Fibrinolytic Enzyme – An Overview

Cardiovascular diseases, like coronary heart disease or artery disorders (arteriosclerosis, including artery solidification), heart failure (myocardial infarction), arrhythmias, congestive heart condition, stroke, elevated vital signs (hypertension), rheumatic heart disorder, and other circulatory system dysfunctions are the most common causes of death worldwide. Cardiovascular disorders are treated with stenting, coronary bypass surgery grafting, anticoagulants, antiplatelet agents, and other pharmacological and surgical procedures; however, these have limitations due to their adverse effects. Fibrinolytic agents degrade fibrin through enzymatic and biochemical processes. There are various enzymes that are currently used as a treatment for CVDs, like Streptokinase, Nattokinase, Staphylokinase, Urokinase, etc. These enzymes are derived from various sources like bacteria, fungi, algae, marine organisms, plants, snakes, and other organisms. This review deals with the fibrinolytic enzymes, their mechanisms, sources, and their therapeutic potential.

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. The current review presents an outline regarding isolation sources, production, features, and thrombolytic potential of fibrinolytic biocatalysts from marine microorganisms identified so far.

Microbial Fibrinolytic Enzymes as Anti-Thrombotics: Production, Characterisation and Prodigious Biopharmaceutical Applications

Cardiac disorders such as acute myocardial infarction, embolism and stroke are primarily attributed to excessive fibrin accumulation in the blood vessels, usually consequential in thrombosis. Numerous methodologies including the use of anti-coagulants, anti-platelet drugs, surgical operations and fibrinolytic enzymes are employed for the dissolution of fibrin clots and hence ameliorate thrombosis. Microbial fibrinolytic enzymes have attracted much more attention in the management of cardiovascular disorders than typical anti-thrombotic strategies because of the undesirable after-effects and high expense of the latter. Fibrinolytic enzymes such as plasminogen activators and plasmin-like proteins hydrolyse thrombi with high efficacy with no significant after-effects and can be cost effectively produced on a large scale with a short generation time. However, the hunt for novel fibrinolytic enzymes necessitates complex purification stages, physiochemical and structural-functional attributes, which provide an insight into their mechanism of action. Besides, strain improvement and molecular technologies such as cloning, overexpression and the construction of genetically modified strains for the enhanced production of fibrinolytic enzymes significantly improve their thrombolytic potential. In addition, the unconventional applicability of some fibrinolytic enzymes paves their way for protein hydrolysis in addition to fibrin/thrombi, blood pressure regulation, anti-microbials, detergent additives for blood stain removal, preventing dental caries, anti-inflammatory and mucolytic expectorant agents. Therefore, this review article encompasses the production, biochemical/structure-function properties, thrombolytic potential and other surplus applications of microbial fibrinolytic enzymes.

Bioevaluation of Pheretima vulgaris Antithrombotic Extract, PvQ, and Isolation, Identification of Six Novel PvQ-Derived Fibrinolytic Proteases

Thrombosis is a disease that seriously endangers human health, with a high rate of mortality and disability. However, current treatments with thrombolytic drugs (such as recombinant tissue-plasminogen activator) and the oral anticoagulants (such as dabigatran and rivaroxaban) are reported to have a tendency of major or life-threatening bleeding, such as intracranial hemorrhage or massive gastrointestinal bleed with non-specific antidotes. In contrast, lumbrokinase is very specific to fibrin as a substrate and does not cause excessive bleeding. It can dissolve the fibrin by itself or convert plasminogen to plasmin by inducing endogenous t-PA activity to dissolve fibrin clots. Therefore, searching for potentially new therapeutic molecules from earthworms is significant. In this study, we first collected a strong fibrinolytic extract (PvQ) from the total protein of the Pheretima vulgaris with AKTA pure protein purification systems; its fibrinolytic bioactivity was verified by the fibrin plate assay and zebrafish thrombotic model of vascular damage. Furthermore, according to the cell culture model of human umbilical vein endothelial cells (HUVECs), the PvQ was proven to exhibit the ability to promote the secretion of tissue-type plasminogen activator (t-PA), which further illustrated that it has an indirect thrombolytic effect. Subsequently, extensive chromatographic techniques were applied to reveal the material basis of the extract. Fortunately, six novel earthworm fibrinolytic enzymes were obtained from the PvQ, and the primary sequences of those functional proteins were determined by LC-MS/MStranscriptome cross-identification and the Edman degradation assay. The secondary structures of these six fibrinolytic enzymes were determined by circular dichroism spectroscopy and the three-dimensional structures of these proteases were predicted by MODELLER 9.23 based on multi-template modelling. In addition, those six genes encoding blood clot-dissolving proteins were cloned from P. vulgaris by RT-PCR amplification, which further determined the accuracy of proteins primary sequences identifications and laid the foundation for subsequent heterologous expression.

