MUTANTS OF TPA AND THEIR CATALYTIC PROPERTIES

1987 ◽  
Author(s):  
B Chaudhuri ◽  
J Heim ◽  
B Meyhack ◽  
J van Oostrum
Keyword(s):  
Catalytic Properties ◽  
Clot Lysis ◽  
In Vitro Mutagenesis ◽  
The Novel ◽  
Single Chain ◽  
Mutant Proteins ◽  
Novel Proteins ◽  
Lysis Activity ◽  
Insight Into

Mutants of tPA were obtained by in vitro mutagenesis to study the effects of N-linked glycosylation, and to gain insight into the properties of pure single-chain tPA. The glycosylation mutants were obtained by modification of the last residue of the sequence Asn.X.Ser/Thr, . known to be specific for N-linked glycosylation, whereas the plasmin sensitive sequence Arg275 Ile276 was modified to Asp 275 Ile276 to obtain a plasmin insensitive tPA. Mutant proteins were expressed in yeast under the control of the repressible acid-phosphatase promoter, and in CHO-cells. The novel proteins were isolated mainly by affinity chromatography using the selective protease inhibitor DE-3. A tPA mutant containing a modification in the sequence Asnl84 Glyl85 Ser 186 showed similar results as compared to recombinant yeast tPA in the clot lysis test, a direct fluoresence assay (FU test), and the indirect double rate assay according to Verheyen (Verheyen test). A mutant with modifications in the sequences Asnll7 Ser 118 Serll9, Asnl84 Glyl85 Serl86, and Asn448 Arg449 Thr450, showed an increased clot lysis activity with no increase in activity in the FU test and Verheyen test. However, the stimulation by fibrinogen fragments in the Verheyen assay was found to be doubled as compared to yeast tPA. An even further increase in stimulation by fibrinogen fragments, upto 500% over yeast tPA, was observed with the plasmin insensitive tPA, although this mutant only showed 20% of the yeast tPA activity in the FU and Verheyen tests

Absence of Synergism Between Tissue-Type Plasminogen Activator (t-PA), Single-Chain UrokinaseType Plasminogen Activator (scu-PA) and Urokinase on Clot Lysis in a Plasma Milieu In Vitro

1986 ◽  
Vol 56 (01) ◽  
pp. 035-039 ◽  
Author(s):  
D Collen ◽  
F De Cock ◽  
E Demarsin ◽  
H R Lijnen ◽  
D C Stump
Keyword(s):  
Human Plasma ◽  
Plasminogen Activator ◽  
Dose Response ◽  
Fibrinolytic System ◽  
Tissue Type ◽  
Clot Lysis ◽  
Single Chain ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator

SummaryA potential synergic effect of tissue-type plasminogen activator (t-PA), single-chain urokinase-type plasminogen activator (scuPA) or urokinase on clot lysis was investigated in a whole human plasma system in vitro. The system consisted of a human plasma clot labeled with 125I-fibrinogen, immersed in titrated whole human plasma, to which the thrombolytic agents were added. Clot lysis was quantitated by measurement of released 125I, and activation of the fibrinolytic system in the surrounding plasma by measurements of fibrinogen and α2-antiplasmin.t-PA, scu-PA and urokinase induced a dose-dependent and time-dependent clot lysis; 50 percent lysis after 2 h was obtained with 5 nM t-PA, 20 nM scu-PA and 12 nM urokinase. At these concentrations no significant activation of the fibrinolytic system in the plasma was observed with t-PA and scu-PA, whereas urokinase caused significant α2-antiplasmin consumption and concomitant fibrinogen degradation. The shape of the dose-response curves was different; t-PA and urokinase showed a log linear dose-response whereas that of scu-PA was sigmoidal.

scholarly journals Therapeutic Potentials of Syzygium fruticosum Fruit (Seed) Reflected into an Array of Pharmacological Assays and Prospective Receptors-Mediated Pathways

