Fibrinolytic Enzyme Research Articles

NON-PLASMIN-THROMBOLYTICS: NECESSITY, OPPORTUNITIES AND PROSPECTS (review of literature data and own research)

State of the problem. Thrombotic complications are a common risk factor for a variety of diseases and are one of the leading causes of death. This leads to a strong interest in finding effective means of prevention and treatment. A characteristic feature of the last decade is the growing interest and numerous attempts to introduce into clinical practice fibrinolytic enzymes that are not functionally related to the hemostasis system. The aim of the work. Investigation of molecular mechanisms that cause the lack of efficiency of native fibrinolysis in relation to fibrin clots with impaired regularity of fibrin structure. Correlation of own achievements in the field of biochemistry and medicine with systematized literary material. The possibilities created by non-plasmin fibrinolytics and the advantages of their use are considered. Discussion and conclusions. The reasons of the complications caused by insufficient efficiency of both own fibrinolytic system, and the entered fibrinolytics are substantiated. It is shown that the leading role in such complications is played by violation of the regularity of the structure of fibrin clots. The mechanisms of action of leading fibrinolytic agents are considered and the expediency of using alternative non-plasmin fibrinolytics is substantiated. The properties and expediency of the use of fibrinolytics based on components of snake venoms and bacterial proteinases as effective means for the breakdown of fibrin with impaired regularity of structure are discussed. Key words: fibrosis, thrombotic therapy, fibrinolytic enzymes, hemostasis system.

Purification and characterization of recombinant nattokinase from Bacillus subtilis R0H1

Nattokinase (NK) is a fibrinolytic enzyme with the potential for fighting cardiovascular diseases (CVD) thanks to its antithrombotic, antihypertensive, anticoagulant, anti-atherosclerotic, and neuroprotective effects. Nattokinase was first discovered and purified from soybean fermented food by Bacillus subtilis natto. To enhance NK’s activity and simplify downstream processes, production of recombinant NK using several microbial expression systems such as Escherichia coli, B. subtilis, and Lactococcus lactic has been studied. Among all of them, B. subtilis is a prominent host for overproduction of functional proteins which can be secreted directly into the culture medium. In this study, recombinant NK from B. subtilis R0H1 was purified using two-step membrane filtration. Results showed 3.2-fold increase in activity and a recovery rate of more than 80%. Molecular weight of NK was approximately 28 kDa and its fibrinolytic degradation capacity was proved according to SDS-PAGE. The optimal pH and temperature of this NK were 8.5 and 55°C, respectively. The enzyme activity was boosted by Mg2+, Ca2+ and obviously inhibited by Co2+, Zn+2, Fe2+, and SDS. The apparent Km and Vmax with fibrin as the substrate were 3.08 mM and 6.7 nmol/min, respectively. The results suggested that membrane filtration is a useful method for purification of recombinant NK from B. subtilis R0H1. Therefore, application of membrane system is proposed to purify NK at the pilot scale. In addition, our findings indicated that recombinant NK produced in B. subtilis R0H1 showed high and stable proteolytic activity in slightly alkaline pH and at high temperature. It also exhibited strong fibrinolytic activity again both substrates: fibrinogen and fibrin.

Effect of pH, temperature, and metalactivator on the activity of fibrinolytic enzymes produced by Pseudomonas aeruginosa Ts 6.4

Enzim fibrinolitik ditemukan pada beberapa mikroorganisme, termasuk Pseudomonas aeruginosa TS 6.4 yang diisolasi dari teripang (Paracaudina australis). Teripang memiliki banyak aktivitas farmakologis seperti antitrombotik dan antihyperlipidemi. Selain itu, Sticophus japonicus salah satu spesies teripang terbukti mengandung enzim fibrinolitik. Aktivitas enzim fibrinolitik dipengaruhi oleh pH, suhu, aktivator, inhibitor, dan konsentrasi enzim-substrat. Penelitian ini bertujuan untuk mengetahui pH dan suhu optimum aktivitas enzim fibrinolitik dari Pseudomonas aeruginosa TS 6.4. pH optimum dicapai pada pH 5 - 6, sedangkan suhu optimum dicapai pada 50oC. Efek dari penambahan aktivator logam CaCl2, MnCl2, MgCl2, ZnCl2, CoCl2 pada aktivitas enzim fibrinolitik juga telah diteliti. Hasil penelitian menunjukkan bahwa aktivitas fibrinolitik Pseudomonas aeruginosa TS 6. dipengaruhi oleh Ca2+, Mg2+, Mn2+, Zn2+, dan Co2+.
 