Fibrinolytic enzymes from extremophilic microorganisms in the development of new thrombolytic therapies: Technological Prospecting

Background: Extremophilic microorganisms from a wide variety of extreme natural environments have been researched, and many biotechnological applications have been carried out, due to their capacity to produce biomolecules resistant to extreme conditions, such as fibrinolytic proteases. The search for new fibrinolytic enzymes is important in the development of new therapies against cardiovascular diseases. Objective: This article aimed to evaluate the patents filed about protease with fibrinolytic activity produced by extremophilic microorganisms whose use is aimed at the development of new drugs for the treatment of cardiovascular diseases. Methods: The prospecting was carried out using data on deposits and patent concessions made available on the technological bases: European Patent Office (EPO), United States Patent and Trademark Office (USPTO), World Intellectual Property Organization (WIPO), Instituto Nacional de Propriedade Industrial – Brazil (INPI), The LENS and Patent Inspiration. The International Patent Classification and subclasses and groups for each document were also evaluated. Results: Although 382 patents were selected using terms related to extreme environments, such as “thermophile” and “acidophiles”, few were related to clinical use and were mainly performed using Bacillus subtilis and Streptomyces megasporus strains. A highlight of nattokinase was produced by Bacillus subtilis GDN and actinokinase by Streptomyces megasporus SD5. Conclusion: The low number of patents on enzymes with this profile (extreme environments) revealed a little-explored field, promising in the development of new microbial thrombolytic drugs, such as fibrinolytic enzymes with less adverse effects.

A brief review on conventional methods for screening, product development, downstream and evaluation of fibrinolytic enzymes from marine microbes

The ocean is a great reservoir of biodiversity and microbial metabolites. Enzymes from marine source have recently gained considerable attention as they have lower side effects and more potency when compared to other existing sources. Fibrinolytic enzymes from microbial sources possess ability to dissolve clots and help to circumvent cardiovascular problems in more efficient and safer way. The complexity of the marine environment involves high salinity, high pressure, low temperature, special lighting conditions. This contributes to the significant differences between the enzymes generated by marine microorganisms and homologous enzymes from terrestrial microorganisms leading to the boosted marine microbial enzyme technology. Further, it is believed that sea water, which is saline in nature and chemically closer to the human blood plasma, could provide biomolecules, in particular enzymes that could have lower or no toxicity or side effects when used for therapeutic applications. However, only a small proportion of fibrinolytic enzymes from marine microbiota has been examined and an even smaller proportion has been exploited. Therefore, much work needs to be done intensively and extensively in terms of potent fibrinolytic enzymes from marine resources.

Role of Fibrinolytic Enzymes in Anti-Thrombosis Therapy

Thrombosis, a major cause of deaths in this modern era responsible for 31% of all global deaths reported by WHO in 2017, is due to the aggregation of fibrin in blood vessels which leads to myocardial infarction or other cardiovascular diseases (CVDs). Classical agents such as anti-platelet, anti-coagulant drugs or other enzymes used for thrombosis treatment at present could leads to unwanted side effects including bleeding complication, hemorrhage and allergy. Furthermore, their high cost is a burden for patients, especially for those from low and middle-income countries. Hence, there is an urgent need to develop novel and low-cost drugs for thrombosis treatment. Fibrinolytic enzymes, including plasmin like proteins such as proteases, nattokinase, and lumbrokinase, as well as plasminogen activators such as urokinase plasminogen activator, and tissue-type plasminogen activator, could eliminate thrombi with high efficacy rate and do not have significant drawbacks by directly degrading the fibrin. Furthermore, they could be produced with high-yield and in a cost-effective manner from microorganisms as well as other sources. Hence, they have been considered as potential compounds for thrombosis therapy. Herein, we will discuss about natural mechanism of fibrinolysis and thrombus formation, the production of fibrinolytic enzymes from different sources and their application as drugs for thrombosis therapy.

Optimization of Cultural Conditions for Maximum Production of Fibrinolytic Enzymes from the Local Marine Bacterial Isolates and Evaluation of their Wound Healing and Clot Dissolving Properties

Fibrinolytic enzymes find necessary applications to treat and prevent cardiovascular diseases. In this study, optimal conditions for enhancing the production of fibrinolytic enzyme from local marine bacterial strains were evaluated. The present study also focuses on screening of wound healing efficacy of the isolated fibrinolytic enzymes.Various physical parameters such as temperature, pH, incubation time and medium components viz. inoculum size, substrate (nitrogen and carbon) concentrations were optimized. A cultivation medium was designed using optimized conditions for mass production of fibrinolytic enzyme and specific activity of enzyme was analyzed. The maximum enzyme production was observed at 37 °C temperature, 8.0 pH,substrate concentration with 3 ml inoculum size and 32 h. of incubation time. Among the different carbon sources tested, Mannitol showed maximum enzyme activity i.e 538 U/ml. yeast extract was found to be the best nitrogen source with an enzyme activity of 498 U/ml. The best substrate for the production fibrinolytic enzyme was found to be kernelwith high activity of 1056U/ml. The crude enzyme displayed potent activity and digested blood clot completely in in vitro condition and exhibited potent activity on wound healing property in macrophages.