Life ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 155
Author(s):  
Jannatul Nasma Rupa Moni ◽  
Md. Adnan ◽  
Abu Montakim Tareq ◽  
Md. Imtiazul Kabir ◽  
A.S.M. Ali Reza ◽  
...  
Keyword(s):  
Bioactive Compounds ◽  
Integrated Approach ◽  
Gas Chromatography Mass Spectrometry ◽  
Clot Lysis ◽  
Lipinski’S Rule ◽  
Lysis Activity ◽  
In Silico Studies ◽  
Immobility Time

Syzygium fruticosum (SF), a valuable Bangladeshi fruit, is considered an alternative therapeutic agent. Mainly, seeds are used as nutritional phytotherapy to ease physical and mental status by preventing chronic diseases. Here, we scrutinized the S. fruticosum seed’s fundamental importance in traditional medicine by following an integrated approach combining in vivo, in vitro, and in silico studies. The SF was fractionated with different solvents, and the ethyl acetate fraction of SF (EaF-SF) was further studied. Mice treated with EaF-SF (200 and 400 mg/kg) manifested anxiolysis evidenced by higher exploration in elevated plus maze and hole board tests. Similarly, a dose-dependent drop of immobility time in a forced swimming test ensured significant anti-depressant activity. Moreover, higher dose treatment exposed reduced exploratory behaviour resembling decreased movement and prolonged sleeping latency with a quick onset of sleep during the open field and thiopental-induced sleeping tests, respectively. In parallel, EaF-SF significantly (p < 0.001) and dose-dependently suppressed acetic acid and formalin-induced pain in mice. Also, a noteworthy anti-inflammatory activity and a substantial (p < 0.01) clot lysis activity (thrombolytic) was observed. Gas chromatography-mass spectrometry (GC–MS) analysis resulted in 49 bioactive compounds. Among them, 12 bioactive compounds with Lipinski’s rule and safety confirmation showed strong binding affinity (molecular docking) against the receptors of each model used. To conclude, the S. fruticosum seed is a prospective source of health-promoting effects that can be an excellent candidate for preventing degenerative diseases.

scholarly journals Exploration of in vitro thrombolytic, anthelminthic, cytotoxic and in vivo anxiolytic potentials with phytochemical screening of flowers of Brassica nigra

2020 ◽  
Vol 6 (1) ◽  
Author(s):  
Mohammad Sarowar Uddin ◽  
Md. Shalahuddin Millat ◽  
Mohammad Safiqul Islam ◽  
Md. Saddam Hussain ◽  
Md. Giash Uddin ◽  
...  
Keyword(s):  
Brassica Nigra ◽  
Anxiolytic Activity ◽  
Clot Lysis ◽  
Dependent Manner ◽  
Standard Drug ◽  
Lead Compounds ◽  
Lysis Activity ◽  
Test Models

Abstract Background Brassica nigra is a plant of Brassicaceae family, which possesses numerous medicinal values. Our present study is intended to assess the potential in vitro thrombolytic, anthelminthic, cytotoxic and in vivo anxiolytic properties of MCE of B. nigra flowers. MCE was fractioned for separating the compound on the basis of polarity by using chloroform, n-hexane and ethyl acetate solvent. Thrombolytic and anthelminthic activities were explained by collecting human erythrocytes and earthworms as test models, respectively. Anxiolytic activity was evaluated by elevated plus maze and hole board models while cytotoxic test was conducted through brine shrimp lethality bioassay. Results MCE revealed the presence of alkaloids, flavonoids, tannin, diterpenes, glycosides, carbohydrates, phenols, fixed oils and fat. In case of thrombolytic test, the MCE, CSF, ASF and n-HSF had produced maximum clot lysis activity at 5 and 10 mg/ml dose conditions. Two different concentrations (10 and 20 mg/ml) of MCE and its fractions showed significant (p < 0.05) anthelminthic activities in a dose-dependent manner. Significant anxiolytic activity was observed for all fractions which was comparable to the standard drug diazepam (p < 0.05). Again, the cytotoxic screening also presented good potentials for all fractions. Conclusion From the findings of present study, we can conclude that MCE of B. nigra flowers and its fraction possess significant anxiolytic, anthelmintic, anticancer and thrombolytic properties which may be a good candidate for treating these diseases through the determination of bio-active lead compounds.