Cloning & expression of SAK enzyme from Staphylococcus aureus in E. coli BL21-CodonPlus.

Staphylokinase (SAK), also known as staphylococcal fibrinolysin, is a protein with a molecular mass of about 15 kDa produced by Staphylococcus aureus. Staphylokinase is synthesized in the late exponential phase, similar to streptokinase. The current study identified and predicted the protein SAK from Staphylococcus aureus. SAK is a fibrinolytic enzyme of the third generation that acts as an indirect activator of plasminogen. The current study cloned and expressed SAK protein isolated from Staphylococcus aureus and used in the form of a grid for enhancement of SAK Catalyst with PCR, disengagement, and change into articulation vector PET24b(+). The recombinant plasmid was changed into E. coli strain BL21 (codon additionally to 440) acceptance with isopropyl β-D-1-thiogalactopyranoside (IPTG).

Role of Plasminogen Activation System in Platelet Pathophysiology: Emerging Concepts for Translational Applications.

Traditionally, platelets have been exclusively considered for their procoagulant and antifibrinolytic effects during normal activation of hemostasis. Effectively, activated platelets secrete coagulation factors, expose phosphatidylserine, and promote thrombin and fibrin production. In addition to procoagulant activities, platelets confer resistance of thrombi to fibrinolysis by inducing clot retraction of the fibrin network and release of huge amounts of plasminogen activator inhibitor-1, which is the major physiologic inhibitor of the fibrinolytic cascade. However, the discovery of multiple relations with the fibrinolytic system, also termed Plasminogen Activation System (PAS), has introduced new perspectives on the platelet role in fibrinolysis. Indeed, the activated membrane surface of platelets provides binding sites on which fibrinolytic enzymes can be activated. This review discusses the evidence of the profibrinolytic properties of platelets through the description of PAS components and related proteins that are contained in or bind to platelets. Our analyses of literature data lead to the conclusion that in the initial phase of the hemostatic process, antifibrinolytic effects prevail over profibrinolytic activity, but at later stages, platelets might enhance fibrinolysis through the engagement of PAS components. A better understanding of spatial and temporal characteristics of platelet-mediated fibrinolysis during normal hemostasis could improve therapeutic options for bleeding and thrombotic disorders.

A Novel Bi-Functional Fibrinolytic Enzyme with Anticoagulant and Thrombolytic Activities from a Marine-Derived Fungus Aspergillus versicolor ZLH-1.

Fibrinolytic enzymes are important components in the treatment of thrombosis-associated disorders. A new bi-functional fibrinolytic enzyme, versiase, was identified from a marine-derived fungus Aspergillus versicolor ZLH-1. The enzyme was isolated from the fungal culture through precipitation with ammonium sulfate at 90% saturation. Additionally, it was further purified by DEAE-based ion-exchange chromatography, with a recovery of 20.4%. The fibrinolytic enzyme presented as one band on both SDS-PAGE and fibrin-zymogram, with a molecular mass of 37.3 kDa. It was elucidated as a member of metalloprotease in M35 family by proteomic approaches. The homology-modeling analysis revealed that versiase shares significant structural homology wuth the zinc metalloendopeptidase. The enzyme displayed maximum activity at 40 °C and pH 5.0. The activity of versiase was strongly inhibited by the metalloprotease inhibitors EDTA and BGTA. Furthermore, versiase hydrolyzed fibrin directly and indirectly via the activation of plasminogen, and it was able to hydrolyze the three chains (α, β, γ) of fibrin(ogen). Additionally, versiase demonstrated promising thrombolytic and anticoagulant activities, without many side-effects noticed. In conclusion, versiase appears to be a potent fibrinolytic enzyme deserving further investigation.

DNA Shuffling of aprE Genes to Increase Fibrinolytic Activity and Thermostability.

