Antithrombotic Activity of Heparinoid G2 and Its Derivatives from the Clam Coelomactra antiquata

Clam heparinoid G2 (60.25 kDa) and its depolymerized derivatives DG1 (24.48 kDa) and DG2 (6.75 kDa) prepared from Coelomactra antiquata have been documented to have excellent fibrinolytic and anticoagulant activity. In this study, to further explore the antithrombotic activity of G2, DG1 and DG2, azure A, sheep plasma, and clot lytic rate assays were used to determine their anticoagulant and thrombolytic activity in vitro. The results indicated that the anticoagulant titer of G2 was approximately 70% that of heparin and the thrombolytic activity of DG2 was greater than G2, DG1, and heparin activities. Moreover, in a carrageenan-induced venous thrombosis model, oral administration of G2 and DG1 each at 20 mg/kg and 40 mg/kg for 7 days significantly reduced blacktail thrombus formation, increased tissue-type plasminogen activator, fibrin degradation products, and D-dimer levels, decreased von Willebrand factor and thromboxane B2 levels, and restored phylum and genus abundance changes of intestinal bacteria. DG2 had no antithrombotic effect. At 20 mg/kg, G2, DG1, and heparin had comparable antithrombotic activities, and DG1 at 40 mg/kg had more muscular antithrombotic activity than G2. Thus, DG1 could be an antithrombotic oral agent owing to its more robust antithrombotic activity and lower molecular weight.

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.

Ethanolic extract of ocimum kilimandscharicum linnleaves: phytochemical and in vitro pharmacological activity

Using an in vitro model, the anti-thrombolytic efficacy of ethanolic extracts of Ocimum kilimandscharicum Linn was investigated. The researchers discovered that different concentrations of the extract had significant anti-thrombolytic activity in a dose-dependent manner , which was comparable to a standard drug. As a result of the presence of flavonoids and polyphenols in the plant extract, it can be concluded that it has a promising future in the treatment of thrombosis. This knowledge will be useful in the clinical development of thrombolytic therapeutics by identifying more potent anti-thrombolytic principles from natural resources..

Thrombolytic Potentials of Some Medicinal Plants Used by the Local People for Cardiovascular Diseases in Bangladesh

Cardiovascular diseases (CVDs) are one of the major causes of death in the world. Medicinal plants with thrombolytic properties may be used as an alternative to modern medicines for CVDs. The present study was aimed to evaluate the thrombolytic potential of six medicinal plants available in Bangladesh using an in vitro clot lysis method where streptokinase and ethanol were used as a positive and negative control, respectively. Ethanolic extract at a dose of 10 mg/ml of Arjun tree (Terminalia arjuna), Garlic (Allium sativum), Elephant apple (Dillenia indica), Amla (Phyllanthus emblica), Yellow mombin (Spondias pinnata) and Burmese grape (Baccaurea ramiflora) showed 14.18 ± 1.23%, 10.72 ± 0.78%, 8.25 ± 0.42%, 7.08 ± 0.64%, 5.42 ± 0.47% and 2.47 ± 0.19% clot lysis, respectively, whereas the standard drug streptokinase lysed 41.11±0.31% clot at a dose of 30,000 IU. From the data, it is evident that ethanolic extracts of six selected medicinal plants possess a moderate to insignificant thrombolytic activities. Among these plants, Arjun tree and Garlic exhibited the highest thrombolytic activity and the Burmese grape showed the lowest thrombolytic activity. Through our study, it could be concluded that Arjun tree, Garlic, and Elephant apple might be used as traditional healing purposes of CVDs. However, further animal studies will prove the scientific justification of their uses. Conservation efforts should be given for Arjun tree, Elephant apple, Yellow mombin, Burmese grape, and Amla to save these plants from extinction in nature. Bangladesh J. Plant Taxon. 28(2): 405-412, 2021 (December)

Robust Thrombolytic and Anti-Inflammatory Action of a Constitutively Active ADAMTS13 Variant in Murine Stroke Models