Formation, translocation and resolution of Holliday junctions during homologous genetic recombination

1995 ◽  
Vol 347 (1319) ◽  
pp. 21-25 ◽  
Keyword(s):  
Molecular Mechanisms ◽  
Genetic Recombination ◽  
Holliday Junctions ◽  
The Novel ◽  
E Coli ◽  
The Past ◽  
Endonucleolytic Cleavage ◽  
Insight Into ◽  
Made In

Over the past three or four years, great strides have been made in our understanding of the proteins involved in recombination and the mechanisms by which recombinant molecules are formed. This review summarizes our current understanding of the process by focusing on recent studies of proteins involved in the later steps of recombination in bacteria. In particular, biochemical investigation of the in vitro properties of the E. coli RuvA, RuvB and RuvC proteins have provided our first insight into the novel molecular mechanisms by which Holliday junctions are moved along DNA and then resolved by endonucleolytic cleavage.

scholarly journals SYNTHESIS OF BENTONITE NANOCLAY AND INCORPORATION OF CASSIA FISTULA LEAF EXTRACT TO FORM ORGANOBENTONITE: CHARACTERIZATION AND ITS BIOMEDICAL APPLICATIONS

2018 ◽  
Vol 11 (9) ◽  
pp. 381
Author(s):  
Anand Raj Lfa ◽  
Jeslin J
Keyword(s):  
Biomedical Applications ◽  
Leaf Extract ◽  
Spherical Shape ◽  
Xrd Analysis ◽  
Antioxidant Property ◽  
Clot Lysis ◽  
Cassia Fistula ◽  
Thrombolytic Activity ◽  
Lysis Activity

Objective: In this work, methanolic leaf extract from Cassia fistula (known as aragvadha) was incorporated into bentonite nanoclay to form organobentonite. This organobentonite of nanosize was further used for its effective biomedical applications since medicinal clay finds its own advantage over decades.Methods: The bentonite nanoclay was produced by energetic stirring followed by centrifugation and was characterized using scanning electron microscopy (SEM), X-ray diffraction (XRD), and Fourier transform infrared (FTIR). The organobentonite was produced using freeze and thaw method. Antioxidant property was studied using Molyneux method, and thrombolytic activity was analyzed using in vitro clot lysis method.Results: The nanosize of bentonite nanoclay between 57 and 82 nm with irregular to spherical shape was confirmed using SEM analysis. The sharp diffraction peak in XRD analysis shows the crystalline nature of bentonite nanoclay, and FTIR results revealed the successful incorporation of the methanolic extract within the bentonite nanoclay. The organobentonite exhibits 84.5% antioxidant property as well as 31% clot lysis activity when compared to the extract and the bentonite nanoclay.Conclusion: Thus, the non-toxic and inexpensive bentonite nanoclay have a high aspect ratio with multifarious applications in medicine, food, cosmetics, and health products. Through this study, the bentonite nanoclay modified using plant alkaloid (organobentonite) is found to possess good biomedical property.

scholarly journals Novel Fibrinolytic Protease Producing Streptomyces radiopugnans VITSD8 from Marine Sponges

Marine Drugs ◽  
2019 ◽  
Vol 17 (3) ◽  
pp. 164 ◽  
Author(s):  
Dhamodharan D ◽  
Jemimah S ◽  
Merlyn S ◽  
Subathra C
Keyword(s):  
Tamil Nadu ◽  
Specific Activity ◽  
Blood Clot ◽  
Exchange Chromatography ◽  
Marine Sponges ◽  
Protease Production ◽  
Clot Lysis ◽  
Carbon And Nitrogen ◽  
Lysis Activity

