Main Article Content

Stalis Norma Ethica Tri Joko Raharjo Dewi Seswita Zilda Nur Hidayati


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.


Download data is not yet available.

Article Details

How to Cite
Ethica, S. N., Raharjo, T. J., Zilda, D. S. and Hidayati, N. . (2023) “In Vitro Anticoagulant Activity of Crude Protease of Bacillus tequilensis HSFI-5”, Indonesian Journal of Medical Laboratory Science and Technology, 5(2), pp. 90–99. doi: 10.33086/ijmlst.v5i2.3791.
Crude Protease, Bacillus tequilensis HSFI-5, Anticoagulant


Putri A. Diathesis Hemorrhagic, Coagulation and Fibrinolytic System. Biomol Heal Sci J. 2022;5(1):54–61. DOI: 10.20473/bhsj.v5i1.35280. DOI:

Palta S, Saroa R, Palta A. Overview of the coagulation system. Indian J Anaesth. 2014;58(5):515–23. DOI: 10.4103/0019-5049.144643. DOI:

WHO. Cardiovascular diseases. 2021. Available from

Virani SS, Alonso A, Aparicio HJ, Benjamin EJ, Bittencourt MS, Callaway CW, et al. Heart disease and stroke statistics-2021 update: a report from the American Heart Association. Circulation. 2021 Feb;143(8):e254–743. Available DOI: 10.1161/CIR.0000000000000950. DOI:

Renú A, Millán M, San Román L, Blasco J, Martí-Fàbregas J, Terceño M, et al. Effect of intra-arterial alteplase vs placebo following successful thrombectomy on functional outcomes in patients with large vessel occlusion acute ischemic stroke: The CHOICE Randomized Clinical Trial. JAMA. 2022 Mar;327(9):826–35. DOI: 10.1001/jama.2022.1645. DOI:

Shi Y, Guo L, Chen Y, Xie Q, Yan Z, Liu Y, et al. Risk factors for ischemic stroke: differences between cerebral small vessel and large artery atherosclerosis aetiologies. Folia Neuropathol. 2021;59(4):378–85. DOI: 10.5114/fn.2021.112007. DOI:

Krishnamurthy A, Belur PD, Rai P, Rekha PD. Production of fibrinolytic enzyme by the marine isolate Serratia marcescens subsp. sakuensis and its in-vitro anticoagulant and thrombolytic potential. J Pure Appl Microbiol. 2017;11(4):1987–98. DOI: 10.22207/JPAM.11.4.41. DOI:

Lu F, Lu Z, Bie X, Yao Z, Wang Y, Lu Y, et al. Purification and characterization of a novel anticoagulant and fibrinolytic enzyme produced by endophytic bacterium Paenibacillus polymyxa EJS-3. Thromb Res. 2010 Nov;126(5):e349-55. Available DOI: 10.1016/j.thromres.2010.08.003. DOI:

Huang S, Pan S, Chen G, Huang S, Zhang Z, Li Y, et al. Biochemical characteristics of a fibrinolytic enzyme purified from a marine bacterium, Bacillus subtilis HQS-3. Int J Biol Macromol. 2013 Nov;62:124–30. DOI: 10.1016/j.ijbiomac.2013.08.048. DOI:

Mahajan PM, Nayak S, Lele SS. Fibrinolytic enzyme from newly isolated marine bacterium Bacillus subtilis ICTF-1: media optimization, purification and characterization. J Biosci Bioeng. 2012 Mar;113(3):307–14. DOI: 10.1016/j.jbiosc.2011.10.023. DOI:

Hidayati N, Nurrahman N, Fuad H, Munandar H, Zilda DS, Ernanto AR, et al. Bacillus tequilensis isolated from fermented intestine of Holothuria scabra produces fibrinolytic protease with thrombolysis activity. IOP Conf Ser Earth Environ Sci. 2021;707(1). DOI: 10.1088/1755-1315/707/1/012008. DOI:

Hidayati N, Fuad H, Munandar H, Zilda DS, Nurrahman N, Fattah M, et al. Proteolytic and clot lysis activity screening of crude proteases extracted from tissues and bacterial isolates of Holothuria scabra. IOP Conf Ser Earth Environ Sci. 2021;755(1). DOI: 10.1088/1755-1315/755/1/012016. DOI:

Choi JH, Sapkota K, Park SE, Kim S, Kim SJ. Thrombolytic, anticoagulant and antiplatelet activities of codiase, a bi-functional fibrinolytic enzyme from Codium fragile. Biochimie [Internet]. 2013;95(6):1266–77. DOI: 10.1016/j.biochi.2013.01.02. DOI:

Wang KH, Li SF, Zhao Y, Li HX, Zhang LW. In vitro anticoagulant activity and active components of safflower injection. Molecules. 2018;23(1):2–9. DOI: 10.3390/molecules23010170. DOI:

Rusyiana R, Lestarini IA, Hamdin CD, Muliasari H. Anticoagulant activity of mangrove (avicennia alba) leaves extract in vitro. Ilmu Kelaut Indones J Mar Sci. 2021;26(2):110–6. DOI: 10.14710/IK.IJMS.26.2.110-116. DOI:

Marmitt DJ, Bitencourt S, Coura CO, Berger M, Faleiro D, Kich DM, et al. In vitro and in vivo anti-inflammatory and anticoagulant activities of Myrciaria plinioides D. Legrand ethanol leaf extract. Inflammopharmacology [Internet]. 2022;30(2):565–77. DOI: 10.1007/s10787-022-00924-0. DOI:

Fuad H, Hidayati N, Darmawati S, Munandar H, Sulistyaningtyas AR, Ernanto AR, et al. Exploration of bacteria isolated from “rusip” fermented tissue of sand sea cucumber Holothuria scabra with fibrinolytic, anticoagulant and antiplatelet activities. AACL Bioflux. 2021;14(3):1242–58.

