Stem Bromelain Proteolytic Machinery: Study of the Effects of its Components on Fibrin (ogen) and Blood Coagulation

Background: Antiplatelet, anticoagulant and fibrinolytic activities of stem bromelain (EC 3.4.22.4) are well described, but more studies are still required to clearly define its usefulness as an antithrombotic agent. Besides, although some effects of bromelain are linked to its proteolytic activity, few studies were performed taking into account this relationship. Objective: We aimed at comparing the effects of stem bromelain total extract (ET) and of its major proteolytic compounds on fibrinogen, fibrin, and blood coagulation considering the proteolytic activity. Methods: Proteolytic fractions chromatographically separated from ET (acidic bromelains, basic bromelains, and ananains) and their irreversibly inhibited counterparts were assayed. Effects on fibrinogen were electrophoretically and spectrophotometrically evaluated. Fibrinolytic activity was measured by the fibrin plate assay. The effect on blood coagulation was evaluated by the prothrombin time (PT) and activated partial thromboplastin time (APTT) tests. Effects were compared with those of thrombin and plasmin. Results: Acidic bromelains and ananains showed thrombin-type activity and low fibrinolytic activity, with acidic bromelains being the least effective as anticoagulants and fibrinolytics; while basic bromelains, without thrombin-like activity, were the best anticoagulant and fibrinolytic proteases present in ET. Procoagulant action was detected for ET and its proteolytic compounds by the APTT test at low concentrations. The measured effects were dependent on proteolytic activity. Conclusion: Two sub-populations of cysteine proteases exhibiting different effects on fibrin (ogen) and blood coagulation are present in ET. Using well characterized stem bromelain regarding its proteolytic system is a prerequisite for a better understanding of the mechanisms underlying the bromelain action.

Identification of lumbrokinase by fibrin-plate method, fibrin-zymography and liquid chromatography mass spectrometry

Lumbrokinase, extracted from the cultured earthworm of Eisenia fetida, has been widely used as biochemical medicine in China to prevent or treat thrombosis. In the present study, the mechanism of lumbrokinase was investigated using the fibrin plate method. The results revealed that lumbrokinase contained both fibrinolytic and kinase components. The method of fibrin-zymography was used to show the existence and activity of lumbrokinase, and we proved that the fibrin-zymogram gel could be adopted as the identification method of earthworm. Subsequently, the components were identified by mass spectrometry. According to the results, fibrinolytic related components existed in the drug. These proteins were further compared with other serine proteins. The result showed that the identified proteins were similar to human trypsin and bovine trypsin. Besides, some also exhibited similar characteristics with human plasminogen activators. The mentioned results demonstrated that lumbrokinase products contained two major groups of protein components, suggesting two different functions.

Fibrinolytic Activity of Two Polypeptide Chains from Human Plasminogen#

Background: Plasminogen is a blood plasma glycoprotein of molecular weight about 92,000 Daltons. Physiologically, it incorporates into blood clots and after its activation by plasminogen activators to plasmin can perform a fibrinolytic function. Microplasmin is truncate polypeptide chain derivate of plasmin may be increase the fibrinolytic activity. Objective: To study the amino acid sequence of two polypeptides chains derivate to the plasminogen with fibrinolytic activity. Methods: he two polypeptides chains were prepared by isoelectric precipitation of human plasma in sodium borate buffer. The sample in a second step was subjected to affinity and ionic interchange chromatography and denaturalized electrophoresis was carried out on the sample previous heat 70ºC. Results: Two polypeptide chains of 29.000 and 35.000 Daltons by autolysis controlled were obtained with 25 UI of fibrinolytic activity in fibrin plate. Conclusion: Microplasmin was obtained with cleavage in different amino acid bounds and rearrangement of amino acids by autolysis with controlled alkaline precipitation.

FIRST REPORT ON FIBRINOLYTIC AND THROMBOLYTIC ACTIVITY OF EUTYPHOEUS GAMMIEI AN EARTHWORM SPECIES COLLECTED FROM TRIPURA, NORTHEAST INDIA

Objective: The present investigation for the first time evaluated the in vitro fibrinolytic and thrombolytic activities of crude extracts from Eutyphoeus gammiei, native, large size earthworm of Tripura, Northeast, India. The present study was designed to evaluate the therapeutic use of the organism E. gammiei as a source of fibrinolytic and thrombolytic agent(s).Methods: The fibrinolytic activity was studied using by fibrin plate and zymography assays. Thrombolytic assay was carried out according to Prasad et al. (2006) using whole blood.Results: The results obtained clearly indicated E. gammiei as a potential source of fibrinolytic and thrombolytic agents. Both in fibrin plate assay and thrombolytic assay with whole blood, E. gammiei crude homogenate showed similar and close results in respect to that of streptokinase. Fibrin zymography also showed antifibrinolytic activity with producing clear bands. Dose and time dependency also is evident from the results.Conclusion: The results of the present study conclude that the studied earthworm species E. gammiei possessed profound fibrinolytic and thrombolytic activity on human blood and E. gammiei might prove to be useful alternative source for the development of new drugs for treatments involving blood coagulation and fibrinolysis.