Fibrinolytic enzymes have received more attention due to their medicinal potential for thrombolytic diseases. The aim of this study is to characterize the in vitro fibrinolytic nature of purified protease producing Streptomyces radiopugnans VITSD8 from marine brown tube sponges Agelas conifera. Three varieties of sponge were collected from the Rameshwaram Sea coast, Tamil Nadu, India. The fibrinolytic activity of Streptomyces sp. was screened and determined by casein plasminogen plate and fibrin plate methods respectively. The crude caseinolytic protease was purified using ammonium sulfate fractionation, affinity and ion-exchange chromatography. Based on the morphological, biochemical, and molecular characterization, the isolate VITSD8 was confirmed as Streptomyces radiopugnans. Maltose and peptone were found to be the best carbon and nitrogen sources for the production of fibrinolytic protease. The carbon and nitrogen source peptone showed (781 U/mL) enzyme activity. The optimum pH and temperature for fibrinolytic protease production was found to be 7.0 and 33 °C respectively. The purified enzyme showed a maximum specific activity of 3891 U. The blood clot lysis activity was compared with the standard, and it was concluded that a minimum of 0.18 U (10 µL) of purified protease was required to dissolve the blood clot. This is the first report which exploits the fibrinolytic protease activity of Streptomyces radiopugnans VITSD8 extracted from a marine sponge. Hence the investigation suggests a potential benefit of purified fibrinolytic protease which will serve as an excellent clot buster alternative.

ANTITHROMBOTIC EFFECTS OF TISSUE-TYPE PLASMINOGEN ACTIVATOR AT PHYSIOLOGICAL BLOOD LEVELS

1987 ◽  
Author(s):  
W Witt ◽  
B Baldus ◽  
P Donner
Keyword(s):  
Plasminogen Activator ◽  
Plasma Levels ◽  
Dose Range ◽  
Tissue Type ◽  
Clot Lysis ◽  
Blood Levels ◽  
Single Chain ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator

Effective thrombolysis in human patients and experimental animals by tissue-type plasminogen activator (t-PA) usually requires t-PA plasma levels in the microgram range. Compared to that physiological plasma levels of t-PA are about 100 - 1000 times lower. To investigate the effects of t-PA at physiological blood levels rat studies were performed in vitro and in vivo employing highly purified recombinant single-chain t-PA (sct-PA: 500,000 IU/mg).t-PA activity in rat whole blood as assessed by dilute blood clot-lysis time (DBC-LT) was increased by addition of sct-PA as low as 3 ng/ml (20 % decrease in DBC-LT). Injection of brady-kinin 10, 100 and 1000 μg/kg i.v. shortened DBC-LT to 54, 23, and 10 % of controls corresponding to the effect of about 10, 30, and 100 ng/ml sct-PA added in vitro. Infusion of sct-PA 15 - 450 μg/kg/h i.v. shortened DBC-LT ex vivo dose-dependently by 20 - 90 % at steady state levels (n = 5). In the same dose range sct-PA inhibited thrombus formation along a silk thread introduced into an arteriovenous shunt in anaesthetized rats. The reduction in thrombus dry weight was dose-dependent amounting to 33 - 67 % of preapplication values (n = 5 - 8) at 15 - 450 μg/kg/h i.v. sct-PA. Already 50 μg/kg/h sct-PA corresponding to a sct-PA activity of about 15 ng/ml displayed a significant (a = 0.05) effect in this model.The results of this study suggest that t-PA present at physiological resting or activation (bradykinin) levels during acute clot formation may have potent antithrombotic efficacy. This study provides further evidence for the importance of a balance coagulation-fibrinolysis which can be influenced on both sides towards thrombophilia as well as to achieve antithrombotic therapy, e.g. by elevating plasma fibrinolytic activity with low-dose t-PA treatment or with drugs which stimulate the endogenous fibrinolytic potential.

scholarly journals Targeting the FMS-like Tyrosin Kinase 3 with the Unicar System: Preclinical Comparison of Murine and Humanized Single-Chain Variable Fragment-Based Targeting Modules

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5614-5614
Author(s):  
Frederick Fasslrinner ◽  
Claudia Arndt ◽  
Anja Feldmann ◽  
Stefanie Koristka ◽  
Liliana Raquel Loureiro ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
The Novel ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
Cell Therapies ◽  
Myeloid Malignancies ◽  
Car T Cell ◽  
Car T

