In Vitro Anticoagulant Activity of Crude Protease of Bacillus tequilensis HSFI-5 Stalis Norma Ethica1.
Tri Joko Raharjo2.
Dewi Seswita Zilda3.
Nur Hidayati4 Master Program of Clinical Laboratory Science.
Universitas Muhammadiyah Semarang.
Semarang.
Indonesia Department of Chemistry.
Universitas Gadjah Mada.
Yogyakarta.
Indonesia Research Center for Deep Sea.
Earth Sciences and Maritime Research Organization.
National Research and Innovation Agency.
Jakarta.
Indonesia Klinik Pratama Subekti Medical.
Pemalang Indonesia Correspondence:
Stalis Norma Ethica.
Perumahan BTN Rejomulyo II/44.
Kediri.
Indonesia Zip Code: 64129 Email: norma@unimus.
Received: January 1, 2023 Revised: April 10, 2023 Accepted: May 15, 2023 Published: May 22, 2023 DOI: 10.
33086/ijmlst.
Abstract Bacillus tequilensis HSFI-5 is a food-grade bacterial isolate obtained from the fermented intestine of Holothuria scabra .
and sea cucumbe.
Strain HSFI-5 had been reported to be able to produce proteases, which had shown several characteristics of an antithrombotic agent, i.
, fibrinolytic and clot-lysis activities.
However, its anticoagulation activity test is yest to be done.
This study aimed to determine the anticoagulant activity of the crude protease HSFI-5 in The study design was a completely randomized design with a sample size of 90 calculated using the Federer The material used was crude protease from B.
tequilensis in skim milk broth.
Prothrombin time (PT), activated partial thromboplastin time .
PTT), and plasma recalcification time (PRT) were carried out to test the anticoagulant activity.
Citrated platelet poor plasma samples were divided into positive control, normal control, direct examination with crude enzyme in volumes of 50 and 100 AAL and pre-incubation at 37AC for 5, 10, and 15 min with crude enzyme volumes of 50 and 100 AAL.
The data normality was tested with the Kolmogorov-Smirnov test and the different tests were analyzed by one-way ANOVA with the Post hoc LSD test.
The results of one-way ANOVA both on PT, aPTT, and PRT examinations showed that there was a significant difference between the treatment groups .
<0.
The longest results of PT, aPTT, and PRT are positive controls, and the shortest results are normal controls for PT, and 15Ao 50 group for aPTT and PRT.
It is clear that crude protease B.
tequilensis HSFI-5 exhibits anticoagulant as well as thrombolytic action, raising the possibility that it could function as an antithrombotic drug.
Keywords Crude Protease.
Bacillus tequilensis HSFI-5.
Anticoagulant.
Citation: Ethica SN.
Raharjo TJ.
Zilda DS.
Hidayati N.
In Vitro Anticoagulant Activity of Crude Protease of Bacillus tequilensis HSFI-5.
Indones J Med Lab Sci Technol.
:90Ae9.
DOI: 10.
33086/ijmlst.
This is an open access article distributed under the Creative Commons Attribution-ShareAlike 4.
0 International License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
A2023 by author.
Stalis Norma Ethica, et al.
fibrinolytic and thrombolytic activities had INTRODUCTION Coagulation is a process involving also been reported, mostly from Bacillus several components, such as blood vessels, subtilis .
Bacillus tequilensis HSFI-5 is platelet cells, and blood coagulation factors.
a bacterial isolate obtained from the Normal fermented intestine of the sand sea cucumber, collaboration between clot formation and clot Holothuria scabra.
The sea cucumber was decay processes.
An imbalance of the isolated from sand sea cucumber from Kodek coagulation system can occur in an illness Gulf Village.
Lombok.
West Nusa Tenggara.
that causes bleeding or thrombosis .
One Strain HSFI-5 was found to have the ability of the diseases causing blood coagulation to produce fibrinolytic protease, which is an disorders is coronary vascular disease important characteristic of an antithrombotic (CVD.
Data from the World Health Organization (WHO) states that 17.
9 million characteristic of the protease of B.
%) in the world die every year due to CVDs.
As many as 85% of people with antithrombotic agent as clot lysis activity.
CVD die from heart attacks and strokes.
The This study represents novelty as the risk of venous thromboembolism (VTE) anticoagulant activity of the protease from B.
increases in the first 3 months after the tequilensis HSFI-5 has not yet been tested.
occurrence of an ischemic stroke .
Such studies are important as a basis for Ischemic stroke can occur due to a sudden further exploring bacterial protease activity reduction in blood flow to the brain, leading through in vivo antithrombotic and toxicity to reduced neurologic function.
Blood flow is .
Another reduced as a result of thromboembolic The commonly used antithrombotic After 4 to 14 days from the time of agents work through the tissue plasminogen commencement, antithrombotic therapy may activator .
PA) mechanism.
