In Vitro Anticoagulant Activity of Crude Protease of Bacillus tequilensis HSFI-5

Bacillus tequilensis HSFI-5 is a food-grade bacterial isolate obtained from the fermented intestine of Holothuria scabra (sand sea cucumber). Strain HSFI-5 had been reported to be able to produce proteases, which had shown several characteristics of an antithrombotic agent, i.e., 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 vitro . The study design was a completely randomized design with a sample size of 90 calculated using the Federer formula. The material used was crude protease from B. tequilensis in skim milk broth. Prothrombin time (PT), activated partial thromboplastin time (aPTT), 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 µL and pre-incubation at 37ºC for 5, 10, and 15 min with crude enzyme volumes of 50 and 100 µL. 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 (p<0.05). The longest results of PT, aPTT, and PRT are positive controls, and the shortest results are normal controls for PT, and 15’ 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.


INTRODUCTION
Coagulation is a process involving several components, such as blood vessels, platelet cells, and blood coagulation factors.
Normal coagulation pathways are a collaboration between clot formation and clot decay processes.An imbalance of the coagulation system can occur in an illness that causes bleeding or thrombosis (1,2).One of the diseases causing blood coagulation disorders is coronary vascular disease (CVDs).Data from the World Health Organization (WHO) states that 17.9 million people (32%) in the world die every year due to CVDs.As many as 85% of people with CVD die from heart attacks and strokes.The risk of venous thromboembolism (VTE) increases in the first 3 months after the occurrence of an ischemic stroke (3,4).
Ischemic stroke can occur due to a sudden reduction in blood flow to the brain, leading to reduced neurologic function.Blood flow is reduced as a result of thromboembolic blockage.After 4 to 14 days from the time of commencement, antithrombotic therapy may be given (5,6).The antithrombotic potential of bacterial proteases can be explored in order to minimize costs (7).

MATERIALS AND METHODS
A , clot lysis and anticoagulant.The anticoagulant activity of bacterial proteases has been reported in several studies(7,8).Proteases with fibrinolytic and thrombolytic activities had also been reported, mostly from Bacillus subtilis(9,10).Bacillus tequilensis HSFI-5 is a bacterial isolate obtained from the fermented intestine of the sand sea cucumber, Holothuria scabra.The sea cucumber was isolated from sand sea cucumber from Kodek Gulf Village, Lombok, West Nusa Tenggara.Strain HSFI-5 was found to have the ability to produce fibrinolytic protease, which is an important characteristic of an antithrombotic agent(11,12).Another important characteristic of the protease of B. tequilensis that underlines its potential as an antithrombotic agent as clot lysis activity.This study represents novelty as the anticoagulant activity of the protease from B. tequilensis HSFI-5 has not yet been tested.Such studies are important as a basis for further exploring bacterial protease activity through in vivo antithrombotic and toxicity assays.The commonly used antithrombotic agents work through the tissue plasminogen activator (tPA) mechanism.Trypsin-like serine protease has been reported to be an anticoagulant agent because it contains sulfated polysaccharides that play a role in anticoagulant, antiangiogenic, and immunostimulating processes (7,13).The presence of thrombolytic activity is one of the potential anticoagulant (11).PT, aPTT, and PRT were used to test anticoagulant activity of crude protease.The PT, aPTT, and PRT 92 assays are the most commonly used tests for the examination of blood coagulation factors (14-16).Discovery of alternative anticoagulants is needed to minimize the risks and costs of drug use.The purpose of this study was to measure the anticoagulant activity of the crude protease HSFI-5 in vitro.