Clinical translation of chimeric antigen receptor (CAR) T cell therapy in myeloid malignancies is progressing slowly compared to its success in treatment of B cell malignancies. Clinical experiences with CAR T cell therapies against the currently investigated tumor-associated antigens (TAA) (e.g. CD33, CD123 and FMS-like tyrosine kinase 3 (FLT3)) were discouraging and severe side effects occurred (cytokine release syndrome, neurotoxicity and myeloid aplasia) (Hoffmann et al. Journal of Clinical Medicine 2019). Probably targeting a single TAA is insufficient to treat high risk myeloid malignancies with CAR T cell therapies. Therefore, combined targeting of two or even more TAAs seems to be a promising approach. In order to implement such a multiple tumor targeting strategy, we developed a modular CAR T cell system termed UniCAR. The system consists of a universal CAR (UniCAR) directed against the La peptide epitope E5B9 combined with single-chain variable fragment (scFv) -based target modules (TM). In contrast to conventional CARs, anti-tumor activity of UniCAR T cells is only turned on in the presence of the TMs. Thus, this approach will allow UniCAR T cell control due to the short half-life of the TM and therefore has a favorable safety profile. Furthermore, different TMs against several TAAs can be administered both sequentially or in parallel to increase the anti-tumor efficacy or face disease relapse due to antigen escape mechanisms. In the field of myeloid malignancies our group developed retargeting strategies against the TAAs CD33 and CD123 (Cartellieri et al. Blood Cancer Journal 2016). In addition, we have developed a new TM for the UniCAR system that is directed against the TAA FLT3. FLT3 is highly expressed on acute myeloid leukemia (AML) cells and also present on CD123low AML samples (Riccioni et al. British Journal of Haematology 2011). The novel FLT3 TM was constructed by fusion of the variable domain of the heavy and the light chain of the murine anti-FLT3 monoclonal antibody (4G8) to the E5B9 UniCAR epitope. In light of a potential clinical application, we in parallel generated a humanized FLT3 TM to further decrease its immunogenicity. Both FLT3 TMs were tested in vitro against different AML cell lines, by using flow cytometry based killing assays as described elsewhere (Fasslrinner et al. British Journal of Haematology 2019). The functionality of the FLT3 TMs in vitro was highly effective. Both FLT3 TMs were able to redirect UniCAR T cells for AML cell lysis already in the picomolar range and were moreover comparable effective than the previously developed CD123 TM. Thus, humanization of the FLT3 TM did not lead to a decrease in anti-tumor efficacy. In summary, we could show that both the novel murine FLT3 TM and the humanized counterpart redirected UniCAR T cells and induced highly effective elimination of AML cells in vitro. Thus, the flexible application of the FLT3-based UniCAR system seems to be a promising tool for cell-based AML therapy alone or even in combination with other AML-specific TMs (e.g. CD33, CD123). Disclosures Koristka: Intellia Therapeutics: Employment. Jung:Synimmune: Other: shareholder interest. Bachmann:GEMoaB Monoclonals: Equity Ownership, Patents & Royalties.

Effect of Standard Heparin and a Low Molecular Weight Heparin on Thrombolytic and Fibrinolytic Activity of Single-Chain Urokinase Plasminogen Activator In Vitro

1989 ◽  
Vol 62 (03) ◽  
pp. 923-926 ◽  
Author(s):  
D M Lourenço ◽  
A M Dosne ◽  
A Kher ◽  
M Samama
Keyword(s):  
Molecular Weight ◽  
Low Molecular Weight Heparin ◽  
Clot Lysis ◽  
Low Molecular Weight ◽  
Single Chain ◽  
Plasma Clot ◽  
Thrombolytic Activity ◽  
Significant Difference ◽  
Plasma Factor