Four aprE genes encoding alkaline serine proteases from B. subtilis strains were used as template genes for family gene shuffling. Shuffled genes obtained by DNase I digestion followed by consecutive primerless and regular PCR reactions were ligated with pHY300PLK, an E. coli-Bacillus shuttle vector. The ligation mixture was introduced into B. subtilis WB600 and one transformant (FSM4) showed higher fibrinolytic activity. DNA sequencing confirmed that the shuffled gene (aprEFSM4) consisted of DNA mostly originated from either aprEJS2 or aprE176 in addition to some DNA from either aprE3-5 or aprESJ4. Mature AprEFSM4 (275 amino acids) was different from mature AprEJS2 in 4 amino acids and mature AprE176 in 2 amino acids. aprEFSM4 was overexpressed in E. coli BL21 (DE3) by using pET26b(+) and recombinant AprEFSM4 was purified. The optimal temperature and pH of AprEFSM4 were similar to those of parental enzymes. However, AprEFM4 showed better thermostability and fibrinogen hydrolytic activity than the parental enzymes. The results indicated that DNA shuffling could be used to improve fibrinolytic enzymes from Bacillus sp. for industrial applications.

Purification and Characterization of a Fibrinolytic Enzyme from Marine Bacillus velezensis Z01 and Assessment of Its Therapeutic Efficacy In Vivo.

Fibrinolytic enzymes are the most effective agents for the treatment of thrombotic diseases. In the present study, we purified and characterized an extracellular fibrinolytic serine metalloprotease (named Velefibrinase) that is produced by marine Bacillus velezensis Z01 and assessed its thrombolysis in vivo. SDS-PAGE and MALDI-TOF-MS analyses showed that the molecular mass of Velefibrinase was 32.3 KDa and belonged to the peptidase S8 family. The optimal fibrinolytic activity conditions of Velefibrinase were 40 °C and pH 7.0. Moreover, Velefibrinase exhibited high substrate specificity to fibrin, and a higher ratio of fibrinolytic/caseinolytic (1.48) values, which indicated that Velefibrinase had excellent fibrinolytic properties. Based on the degradation pattern of fibrin and fibrinogen, Velefibrinase could be classified as α/β-fibrinogenase. In vitro, Velefibrinase demonstrated efficient thrombolytic ability, anti-platelet aggregation, and amelioration of blood coagulation (APTT, PT, TT, and FIB), which were superior to those of commercial anticoagulant urokinase. Velefibrinase showed no hemolysis for erythrocyte in vitro and no hemorrhagic activity in vivo. Finally, Velefibrinase effectively prevented mouse tail thrombosis in a dose-dependent (0.22–0.88 mg/kg) manner. These findings suggested that Velefibrinase has the potential to becoming a new thrombolytic agent.

Exploring Agaricomycetes from the Paranaense rainforest (Misiones, Argentina) as an unconventional source of fibrinolytic enzymes

ABSTRACT Fungal fibrinolytic enzymes, secreted by some Agaricomycetes, are recognized as important thrombolytic agents due to their ability to rapidly dissolve thromboembolic clots. The present work evaluated fibrinolytic and proteolytic secretion abilities of 35 Agaricomycetes isolates from the Paranaense rainforest (Misiones, Argentina). We detected proteolytic activity in 40% of the strains while nine strains showed fibrinolytic activity. Schizophyllum commune LBM 026, Schizophyllum commune LBM 223, and Hornodermoporus martius LBM 224 exhibited the highest levels of fibrinolytic activity. Fibrin zymography from S. commune LBM 026 and LBM 223 showed an enzyme of 27.5 kDa, while H. martius LBM 224 presented an enzyme of 29 kDa. The evaluation of the enzymatic stability of culture supernatant of these strains revealed that the fibrinolytic activity was highly stable over a wide temperature and pH range. Long-term stability of fibrinolytic activity at physiological conditions evidenced that the strains had a half­life of at least 72 h. Fibrinolytic enzymes produced by S. commune LBM 026 and LBM 223 were inhibited in the presence of EDTA indicating that they are metalloproteases. This work reveals the potential of S. commune LBM 026, S. commune LBM 223, and H. martius LBM 224 as an unconventional source of thrombolytic agents.