Wang J. Preliminary blood compatibility comparison of silk fibroin dissolved by different solvents. Biomed J Sci Tech Res. 2018;12(2):9160–3. DOI: 10.26717/bjstr.2018.12.002233. DOI:

Usama Mazhar M, Muneer K, Anwar F, Ahmed Siddiqui W, Qureshi A, Bilal R, et al. In-vitro anticoagulant activity of three extracts of Astragalussarcocolla. Pakistan J Med Heal Sci. 2020;14(2):393–6.

Bianchini EP, Auditeau C, Razanakolona M, Vasse M, Borgel D. Serpins in hemostasis as therapeutic targets for bleeding or thrombotic disorders. Front Cardiovasc Med. 2021;7(January):1–7. DOI: 10.3389/fcvm.2020.622778. DOI:

Hassan SWM. Antibacterial, anticoagulant and anti-inflammatory activities of marine Bacillus cereus s1. J Pure Appl Microbiol. 2016;10(4):2593–606. DOI: 10.22207/JPAM.10.4.15. DOI:

Lee W, Yoo H, Ku SK, Kim JA, Bae JS. Anticoagulant activities of piperlonguminine in vitro and in vivo. BMB Rep. 2013;46(10):484–9. Available from: DOI: 10.5483/BMBRep.2013.46.10.028. DOI:

Ayodele OO, Onajobi FD, Osoniyi O. In vitro anticoagulant effect of Crassocephalum crepidioides leaf methanol extract and fractions on human blood. J Exp Pharmacol. 2019;11:99–107. DOI: 10.2147/JEP.S218261. DOI:

Zhou Y, Chen H, Yu B, Chen G, Liang Z. Purification and characterization of a fibrinolytic enzyme from marine Bacillus velezensis Z01 and assessment of its therapeutic efficacy in vivo. Microorganisms. 2022;10(5):1–17. DOI: 10.3390/microorganisms10050843. DOI:

Chester K, Zahiruddin S, Ahmad A, Khan W, Paliwal S, Ahmad S. Bioautography-based identification of antioxidant metabolites of Solanum nigrum L. and exploration its hepatoprotective potential agchester, K. et al. Bioautography-based Identification of antioxidant metabolites of Solanum nigrum L. and explorati. Pharmacogn Mag. 2017;13 (Suppl(62):179–88. DOI: 10.4103/

Pontarollo G, Acquasaliente L, Peterle D, Frasson R, Artusi I, De Filippis V. Non-canonical proteolytic activation of human prothrombin by subtilisin from Bacillus subtilis may shift the procoagulant–anticoagulant equilibrium toward thrombosis. J Biol Chem. 2017;292(37):15161–79. DOI: 10.1074/jbc.M117.795245. DOI:

Gogoi D, Arora N, Kalita B, Sarma R, Islam T, Ghosh SS, et al. Anticoagulant mechanism, pharmacological activity, and assessment of preclinical safety of a novel fibrin(ogen)olytic serine protease from leaves of Leucas indica. Sci Rep [Internet]. 2018;8(1):1–17. DOI: 10.1038/s41598-018-24422-y. DOI:

Lakhani CM, Benjamin M. Davis, Glen F. Rall MJS. Antithrombin: an anticoagulant, anti-inflamatory and anti bacterial serpin. Physiol Behav. 2017;176(3):139–48. DOI: 10.1111/jth.14724.Antithrombin.

Águila S, Izaguirre G, Martínez-Martínez I, Vicente V, Olson ST, Corral J. Disease-causing mutations in the serpin antithrombin reveal a key domain critical for inhibiting protease activities. J Biol Chem. 2017;292(40):16513–20. DOI: 10.1074/jbc.M117.787325. DOI:

Yaron JR, Zhang L, Guo Q, Haydel SE, Lucas AR. Fibrinolytic serine proteases, therapeutic serpins and inflammation: fire dancers and firestorms. Front Cardiovasc Med. 2021;8(March):1–21. DOI: 10.3389/fcvm.2021.648947. DOI:

Alkan C, Coe P, Eichler E. Serpins in thrombosis, hemostasis, and fibrinolysis. J Thromb Haemost [Internet]. 2011;23(1):1–7. Available from:

Stalis Norma Ethica, Master Program of Clinical Laboratory Science, Universitas Muhammadiyah Semarang, Semarang, Indonesia

Tri Joko Raharjo, Department of Chemistry, Universitas Gadjah Mada, Yogyakarta, Indonesia

Dewi Seswita Zilda, Research Center for Deep Sea, Earth Sciences and Maritime Research Organization, National Research and Innovation Agency, Jakarta, Indonesia

Nur Hidayati, Klinik Pratama Subekti Medical, Pemalang Indonesia