150, 300, 600 µg/kg body weight (BW) was administered to rats, then induction of carrageenan was given intravenously to the rats' tails 30 min after injection of B thuringiensis HSFI-12 protease concentrate. The average length of the infarct area in the tail of the rat was shorter in the rats that were given various doses of B thuringiensis HSFI-12 protease concentrate compared to the negative control (rats induced by carrageenan 20 mg/kg BW). Results: The PT examination results showed a prolonged PT time at 300 µg/kg BW dose, while there was at risk of bleeding at 600 µg/kg BW dose. The activated partial thromboplastin time (aPTT) examination results showed that time elongation beyond the normal range did not occur in rats after treatment. The amount leukocytes (WBC) and erythrocytes (RBC) after treatment were within the normal range indicating that they did not affect the haemostasis mechanism, while the platelet count (PLT) assay showed decrease in the number of platelets (thrombocytopenia). However, after treatment the number of platelets (PLT) showed a positive response as seen from an increase in values close to normal range. As conclusion, induction of carrageenan conducted had successfully caused thrombosis in R norvegicus' tail used as the thrombosis model. Conclusion: Concentrated protease of B thuringiensis HSFI-12 showed in vivo antithrombotic potential with an effective dose of based on PT, aPTT and blood count evaluation at 150 µg/kg BW.

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Prospective In Vivo Assays on the Antithrombotic Potential of Protease Extracted from Bacillus sp. HSFI-12

Cited by 2 publications

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Characteristics and Substrate Specificity of Semi-Purified Bacterial Protease ofBacillusthuringiensisHSFI-12 with Potential as Antithrombotic Agent

Characteristics and Substrate Specificity of Semi-Purified Bacterial Protease ofBacillusthuringiensisHSFI-12 with Potential as Antithrombotic Agent

In vivo antithrombotic potential of protease from Bacillus thuringiensis HSFI-12

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