A Review Article: Protein Engineering of Therapeutic Enzymes

Through the development of advanced, stimulus-responsive pharmacological systems, protein engineering has the potential to alter the metabolic drug landscapes. Protein therapies are a fast growing category of FDA-approved medications that have the potential to improve clinical consequences in the long run. Protein therapeutics engineering is still in its preliminary phase; however recent advancements in protein engineering skills are being used to gain direct monitoring over pharmacodynamics. Drugs that are intended to be metabolized under specific conditions are known as stimulus-responsive protein medicines. Protein engineering is being used to create biochemically based smart therapies that are personalized to the patient's needs. Protein engineering brings a new features and functionality to the bio-chemical and bio-physical modification of proteins to meet the necessities of therapeutic applications. Provided the enormous capability of protein engineering approaches to modify the activity of bio-catalysts, this review summarizes the current studies are showing the advancements and limitations in using these methods to therapeutic enzyme engineering. The current review will concentrate mainly on three types of therapeutic based enzymes: Diagnostic enzymes, Fibrinolytic enzymes, and pharmaceutical enzymes, in which the protein is the restorative agent, prodrug-activating enzymes, provokes a therapeutic effects, and diagnostic enzymes, in which the remarkable specificity and selectivity of a protein offer advantages compared to conventional analytical techniques.

In vitro thrombolytic potential of fibrinolytic enzyme from Brevibacterium sp. isolated from the root of the plant, Aloe castellorum

Cardiovascular diseases (CVDs) like stroke and heart attack are leading causes of mortality worldwide, resulting in about 30% of deaths. The proportion in the Kingdom of Saudi Arabia was 46%. The commercial thrombolytic agents are thus associated with a range of side effects, while bacterially derived fibrinolytic enzymes have no or little side effects. Hence, there is an urgent need to identify novel fibrinolytic enzymes to treat or prevent CVDs. An endophytic bacterial strain producing fibrinolytic enzyme was isolated from the root of the plant Aloe castellorum. Enzyme production was enhanced through a conventional method and a statistical approach. The in vitro lytic activity of blood clots was also assessed. Endangered plant of Aloe castellorum roots, researchers isolated an endophytic strain producing fibrinolytic enzymes. It was identified as Brevibacterium sp. The culture medium was also optimized by a conventional method to screen the factors significant for successful culture. Fibrinolytic enzyme production peaked when Brevibacterium sp. was cultured in the presence of maltose (613 ± 12 U/mL), followed by starch (576 ± 13 U/mL). Among the selected nitrogen sources, yeast extract (642 ± 5.9 U/mL) and beef extract (610 ± 13 U/mL) enhanced enzyme yield relative to that in the control (487 ± 7.2 U/mL). Ionic sources such as Mg2+ (672 ± 10.3 U/mL) and Ca2+ (605 ± 12.3 U/mL) showed enhanced fibrinolytic enzyme production relative to the control (405 ± 13.2 U/mL). Upon employing a two-level full factorial design for examining the significant factors, it was revealed that maltose, yeast extract, and Mg2+ were important (p < 0.05). To enhance thrombolytic agent production using central composite design and response surface methodologies. The fibrinolytic enzyme obtained from Brevibacterium sp. has great potential to lyse blood clots in vitro.

Systematic analysis on the obtaining of fibrinolytic fungi enzymes

Fibrinolytic proteases operate directly on fibrin clot and are able to maintain blood flow. Fungi show up as viable sources for obtaining this enzyme. The purpose of this systematic review is to is to unveil all the information concerning production, purification and characterization of fibrinolytic proteases by fungi. The search was conducted in ScienceDirect, PubMed and Scopus databases, using as keywords “(Fibrinolytic enzyme) OR (Fibrinolytic protease) AND (Fungal or Fungus or Fungi)”. Delimiting period of 10 years (2011-2021). The results obtained were filtered by selection criteria, and review articles or articles outside the scope of this work were excluded. The articles were evaluated and scored (0-10) according to pre-established criteria. None of the studies obtained the score 10, however the study with the highest score (9) presented relevant data in all criteria analyzed, obtaining fibrinolytic enzyme from Xylaria curta. Among the 21 selected articles, 12 different genera appear and Submerged Fermentation and purification of Serino-proteases were more described. This work also observed a greater representation of purification and characterization steps, indicating the need for attention to cultivation process and enzymatic application. It is clear that the production of these enzymes by fungi is pertinent towards the high recovery observed even after purification and its tendency for pharmaceutical application.

Production and market comparison of urokinase and streptokinase as effective and cheap fibrinolytic agents for treatment of cardiovascular diseases

Failure of hemostasis and the formation of blood clots in the arteries are the main reasons that provoke the onset of cardiovascular diseases (CVDs) such as myocardial infarction and ischemic stroke. Cardiovascular diseases have become the primary cause of deaths and disabilities across the globe. Therefore, this problem needs to be addressed with urgency. The disintegration of blood clots requires fibrinolytic agents, which are involved in thrombolysis. Streptokinase and urokinase are fibrinolytic enzymes; the former is primarily produced from microbial sources and the latter is isolated from urine, respectively. Streptokinase and urokinase have been in use for a long time to treat cardiovascular diseases. This review explains in detail the comparison of employing streptokinase and urokinase for the said purpose in a cost-effective manner. The recombinant production of both the agents has been discussed in detail. Furthermore, the efficacy of both the agents has been compared based upon their side effects and retention time in the body. A thorough study has been made to compare the influence of using both the agents on the health of cardiovascular patients in the last decade.

