Spectrophotometric analysis of thrombolytic activity: SATA assay

Abstract

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).

Streptokinase Loading Fabrication Magnetic Nanoparticle Supported With Tannic Acid as a Modified Thrombolytic Agent

“…The lowest value of entrapped efficiency was 25% with (0.05 mg/ml) concentration of NpFeSiPrTa as in Figure (11). Loading capacity study was given highest value 51% with (0.1mg/ml) concentration of NpFeSiPrTa and this value decreasing with increasing or decreasing the concentration from 0.1 mg/ml as show in Figure (12).…”

“…Incubation at 37°C for 120min, D-dimer was measured every 60 minutes(for two times) as indicator of thrombolytic agent activity 12 . Then the fluidis removed completely, weight of blood clot in these samples weremeasured using a sensitive balance depending on the equation (3).…”

Clot‐targeted magnetic hyperthermia permeabilizes blood clots to make them more susceptible to thrombolysis

“…Additionally, absorbance readings of plasma samples were used to assess the release of red blood cells from the clots as well as hemoglobin from damaged red blood cells using a previously described method. [30] Plasma samples from clots treated with both tPA and MH exhibited significantly higher values of absorbance at 415 nm (275 ± 33% of control) than those treated with tPA alone (213 ± 23% of control; n = 15; P < 0.05; Figure 3B). These data demonstrate that there was greater release of red blood cells and hemoglobin from the clots into the surrounding plasma.…”

Development and Testing of Thrombolytics in Stroke

“…[141][142][143][164][165][166][167][168][169][170] Thrombolytics-induced lysis can be roughly monitored visually to report the complete clot lysis time 141,142,[162][163][164] or more accurately, by the change in clot-related light scattering. 141,143,169,171 Other options include measuring (1) the release of red blood cells (RBCs) from whole blood clots, 165,166,172,173 (2) changes in the clot mass, 167,168 (3) the viscoelastic properties using thromboelastography, 174,175 or (4) fluorescence change upon dissolution of labeled clots. 176,177 Unlike the chromogenic/fluorogenic substrate assay and the fibrin plate assay, these assays use macroscopic clots, thereby better mimicking in vivo biology.…”