Trypsin-like be given .
The antithrombotic potential serine protease has been reported to be an of bacterial proteases can be explored in anticoagulant agent because it contains order to minimize costs .
sulfated polysaccharides that play a role in Antithrombotic activities of bacterial immunostimulating processes .
The characteristics: fibrinolysis, clot lysis and presence of thrombolytic activity is one of the The anticoagulant activity of potential anticoagulant .
PT, aPTT, and bacterial proteases has been reported in PRT were used to test anticoagulant activity of crude protease.
The PT, aPTT, and PRT .
Proteases Ina.
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assays are the most commonly used tests for crude protease .
Ao .
10 mins the examination of blood coagulation factors pre incubation with addition of 100 AAL .
Ae.
crude protease .
Ao .
15 mins anticoagulants is needed to minimize the pre incubation with addition of 50 AAL risks and costs of drug use.
The purpose of crude protease .
Ao .
15 mins this study was to measure the anticoagulant pre incubation with addition of 100 AAL activity of the crude protease HSFI-5 in vitro.
crude protease .
Ao .
Each experiment Discovery had three replicate plants per treatment.
The was used in this research.
The study The blood samples used for namely PT, aPTT, and PRT.
The study the study were taken from a healthy volunteer who was MATERIALS AND METHODS Laboratory The willing to be a Hematology of Universitas Muhammadiyah The research was conducted Semarang The with permission from the ethics committee controlled variables of this study included of the Faculty of Public Health.
University of Muhammadiyah Semarang with number October from B.
tequilensis HSFI-5 .
f known 377/KEPK-FKM/UNIMUS/2020.
and length of incubation Isolation of Crude Protease Enzyme tequilensis HSFI-5 tequilensis HSFI-5 isolates were the FedererAos formula.
The total number subcultured on skim milk broth (SMB) medium and incubated at 37AC for 72 group, namely .
positive control.
Bacterial cultures were centrifuged at 3000 rpm for 15 min at 4A C.
The .
with addition of 50 AAL crude protease The (D .
direct incubation with addition harvested from the centrifugation results of 100 AAL crude protease (D .
5 mins pre incubation with addition of 50 AAL anticoagulation assay .
In Vitro Assays .
Ao .
rude pre incubation with addition of 100 AAL PT, aPTT, and PRT were performed to crude protease .
Ao .
10 mins examine the anticoagulant activity of crude pre incubation with addition of 50 AAL HSFI-5.
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Anticoagulant heparin was used as a Coatron M1 device.
As much as 50 AAL positive control.
of p was reacted with 50 AAL of tequilensis HSFI-5 Crude Protease aPTT reagent and then incubated for 2 min Treatment at 37A C.
The preparation of the test Normal and positive controls were sample was carried out like the sample treatment for the PT test, and then the direct assay treatments with crude protease B.
clotting time was expressed in seconds tequilensis HSFI-5 50 AAL and 100 AAL .
was carried out by means of 50 AAL p with manufacturing procedure.
50 AAL/100 AAL crude protease, which Plasma recalcification time (PRT) was analyzed for clotting that occurred Test TEclot (German.
PRT examination was carried out NaCl 37AC.
The were prepared at 50 L and 100 L raw protease volumes with pre-incubation times of 5, 10, and 15 minutes.
was carried out as in the treatment for CaCl2 PT and aPTT examinations.
As much as Prothrombin Time (PT) 100 AAL p was added to 100 AAL The PT examination used a sample NaCl and incubated of citrated platelet poor plasma .
1 min.
The mixture was then added to 100 AAL As much as 50 AAL of p was added with CaCl2 0.
025 M and incubated for 90 s.
The 50 AAL of PT reagent (TEclot PT-S.
German.
clot that appeared every 30 seconds was and incubated at 37AC for 2 min before PRT results expressed in seconds reading photometrically on a coagulometer were recorded .
(Coatron M1.
German.
PT results were Statistical Analysis Data were analyzed using SPSS 25 clotting was analyzed using a Coatron software (IBM.
USA).
Kolmogorov-Smirnov test for analyzing normality of data.
Normal
PT-S,
data were followed by the one-way Anova The (TEclot German.
difference test.
A post hoc LSD test was Activated Partial Thromboplastin Time performed to determine the differences .
PTT) between the treatment groups in the study.
Coagulation analysis with aPTT was Ina.
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05 was Stalis Norma Ethica, et al.
lowest mean was in the normal control
RESULTS
The bacterial isolate of B.
The highest and lowest aPTT test HSFI-5 used in this study was from result were determined in the positive a previous study originally deposited in control and 15Ao 50 groups, respectively.
a microbank of the microbiology laboratory The highest and lowest mean PRT values of Universitas Muhammadiyah Semarang .
After subculturing in 250-mL of SMB groups, respectively.