Microscale structural changes of individual fibrin fibers during fibrinolysis

Fibrinolysis is the enzymatic digestion of fibrin, the primary structural component in blood clots. Mechanisms of fibrin fiber digestion during lysis have long been debated and obtaining detailed structural knowledge of these processes is important for developing effective clinical approaches to treat ischemic stroke and pulmonary embolism. Using dynamic fluorescence microscopy, we studied the time-resolved digestion of individual fibrin fibers by the fibrinolytic enzyme plasmin. We found that plasmin molecules digest fibers along their entire lengths, but that the rates of digestion are non-uniform, resulting in cleavage at a single location along the fiber. Using mathematical modeling we estimated the rate of plasmin arrival at the fiber surface and the number of digestion sites on a fiber. We also investigated correlations between local fiber digestion rates, cleavage sites, and fiber properties such as initial thickness. Finally, we uncovered a previously unknown tension-dependent mechanism that pulls fibers apart during digestion. Taken together these results promote a paradigm shift in understanding mechanisms of fibrinolysis and underscore the need to consider fibrin tension when assessing fibrinolytic approaches. Statement of significanceWe developed a method for interrogating lysis of individual fibrin fibers, enabling the time-resolved observation of individual fiber digestion for the first time. Our results resolve longstanding disagreements about fibrinolytic processes and reveal previously unknown mechanisms that also play a role. Also, we developed the first microscale mathematical model of plasmin-fibrin interaction, which predicts the number of plasmin molecules on each fiber and can serve as a framework for investigating novel therapeutics.

Marine Microbial Fibrinolytic Enzymes: An Overview of Source, Production, Biochemical Properties and Thrombolytic Activity.

Cardiovascular diseases (CVDs) have emerged as a major threat to global health resulting in a decrease in life expectancy with respect to humans. Thrombosis is one of the foremost causes of CVDs, and it is characterized by the unwanted formation of fibrin clots. Recently, microbial fibrinolytic enzymes due to their specific features have gained much more attention than conventional thrombolytic agents for the treatment of thrombosis. Marine microorganisms including bacteria and microalgae have the significant ability to produce fibrinolytic enzymes with improved pharmacological properties and lesser side effects and, hence, are considered as prospective candidates for large scale production of these enzymes. There are no studies that have evaluated the fibrinolytic potential of marine fungal-derived enzymes. The current review presents an outline regarding isolation sources, production, features, and thrombolytic potential of fibrinolytic biocatalysts from marine microorganisms identified so far.

Histomorphometric analysis of the lung of Swiss mice treated with a fibrinolytic protease

Fibrinolytic enzymes are considered promising alternative in the treatment of cardiovascular diseases by preventing fibrin clots. A protease from Mucor subtilissimus UCP 1262 was obtained by solid state fermentation and purified by ion exchange chromatography. The purified extract was administered at an acute dose of 2000 mg/mL to evaluate its toxic effects to the lungs of mice. After 14 days of treatment, a histomorphometric study was performed by the type 1 and 2 pneumocyte count and the evaluation of the lung area. As result, the experimental group showed a significant decrease of type 2 pneumocyte and although a decrease in the alveolar area was observed in relation to the control group, no significant pulmonary toxicity, emphysema, and fibrosis characteristics were detected. The in vitro tests suggest possible clinical applications for the enzyme.