Advances in our understanding of ADAMTS13 structure, and the conformation changes required for full activity, have rejuvenated the possibility of its use as a thrombolytic therapy. We have tested a novel Ala1144Val ADAMTS13 variant (caADAMTS13) which exhibits constitutive activity, characterised using in vitro assays of ADAMTS13 activity, and greatly enhanced thrombolytic activity in two murine models of ischaemic stroke, the distal FeCl3 MCAo model and tMCAO with systemic inflammation and ischaemia/reperfusion injury. The primary measure of efficacy in both models was restoration of rCBF to the MCA territory which was determined using laser speckle contrast imaging. The caADAMTS13 variant exhibited a constitutively active conformation and a 5-fold enhanced activity against FRETS-VWF73 compared to wild type (wt)ADAMTS13. Moreover, caADAMTS13 inhibited VWF-mediated platelet capture at sub-physiological concentrations and enhanced t-PA/plasmin lysis of fibrin(ogen), neither of which were observed with wtADAMTS13. Significant restoration of rCBF and reduced lesion volume was observed in animals treated with caADAMTS13. When administered 1 h after FeCl3 MCAo the caADAMTS13 variant significantly reduced residual VWF and fibrin deposits in the MCA, platelet aggregate formation and neutrophil recruitment. When administered 4 h after reperfusion in the tMCAo model the caADAMTS13 variant induced a significant dissolution of platelet aggregates and a reduction in the resulting tissue hypoperfusion. The caADAMTS13 variant represents a potentially viable therapeutic option for the treatment of acute ischaemic stroke, amongst other thrombotic indications, due to its enhanced in vitro and in vivo activities that result from its constitutively active conformation.

Evaluation on In Vitro Blood Clot Dissolving Potential of Aqueous Extract of Sida acuta Burm. F. Leaves

Clotting of blood is the vital processes and a perplexing interaction of various mechanisms of circulatory system due of failure of which is sometimes considered as a concern within the circulatory system causing acute myocardial or cerebral infarction which might cause demise. Sida acuta burm. f (Malvaceae) is abundantly growing small perennial shrub utilized by natives for diuretic, anthelmintic, calmative and wound healing properties, and are utilized in treating disorders like blood, bile, liver, nervous, urinary diseases and rheumatism. The present study was intended to evaluate the blood clot dissolving potential of Sida acuta leaf aqueous extract in vitro. The plant material as leaves were locally collected and subjected to phytochemical extraction with distilled water. The preliminary phytochemical tests total phenolic content was estimated by Folin-Ciocalteu’s method. In vitro thrombolytic activity of 3 different concentrations of aqueous extract was estimated on goat blood clot compared to the activity of streptokinase. The aqueous extract of S. acuta leaves are reported to be rich in alkaloids, flavonoids, tannins, terpenoids and glycosides while the total phenolic content was estimated to be 17.48 % in extract which are mostly responsible for any pharmacological activity. Compared to the thrombolytic activity of standard streptokinase which was 73 %, the aqueous leaf extract of S. acuta displayed considerable blood clot dissolving activity at concentration 10 mg/100µl, 5.0 mg/100µl, and 2.5 mg/100µl as 41 %, 34 % and 12 % respectively. This property of plant extract is promising which could be could be exploited in development of new biopharmaceutical and therapeutic agents after stringent further physiological compatibility and in vivo pharmacological studies. Keywords: Sida acuta, phytochemical extract, thrombolytic activity, streptokinase

Synthesis of the Polyhydroquinoline Derivative 4-(2-Chloro-Phenyl)-2,7,7-Trimethyl5-oxo-1,4,5,6,7,8-Hexahydroquinoline-3-Carboxylic Acid Ethyl Ester: Antimicrobial and Enzyme Modulator

Multicomponent reactions are extremely relevant in green chemistry. They offer better conditions than traditional synthesis and are, therefore, used for many organic modifications. Recently, the synthesis of polyhydroquinolines has received much attention for its high pharmacological potential. In the present study, a polyhydroquinoline derivative was synthesized without the use of catalysts or solvents. The results of nuclear magnetic resonance and infrared spectroscopy demonstrated that the molecule was successfully synthesized. The molecule presents significant results of antimicrobial activity for the bacteria tested in the serial dilution method. It also increased the clotting time by 25.66 seconds for the highest dose and 12.66 seconds for the other doses tested. Prior incubation with the dose of 125 mg reduced the thrombolytic activity to 73%. The 125, 100, and 50 mg doses previously incubated with Bothrops moojeni venom inhibited approximately 30% of the phospholipase activity. The molecule was also able to reduce the cytotoxicity induced by proteases significantly. In conclusion, the molecule presents several biological properties, which highlights its pharmaceutical potential.

