Modeling of the fibrin agarose plate assay and its application for thrombolytic analysis

Wang, MingQi; Yang, W P; Wu, Qiang; Gu, Hongchen

doi:10.1007/s11434-012-5297-6

Cited by 4 publications

(4 citation statements)

References 18 publications

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“…The measurement of thrombolytic activity by fibrin agarose plates have already been introduced however, it demands expensive materials and is not an exact quantitative analysis. 19 The concentration of fibrin degraded particles released after clot lysis has been measured by D-dimer assay which is highly accurate, but it is an expensive approach. 16 The occluded tube model, which simulates the obstruction of a vessel by a clot, was shown to be unable of discriminating the lower doses of thrombolytic agents while requiring expensive materials.…”

Section: Discussionmentioning

confidence: 99%

Spectrophotometric analysis of thrombolytic activity: SATA assay

Zamanlu¹,

Eskandani²,

Mohammadian³

et al. 2017

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Introduction: Measurement of thrombolytic activity is crucial for research and development of novel thrombolytics. It is a key factor in the assessment of the effectiveness of conventionally used thrombolytic therapies in the clinic. Previous methods used for the assessment of thrombolytic activity are often associated with some drawbacks such as being costly, time-consuming, complex with low accuracy. Here, we introduce a simple, economic, relatively accurate and fast method of spectrophotometric analysis of thrombolytic activity (SATA) assay, standardized by tissue plasminogen activator (tPA), which can quantitatively measure in vitro thrombolytic activity. Methods: Blood clots were formed, uniformly, by mixing citrated whole blood with partial thromboplastin time (PTT) reagent, together with calcium chloride. Then, designated concentrations of tPA were added to the samples, and the released red blood cells from each clot were quantified using spectrophotometry (λmax=405nm) as an indicator of thrombolytic activity. The accuracy of the method was tested by assessment of dose-responsibility against R2 value obtained by linear equation and measurement of the limit of detection (LOD) and limit of quantification (LOQ). The SATA assay was validated in comparison with some currently used techniques. Results: A linear relationship was obtained between different concentrations of tPA versus the spectrophotometric absorbance of the related dilutions of lysed clots, at λmax=405nm. Calculated R2 values were greater than 0.9; with LOD of 0.90 µg/mL of tPA (436.50IU) and LOQ of 2.99 µg/mL of tPA (1450.15IU). Conclusion: Conclusively, the SATA assay is a very simple quantitative method with repeatable and reproducible results for estimating the potency of an unknown thrombolytic agent, and calculating the activity as delicate as 1 µg/mL of tPA (485 IU/mL of thrombolytic dose).

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Section: Discussionmentioning

confidence: 99%

Spectrophotometric analysis of thrombolytic activity: SATA assay

Zamanlu¹,

Eskandani²,

Mohammadian³

et al. 2017

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“…After polymerization, 3 mm holes were punctured, filled with 10 μl of purified AprE127 or urokinase (Sigma U4010) with final concentrations of 5.30, 0.60, 0.07, and 0.01 mg/ml, and incubated at 37°C for 2 h. Images of the halos in the plate were taken at different times to measure the diameter of hydrolysis. The thrombolytic activity was calculated using the equation

where R is the radius of the halo, and t is the diffusion time [ 30 ]. The plasminogen activation assay was performed by reacting 71 μg purified S127e with 2.5 μg plasminogen (Sigma P7999) in fibrin plates and comparing with the same amount of urokinase.…”

Section: Methodsmentioning

confidence: 99%

Purification and Characterization of a Thrombolytic Enzyme Produced by a New Strain ofBacillus subtil