Baseline values for the PT assay were 10-14 seconds, aPTT 35 mL of crude protease was obtained 22-30 seconds, and PRT 90-250 seconds.
The Kolmogorov-Smirnov normality test This one-way ANOVA test was used and yielded a nano drop value of 7,7 mg/mL.
<0.
An LSD post hoc test was Hematology used to determine differences in each group.
highest mean PT examination was in The results of the post hoc LSD test are shown in Figure 1.
p-value of the protein content of the crude protease The 15Ao Initial then subjected to PT, aPTT.
PRT and Ina.
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Figure 1.
Post hoc LSD test results of (A) PT, (B) aPTT, (C) PRT.
*significant.
clot-lytic The crude protease B.
tequilensis HSFI-5 Crude was recovered from the bacterial culture supernatant of skim milk broth medium and previous studies based on fibrin potential development as antithrombotic plates and gravimetric analysis reported agents .
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DISCUSSION
Stalis Norma Ethica, et al.
proteases by performing PT, aPTT and PRT.
The existence of side effects of presence of elongations in PT indicates available anticoagulants has led researchers that the crude protease B.
to investigate the anticoagulant activity HSFI-5 processes in the extrinsic and common HSFI-5 .
Anticoagulant activity of The pathways .
crude protease B.
Techylensis was analyzed aPTT testing is commonly used to detect by direct examination of PT, aPTT, and coagulation factor deficiencies in intrinsic PRT .
o dela.
, and by varying incubation signaling pathways.
The results showed times and crude protease volumes.
that the addition of 100 AAL of crude protease The results of the descriptive analysis significantly prolonged the aPTT results of the PT studies showed a trend for for the same pre-incubation time.
However, the studied proteases to prolong the PT the addition of 50 AAL compared to 100 AAL time, consistent with the increased volume of crude protease reduced the sample.
This of HSFI-5 crude protease.
The highest was probably due to the low protease yield was obtained in the group with concentration, which required a larger 100 AAL of crude protease B.
HSFI-5 added and p incubated for Prolongation of the aPTT results 15 min.
Research data show that the compared to the normal controls resulted in longer the pre-incubation period and the up to 2.
75-fold time prolongation.
This more crude protease added, the longer the PT time.
The most significant result HSFI-5 is of the PT examination was a 57.
the coagulation process.
the potency of clotting factors in the able to All groups had extended PRT test The difference in PT assay times, but a pattern was observed in which the addition of 100 AAL of crude addition of 100 AAL of crude protease, protease significantly increased the number suggesting that the addition of 100 AAL of samples.
gave optimal PT result at 37A C and 15 observed in the 15 min pre-incubation group pre-incubation time.
These results are in line with studies suggesting that demonstrate that the optimal anticoagulant proteases can cause prolongation of PT .
activity of crude protease is preincubated The PT test is a coagulation test to assess for 15 minutes in a volume of 100 AAL.
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Extending the PRT results, we showed smaller volumes, and minimal pre-incubation times requires the use of concentrated As a result, it can be used HSFI-5 Longer incubation times and thrombosis .
,29,.
activity because the low concentration of crude protease requires longer volumes CONCLUSIONS and times to work optimally.
A lower The concentration of crude protease increases HSFI-5 based on PT, aPTT, and PRT vacuum the anticoagulant mechanism through the and may be an alternative anticoagulant serpin mechanism.
in the future.
The longest results for PT.
The aPTT, and PRT represent the positive the crude protease HSFI-5 is believed to be controls, and the shortest results were obtained from the normal control for PT.
Coagulation and fibrinolysis are primarly protease for aPTT and PRT.
However, 15-min AAL serpine superfamily.
Antithrombin (AT) is a physiological anticoagulant that targets are required to confirm the results of these procoagulant enzymes, particularly factor in vitro anticoagulation studies.
Xa and thrombin .
Ae.
Serpine participate in the anticoagulant mechanism by carrying AUTHOR CONTRIBUTIONS out the proteolytic activity of clotting pathways .
Ae.
Antithrombin inhibits the activity of factor Xa and thrombin .
Ae.
Serpines have been identified in bacteria, archaea, eukaryotes, and viruses.
Several papain, and caspase families.
The PCI/AT inhibited .
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Dewi Seswita Zilda:
Performed enzyme preparation.
Tri Joko Raharjo Stalis Norma Ethica Nur Hidayati:
assisted in interpreting the results and worked on the manuscript.
ACKNOWLEDGEMENTS
This study was funded by National Competitive Applied Research Grant serpine-protease Stalis Norma Ethica: Were involved Stalis Norma Ethica, et al.
(Penelitian terapan Kompetitif Nasional.
CONFLICT OF INTEREST PTKN) 2022 from Indonesian Ministry of Education.
Culture.
Research Technology Higher Education Authors declare that no conflict of interest in this study.
(Kemendikbud Riste.
REFERENCES