Size exclusion chromatography (SEC-HPLC) as an alternative to study thrombin inhibition

Abstract

In vitro analysis of anticoagulant compounds with a potential use as antithrombotic drugs, has been traditionally performed using techniques like spectrophotometry, turbidimetry, as well as electrochemical and clinical assays. Although, these techniques have some disadvantages such as: the inability to measure the total biological activity of thrombin, interferences and, sometimes, the quantitative determination of the inhibition ratio is not accurate. In the present work, the conversion of fibrinogen to fibrin was monitored by molecular exclusion chromatography (SEC-HPLC) in three different reaction systems. An inhibition percentage of 43.19±2.02% was obtained using heparin as an anticoagulant, in addition to the determination of the percentage of heparin bonded to thrombin. This methodology has not been previously described and has high potential for the determination of anticoagulant capacity with higher precision, the determination of thrombin's total biological activity and the quantitative determination of the inhibition ratio.

Keywords: Fibrinogen; Molecular exclusion chromatography; Thrombin inhibition.