Hematology 94 RESULTS
completely randomized design was used in this research.The study used three examination parameters, namely PT, aPTT, and PRT.The study was conducted at the Laboratory of this study included a varied volume of crude protease from B. tequilensis HSFI-5 (of known concentration) and length of incubation time.The number of samples used in the study was calculated using the Federer's formula.The total number of treatment in this study was 10 group, namely (1) positive control; (2) normal control; (3) direct incubation with addition of 50 µL crude protease (D 50); (4) direct incubation with addition of 100 µL crude protease (D 100); (5) 5 mins pre incubation with addition of 50 µL crude protease (5' 50); (6) 5 mins pre incubation with addition of 100 µL crude protease (5' 100); (7) 10 mins pre incubation with addition of 50 µL crude protease (10' 50); (8) 10 mins pre incubation with addition of 100 µL crude protease (10' 100); (9) 15 mins pre incubation with addition of 50 µL crude protease (15' 50); (10) 15 mins pre incubation with addition of 100 µL crude protease (15' 100).Each experiment had three replicate plants per treatment.The total study sample for each study parameter was 30.The total number of study samples was 90 samples.The blood samples used for the study were taken from a healthy volunteer who was willing to be a respondent.The research was conducted with permission from the ethics committee of the Faculty of Public Health, University of Muhammadiyah Semarang with number 377/KEPK-FKM/UNIMUS/2020. Isolation of Crude Protease Enzyme B. tequilensis HSFI-5 B. tequilensis HSFI-5 isolates were subcultured on skim milk broth (SMB) medium and incubated at 37ºC for 72 hours.Bacterial cultures were centrifuged at 3000 rpm for 15 min at 4º C. The supernatant (crude protease) was harvested from the centrifugation results and could be used for in vitro anticoagulation assay (17).In Vitro Assays PT, aPTT, and PRT were performed to examine the anticoagulant activity of crude protease B. tequilensis HSFI-5.Anticoagulant heparin was used as a positive control.B. tequilensis HSFI-5 Crude Protease Treatment Normal and positive controls were treated as standard procedures, while direct assay treatments with crude protease B. tequilensis HSFI-5 50 µL and 100 µL was carried out by means of 50 µL PPP with 50 µL/100 µL crude protease, which was analyzed for clotting that occurred as standard procedure.Test samples were prepared at 50 μL and 100 μL raw protease volumes with pre-incubation times of 5, 10, and 15 minutes.Prothrombin Time (PT) The PT examination used a sample of citrated platelet poor plasma (PPP).As much as 50 µL of PPP was added with 50 µL of PT reagent (TEclot PT-S, Germany) and incubated at 37ºC for 2 min before reading photometrically on a coagulometer (Coatron M1, Germany).PT results were expressed in seconds.The resulting clotting was analyzed using a Coatron M1 coagulometer according to its manufacturing method (TEclot PT-S, Coagulation analysis with aPTT was performed photometrically with the TEclot aPTT-S reagent and read with a Coatron M1 device.As much as 50 µL of PPP was reacted with 50 µL of aPTT reagent and then incubated for 2 min at 37º C. The preparation of the test sample was carried out like the sample treatment for the PT test, and then the clotting time was expressed in seconds (s) following the TEclot (Germany) manufacturing procedure.Plasma recalcification time (PRT) PRT examination was carried out manually with the addition of NaCl and CaCl2 0.025 M at 37ºC.The preparation of examination samples was carried out as in the treatment for PT and aPTT examinations.As much as 100 µL PPP was added to 100 µL physiological NaCl and incubated for 1 min.The mixture was then added to 100 µL CaCl2 0.025 M and incubated for 90 s.The clot that appeared every 30 seconds was observed.PRT results expressed in seconds were recorded (18).Statistical Analysis Data were analyzed using SPSS 25 software (IBM, USA).Kolmogorov-Smirnov test for analyzing normality of data.Normal data were followed by the one-way Anova difference test.A post hoc LSD test was performed to determine the differences between the treatment groups in the study.A significant value of less than 0.05 was expressed as the result with a significant difference.The bacterial isolate of B. tequilensis HSFI-5 used in this study was from a previous study originally deposited in a microbank of the microbiology laboratory of Universitas Muhammadiyah Semarang (11).After subculturing in 250-mL of SMB medium, culturing and centrifugation, 35 mL of crude protease was obtained as a supernatant.This sample was then subjected to PT, aPTT, PRT and hematology assays.Initial analysis of the protein content of the crude protease yielded a nano drop value of 7,7 mg/mL.Hematology results showed that the highest mean PT examination was in the positive control group, and the lowest mean was in the normal control group.The highest and lowest aPTT test result were determined in the positive control and 15' 50 groups, respectively.The highest and lowest mean PRT values in the positive control and 15' 50 groups, respectively.Baseline values for the PT assay were 10-14 seconds, aPTT 22-30 seconds, and PRT 90-250 seconds.The Kolmogorov-Smirnov normality test was normal for all groups, so the one-way ANOVA test was used and performed.The p-value was 0.000 (p<0.05).An LSD post hoc test was used to determine differences in each group.The results of the post hoc LSD test are shown in Figure 1.