SummaryThe effect of unfractioned heparin (UH) and low molecular weight heparin (LMWH) (Kabi 2165 - Fragmin®) on in vitro scu-PA thrombolytic and fibrinogenolytic activity was investigated. Thrombolytic activity was evaluated by following lysis of radiolabeled plasma clot immersed in plasma in presence of scu-PA alone or with either form of heparin. A 200 IU/ml scu-PA concentration produced clot lysis within 7 hr. UH or LMWH led to a slightly faster clot lysis which was statistically significant only at the 2nd and 3rd hour. No significant difference could be evidenced between UH and LMWH effect. During clot lysis, plasmin, generated within the clot led to a gradual transformation of scu-PA to tcu-PA, specially after a 4-hr incubation. Appearance of tcu-PA activity in the plasma surrounding the clot was significantly inhibited by either form of heparin. This finding contrasts with results observed in purified systems and suggests the presence of heparin-dependent plasma factor(s) inhibiting tcu-PA formation or its activity. Possible candidates might be anti-thrombin III and PAI-3.No fibrinogen breakdown was observed when plasma was incubated for 7 hr at 37° C in presence of scu-PA alone (200 IU/ ml) or with either form of heparin. However, in presence of a plasma clot, an important fibrinogen breakdown was observed during clot lysis reflecting the action of plasmin and/or tcu-PA generated within the clot, in the surrounding plasma. Fibrinogenolysis was less pronounced in the presence of both heparin preparations possibly as a consequence of the reduction in the tcu-PA level. These results underline the importance of plasma factors in the interaction of heparin with plasminogen activators such as scu-PA.

EVIDENCE FOR SYNERGISM BETWEEN TISSUE-TYPE PLASMINOGEN ACTIVATOR AND SINGLE CHAIN UROKINASE ON IN VITRO PLASMA CLOT LYSIS

1987 ◽  
Author(s):  
R S Rappaport ◽  
M R Blume ◽  
R L Vogel ◽  
M H Levner ◽  
P P Hung
Keyword(s):  
Plasminogen Activator ◽  
Tissue Type ◽  
Clot Lysis ◽  
Single Chain ◽  
Plasma Clot ◽  
Tissue Type Plasminogen Activator ◽  
Molar Ratios ◽  
Type Plasminogen Activator

There is mounting evidence from animal models and the clinic that combination thrombolytic therapy with tissue-type plasminogen activator (tPA) and single chain urokinase (scuPA) is synergistic. Yet, efforts to demonstrate synergism between these two plasminogen activators in vitro have met with discordant results. Collen et al (Thromb. Haemostasis, 56:35, 1986) reported an absence of synergism between these two agents on clot lysis in an in vitro plasma milieu when they were evaluated at molar ratios of 1:4 (tPA:scuPA and vice versa). Gurewich and Pannell (Thromb. Res., 44:217, 1986), however, reported a synergistic effect on fibrin-specific clot lysis in vitro when the agents were combined in concentrations exceeding molar ratios of 1:4 (tPA:scuPA). Here, we present evidence that synergism between tPA and scuPA may be demonstrated in vitro provided that the molar ratio of tPA to scuPA exceeds 1:4 and that the concentration of clot bound or unbound tPA is minimized. In order to achieve this experimental condition, the standard in vitro plasma clot lysis assay was modified. Human plasma clots were incubated first for a short time in plasma containing varying amounts of tPA. After incubation, the clots were washed thoroughly and reimmersed in plasma alone or in plasma containing varying amounts of scuPA or tPA. Under these conditions, lysis proceeded at a greater rate and to a greater extent when tPA clots were immersed in plasma containing an appropriate amount of scuPA than when they were immersed in plasma alone or in plasma containing appropriate amounts of tPA. Lysis of untreated clots or clots exposed first to scuPA and then to plasma containing varying amounts of scuPA proceeded far less efficiently with a characteristic lag. The enhanced lysis produced by tPA and scuPA obeyed the classical definition of synergy: the same biological effect can be obtained with two drugs together at algebraic fractional combinations of less than 1 (Berenbaum, M.C., Clin. Exp. Immunol., 28:1-18, 1977). Thus, conditions that more closely mimic the in vivo situation resulting from a bolus injection of tPA followed by infusion with scuPA, may provide a system for duplication of in vivo synergism in. vi tro and investigation of the mechanism thereof.

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