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 (?max=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 ?max=405nm. Calculated R2 values were greater than 0.9; with LOD of 0.90 آµg/mL of tPA (436.50IU) and LOQ of 2.99 آµg/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 آµg/mL of tPA (485 IU/mL of thrombolytic dose).