ISOLATION OF THERMOTOLERANT BACTERIA PRODUCING FIBRINOLYTIC ENZYME

Fibrinolytic enzymes were widely used in the treatment of cardiovascular diseases. However, the efficiency of the commercial enzymes are still lack of perfection because there are many side effects as well as not tolerant to downstream processing such as heat sensitive during spray drying process. Therefore, this study presents newly isolated thermophiles bacteria producing fibrinolytic enzyme. Sample was collected from Hot Spring Selayang at Selayang Selangor. Spread plate agar containing skim milk powder growth at pH 7, 53 for 24 hours was utilized to isolate thermotolerant bacteria producing protease. Further isolation on bacteria producing fibrinolytic enzyme was carried out using fibrin plate. 16S rDNA gene sequence analysis was used to identify the genotype of the isolates. 27 colonies of thermotolerant bacteria were isolated, however, only 19 of them showing proteolytic activity. All of the 19 isolates are motile and cocci in shapes, with 4 types of arrangement, which are single, diplo (pair), strepto (chain) and staphylo (cluster). HSP04 and HSP11 are gram positive bacteria and others are gram negative. From 19 isolates only 6 were chosen for further analysis. HSP23 showed the highest fibrinolytic activity compared with others. HSP23 was identified as Bacillus licheniformis with 98 % similarity to Bacillus licheniformis DCM 13 and Bacillus licheniformis strain ATCC 14580.

Signs of Hypofibrinolysis in Patients with Unprovoked Idiopathic Venous Thromboembolism

Background: The causes for venous thromboembolism (VTE) remain undetermined in at least 30% of patients with unprovoked VTE. Hypofibrinolysis may be associated to VTE, however the occurrence of hypofibrinolysis in patients with unprovoked, idiopathic, VTE is not well stablished. Aims: To evaluate whether hypofibrinolysis would be associated with unprovoked idiopathic VTE. Methods: Patients with a history of unprovoked VTE without acquired or inherited thrombophilia were included. Global tests of fibrinolysis, such as euglobolin lysis time (ELT) and lysis area on fibrin plate (LAFP), and specific tests of fibrinolysis, such as plasma activity plasminogen, a-2 antiplasmin (a2AP), plasminogen activator inhibitor-1 (PAI-1), and thrombin activatable fibrinolysis inhibitor (TAFI), were performed in patients and healthy controls. We also analyzed the plasma activity of factor (F) XIII. Results: Thirty-one patients and fifty healthy controls were included. ELT results were higher in patients than in controls (median= 295 and IQ= 205-355 minutes vs. median=250 and IQ= 167-295 minutes, respectively, p = 0.006) and LAFP values were lower in patients compared to controls (median=81 and IQ= 56-110 vs. median= 95 and IQ 72-132 respectively, p = 0.0014), suggesting that they were experiencing a hypofibrinolytic state. Plasma activity of plasminogen (median= 131 and IQ= 119-141 vs. median=120 and IQ=111-137, respectively, p = 0.045) and FXIII (median= 103 and IQ 89-127 vs. median= 96 and IQ=80-105, respectively, p = 0.050), were higher in patients than in controls, whereas plasma activity of α-2AP was lower in patients (median= 124 and IQ 114-128 vs. median= 127 and IQ=121-133, p≤0.001). Interestingly, patient's median TAFI activity was lower than in controls (median=16 and IQ 13-18μg/mL vs. median= 19 and IQ= 17-21g/mL, p≤0.001). However, PAI-1 activity did not differ between groups. Conclusions: Hypofibrinolysis may occur in patients with unprovoked idiopathic VTE and may be detected by global tests of fibrinolysis. It is possible that hypofibrinolysis contribute to the pathogenesis of thrombosis in these selected cases. Disclosures No relevant conflicts of interest to declare.

Cloning and Expression of a Novel Protease with Fibrinolytic Activity from Arenicola cristata

cDNA of a novel protease, designated as AFEI, was cloned from digestive tract of Arenicola cristata by RACE. The cDNA of AFEIcomprised 897bp and an open reading frame that encoded polypeptides of 264 amino acid residues. AFEIshowed similarity to serine protease family and contained the conserved catalytic amino acid residues. The gene encoding the active form of AFEIwas expressed in E.coli and the purified recombinant protein could dissolve an artificial fibrin plate with plasminogen, which indicated the recombinant protein might be a plasminogen activator for thrombosis therapy.

DLBS1033, A Protein Extract fromLumbricus rubellus, Possesses Antithrombotic and Thrombolytic Activities

The medicinal value of earthworm has been widely known since the history of Asian ancient medicine. This present study aims to determine the mechanism of action and effect of a standardized extract ofLumbricus rubellusnamed as DLBS1033. The fibrinogen degradation, antiplatelet aggregation, andex vivoantithrombotic assay using human blood were performed to study antithrombotic activity. Fibrin plate and clot lysis assay were also done to examine thrombolytic properties. DLBS1033 was found to possess fibrinogenolytic activity onα-,β-, andγ-chain of fibrinogen. It also induced antiplatelet aggregation and prolonged blood clotting time, which further confirmed its antithrombotic properties. In addition, thrombolytic properties of DLBS1033 were shown with its fast and long-acting fibrinolytic activity, as well as its effective blood clot lysis activities. In conclusion, DLBS1033 conferred antithrombotic and thrombolytic action which could be used as a safe and promising oral thrombolytic drug.

Molecular Cloning and Characterization of cDNA Encoding Fibrinolytic Enzyme-3 from Earthworm Eisenia foetida

The earthworm fibrinolytic enzyme-3 (EFE-3, GenBank accession No: AY438622), from the earthworm Eisenia foetida, is a component of earthworm fibrinolytic enzymes. In this study, cDNA encoding the EFE-3 was cloned by RT-PCR. The cDNA contained an open reading frame of 741 nucleotides, which encoded a deduced protein of 247 amino acid residues, including signal sequences. EFE-3 showed a high degree of homology to earthworm (Lumbricus rebullus) proteases F-III-1, F-III-2, and bovine trypsin. The recombinant EFE-3 was expressed in E. coli as inclusion bodies, and the gene encoding the native form of EFE-3 was expressed in COS-7 cells in the medium. Both the refolding product of inclusion bodies and the secreted protease could dissolve the artificial fibrin plate.