D-dimer - uloga u hemostaznom sistemu i primena u specifičnim kliničkim stanjima

D-dimer is a soluble fibrin degradation product, which is formed by the breakdown of cross-linked fibrin monomers, previously created under thrombin activity, formed as a result of hemostatic activation. The degradation of cross-linked fibrin occurs as part of the activation of the fibrinolysis system, whose task is to remove the created coagulum and ensure the patency of the blood vessel. This process takes place under the influence of the most important fibrinolytic enzyme, plasmin. D-dimer can, therefore be considered a biomarker of coagulation activation and fibrinolysis, and is routinely used to rule out venous thromboembolism (VTE). D-dimer is increasingly used to assess the risk of VTE recurrence and to help define the optimal duration of anticoagulant therapy in the treatment of patients with VTE, to diagnose disseminated intravascular coagulation (DIC), to screen medical patients at increased risk of VTE. In particular, the use of D-dimer, which is sometimes unjustifiably high, should be emphasized, as part of the monitoring and treatment of patients with COVID-19 infection. This review article aims to: (1) define the origin and role of D-dimer in the hemostatic system, (2) the method of performing the analysis, the selection of adequate tests and interpretation in accordance with the consideration of possible preanalytical variables that affect the measurement of D-dimer; (3) consideration of its application in specific clinical settings or diseases.

Clinical Pharmacokinetics and Pharmacodynamics of Desmoteplase.

Desmoteplase is a bat (Desmodus rotundus) saliva-derived fibrinolytic enzyme resembling a urokinase and tissue plasminogen activator. It is highly dependent on fibrin and has some neuroprotective attributes. Intravenous administration of desmoteplase is safe and well tolerated in healthy subjects. Plasma fibrinolytic activity is linearly related to its blood concentration, its terminal elimination half-life ranges from 3.8 to 4.92 h (50 vs. 90 μg/kg dose). Administration of desmoteplase leads to transitory derangement of fibrinogen, D-dimer, alpha2-antiplasmin, and plasmin and antiplasmin complex which normalize within 4–12 h. It does not alter a prothrombin test, international normalized ratio, activated partial thromboplastin time, and prothrombin fragment 1.2. Desmoteplase was tested in myocardial infarction and pulmonary embolism and showed promising results versus alteplase. In ischemic stroke trials, desmoteplase was linked to increased rates of symptomatic intracranial hemorrhages and case fatality. However, data from “The desmoteplase in Acute Ischemic Stroke” Trials, DIAS-3 and DIAS-J, suggest that the drug is well tolerated and its safety profile is comparable to placebo. Desmoteplase is theoretically a superior thrombolytic because of high fibrin specificity, no activation of beta-amyloid, and lack of neurotoxicity. It was associated with better outcomes in patients with significant stenosis or occlusion of a proximal precerebral vessels. However, DIAS-4 was stopped as it might have not reached its primary endpoint. Due to its promising properties, desmoteplase may be added into treatment of ischemic stroke with extension of the time window and special emphasis on patients presenting outside the 4.5-h thrombolysis window, with wake-up strokes and strokes of unknown onset.

Utilization of sugarcane bagasse for enhancement production of fibrinolytic enzyme using statistical approach

The purpose of this investigation was to produce fibrinolytic enzyme by utilizing sugarcane bagasse as low cost substrate through solid state fermentation in an effective way. The potential producer of microorganism, Alcaligenes aquatilis PJS_1 was isolated and identified from slaughter house soil samples. In small scale level, higher production of microbial fibrinolytic enzyme was observed when the conditions provided like Glucose for carbon, Casein for nitrogen and (NH 4 ) 2 SO 4 for mineral sources when maintained at 60 h with 100% moisture. The sugarcane bagasse medium with glucose and casein was found to have optimum production when incubated for 60 h at 35°C. The culture conditions were optimized to improve the production of fibrinolytic enzyme (3832 U ml −1 ) from Alcaligenes aquatilis PJS_1 through statistical approach. Blood clotting assay was performed to determine its anticoagulant property. The fibrinolytic property of the synthesised enzyme was confirmed when it dissolved blood clot sample effectively. Thus, the enzyme it could be used in medicine to produce as a thrombolytic agent.

Produksi Enzim Fibrinolitik Tempe oleh Rhizopus oryzae FNCC 6078

ackground Rhizopus oryzae FNCC 6078 had been evaluated producing fibrinolytic enzyme under solid state fermentation. Soybean had been used to produce fibrinolytic enzyme through fermentation in tempeh. The main purpose of this study was to reveal optimum condition for fermentation. The parameters of the condition were inoculum volume, incubation period and temperature. Optimum condition was defined by maximum fibrinolytic activity. Methode Fibrinolytic activity was measured using spectrophotometry at 274 nm. Result optimum condition for producing fibrinolytic enzyme was 1,5 mL volume of inoculum of Rhizopus oryzae suspension in 25%T, 42 hours for incubation period and 35oC temperature incubation.