Frías

Toubarro

Fraga

et al. 2021

J. Microbiol. Biotechnol.

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Fibrinolytic enzymes with a direct mechanism of action and safer properties are currently requested for thrombolytic therapy. This paper reports on a new enzyme capable of degrading blood clots directly without impairing blood coagulation. This enzyme is also non-cytotoxic and constitutes an alternative to other thrombolytic enzymes known to cause undesired side effects. Twenty-four Bacillus isolates were screened for production of fibrinolytic enzymes using a fibrin agar plate. Based on produced activity, isolate S127e was selected and identified as B. subtilis using the 16S rDNA gene sequence. This strain is of biotechnological interest for producing high fibrinolytic yield and consequently has potential in the industrial field. The purified fibrinolytic enzyme has a molecular mass of 27.3 kDa, a predicted pI of 6.6, and a maximal affinity for Ala-Ala-Pro-Phe. This enzyme was almost completely inhibited by chymostatin with optimal activity at 48 o C and pH 7. Specific subtilisin features were found in the gene sequence, indicating that this enzyme belongs to the BPN group of the S8 subtilisin family and was assigned as AprE127. This subtilisin increased thromboplastin time by 3.7% (37.6 to 39 s) and prothrombin time by 3.2% (12.6 to 13 s), both within normal ranges. In a whole blood euglobulin assay, this enzyme did not impair coagulation but reduced lysis time significantly. Moreover, in an in vitro assay, AprE127 completely dissolved a thrombus of about 1 cc within 50 min and, in vivo, reduced a thrombus prompted in a rat tail by 11.4% in 24 h compared to non-treated animals.

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“…The processed earthworm powders were used as assaying fibrinolytic activity was determined by following the protocols of the fibrin plate method [2]. Briefly, the materials were prepared: in a 3% fibrinogen solution, a thrombin solution (1,000 U of thrombin in 1000 μl of PBS), and a plasmin solution (100 μg of plasmin in 1 ml of PBS), and then in a 10% sample solution (50 mg of sample in 0.5 ml of distilled water).…”

Section: Experimental Design Materials and Methodsmentioning

confidence: 99%

Data on the potent fibrinolytic effects of the Lumbricus rubellus earthworm and the Perinereis linea lugworm

et al. 2019

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There is technology available for anti-thrombus with earthworms, but the procedure is complex and extracts protein with inferior purity. In order to develop a simplified process with a stronger purity of protease, we investigated the Lumbricus rubellus earthworm and Perinereis linea lugworm. We purified water extracts cut off at 10 kDa of molecular weight using ultrafiltration because proteins are large biomolecules, or macromolecules, consisting of one or more long chains of amino acid residues. We purified EW1 (raw earthworm extract), EW2 (molecular weight (m.w) > 10 kDa of earthworm extract), and EW3 (m.w < 10 kDa) from the Lumbricus rubellus earthworm. Likewise, we purified LW1 (wild lugworm extract), LW2 (m.w > 10 kDa), and LW3 (m.w < 10 kDa) from the Perinereis linea lugworm. Using a fibrin assay, we found that fibrinolytic activity of the specimens had a rank order of clear zone diameter: EW2 > EW1 > EW3 > LW2 > LW1 > LW3. In particular, EW2 and LW2 showed a potent fibrinolytic effect in two different worm specimens. The protein content of each sample was detected as 2.34 (EW1), 3.03 (EW2), 2.80 (LW1), 3.71 (LW2) mg/ml respectively, and their molecular weights were measured using SDS-PAGE. The samples contained the following amounts of total fatty acids: EW1, 3.61%; EW2, 0.48%; LW1, 4.96%; and LW2, 0.23%.We developed a process to increase the thrombolytic effect with a higher purity protein. The study results demonstrate this procedure and provide basic data for developing an anti-thrombolytic agent.

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Modeling of the fibrin agarose plate assay and its application for thrombolytic analysis

Cited by 4 publications

References 18 publications

Spectrophotometric analysis of thrombolytic activity: SATA assay

Spectrophotometric analysis of thrombolytic activity: SATA assay

Purification and Characterization of a Thrombolytic Enzyme Produced by a New Strain ofBacillus subtil

Data on the potent fibrinolytic effects of the Lumbricus rubellus earthworm and the Perinereis linea lugworm

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