A Simplified Method for Preparation of Highly Purified Human Thrombin

Increasing use of synthetic substrates for potency estimation of biological activities within the coagulation field has raised a demand for a supply of highly purified thrombin. We have simplified a thrombin preparation procedure using clinical factor IX concentrates as starting material. Activation was performed using fresh brain thromboplastin prepared in a simplified way. A first ionic exchange chromatography step was used, which got rid of 80% of the impurities. The thrombin was further purified on SP Sephadex comparatively following two different pathways. In one case the whole purification procedure was performed at pH 6.5 which led to a thrombin with proposed high stability but which gives a relatively low-yield preparation. In another purification procedure the preparation was performed at pH 7.4 which gives a considerably better yield but possibly poorer stability upon storage. In both cases a final purification step on heparin gel was performed. The pH 6.5 thrombin could be separated into two fractions, one with a specific activity of 2500 iu/mg and another with a specific activity of 6000 iu/mg. A protein peak corresponding to the highly active thrombin was obtained also from the pH 7.4 thrombin, but that material had lost its biological activity. The different thrombin preparations have been carefully ampouled and sealed under nitrogen in order to faciliate further stability studies and comparative studies on bio-assays using clotting methods as well as synthetic substrate methods.

 

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