Isolation and Purification of Thrombolytic Enzyme Extracted from Earthworm Punjab, Pakistan

The Cardiovascular disease due to thrombus (clot) formation is the major factor of death throughout the world. Earthworms being the eco engineers has thrombolytic enzyme that can be used for thrombolysis. The thrombolytic enzyme was isolated and purified from supernatant of earthworm Apporectodea longa by column chromatography. Six Strain BKT 11, BKT 15, BKT 17, BKT 26, BKT 27 and BKT 28 shows the thrombolytic activity 791.64 U/mg, 1362.39 U/mg, 1205.4 U/mg, 710.63 U/mg, 529.66 U/mg and 625.00 U/mg respectively. Thrombolytic activity was confirmed by blood clot lysis method. Different concentrations 50 ?l,100 ?l, 150 ?l, 200 ?l and 250 ?l of extracted enzyme were applied on 25mg of wet blood clot along with control where distill water used. These fractions of extracted enzymes represent the dissolution of clot (thrombolysis). The molecular weight 32 KDa was determined by sodium dodecyl sulphate gel electrophoresis (SDS-PAGE). Results show that extracted elute have potential of fibrinolytic activity in this specie of earthworm and it can serve as a suitable therapeutic agent. Keywords: Thrombolytic activity, Casein plate assay, Blood clot lysis, spectrophotometry, Gel electrophoresis.

Phytochemical and pharmacological investigations of different extracts of leaves and stem barks of Macropanax dispermus (Araliaceae): a promising ethnomedicinal plant

Background Macropanax dispermus is traditionally used to treat various diseases by ethnic people. The present research reports the pharmacological properties with phytochemical profiling of the crude extracts of M. dispermus leaves (MDML), its n-hexane (MDHL), carbon tetrachloride (MDTL), chloroform (MDCL), ethyl acetate (MDEL), and aqueous (MDAL) fractions, and crude methanol extracts of its stem barks (MDMS). The in vitro thrombolytic activity was done on human erythrocytes whereas the cytotoxic activity was done by brine shrimp lethality assay. The in vivo analgesic activity was examined by acetic acid-induced writhing, tail immersion, and formalin-induced paw licking method. In contrast, antipyretic activity was done by the brewer’s yeast-induced pyrexia method. Results MDHL and MDMS showed 37.05% and 42.21% of significant (p < 0.01) thrombolytic activity, respectively. MDCL and MDMS showed the lower LC50 values of 23.15 and 37.11 µg/ml during cytotoxicity test, respectively. In acetic acid writhing method, MDTL and MDEL showed significant (p < 0.001) inhibition of writhing by 79.34% and 80.17%, respectively. MDMS showed significant (p < 0.001) maximal possible effect (%MPE) of 45.95%, 62.26%, 65.79%, 89.69% and elongation of time in pain reaction of 48.53%, 60.28%, 58.76%, and 70.14% at 30, 60, 90, and 120 min intervals, respectively. MDML at 400 mg/kg exhibited significant (p < 0.001) 82.72% of inhibition of pain at the late phases. MDEL at 400 mg/kg of dose exhibited significant (p < 0.001) reduction of rectal temperature by 36.31%, 62.42%, 89.81%,, and 96.82% at 1, 2, 3, and 4 h intervals, respectively. Conclusion The current research suggests that the plant extracts possess potential thrombolytic, cytotoxic, analgesic, and antipyretic activities.

Heterologous expression of nattokinase from B. subtilis natto using Pichia pastoris GS115 and assessment of its thrombolytic activity

Background Nattokinase is a fibrinolytic enzyme that has huge market value as a nutritional supplement for health promotion. In order to increase nattokinase yields, fermentation conditions, strains, cultivation media, and feeding strategies have been optimized. Nattokinase has been expressed using several heterologous expression systems. Pichia pastoris heterologous expression system was the alternative. Results This report aimed to express high levels of nattokinase from B. subtilis natto (NK-Bs) using a Pichia pastoris heterologous expression system and assess its fibrinolytic activity in vivo. Multicopy expression strains bearing 1–7 copies of the aprN gene were constructed. The expression level of the target protein reached a maximum at five copies of the target gene. However, multicopy expression strains were not stable in shake-flask or high-density fermentation, causing significant differences in the yield of the target protein among batches. Therefore, P. pastoris bearing a single copy of aprN was used in shake-flask and high-density fermentation. Target protein yield was 320 mg/L in shake-flask fermentation and approximately 9.5 g/L in high-density fermentation. The recombinant nattokinase showed high thermo- and pH-stability. The present study also demonstrated that recombinant NK-Bs had obvious thrombolytic activity. Conclusions This study suggests that the P. pastoris expression system is an ideal platform for the large-scale, low-cost preparation of nattokinase.