Decreased Immunogenicity of Purified Topical Bovine Thrombin Preparations.

Abstract 4209

It has been reported that severe coagulopathy following exposure to topical bovine thrombin may be attributed to the impurities in bovine thrombin preparations. Prepared through the activation of bovine prothrombin by thromboplastin, a crude thrombin preparation was further purified using ion-exchange chromatography and membrane filtration steps to yield thrombin 4A and 4B preparations. The aim of this study was to compare the relative purity of these bovine thrombin preparations by virtue of the detection of bovine prothrombin-related antigens. Bovine prothrombin was administered to three individual rabbits on days 0, 21, 42, 91, 123 and 151 using standard immunologic method. Blood was drawn on 30, 50, 105, 137 and 165 days and the pooled antisera from three rabbits were purified to obtain IgG using protein G affinity columns. Utilizing western blotting, serial diluted bovine crude thrombin, 4A and 4B preparations were probed using the prothrombin IgGs from each time point to explore prothrombin-related antigens in these preparations. Compared with the IgG collected on day 30, the prothrombin IgGs collected from day 50 to 165 showed a time-dependent increased detecting ability for the prothrombin antigens in the bovine thrombin preparations. The lowest amount of crude thrombin, 4A and 4B preparations that prothrombin IgG could detect was 0.125U, 10U and 20U, respectively. The rank order of the number of immunoreactive bands in three bovine thrombin preparations probed by the prothrombin IgGs collected from any given time point was consistant and reproducible: crude thrombin > thrombin 4A > thrombin 4B. The results indicate that thrombin 4B represents the most purified thrombin preparation and relatively non-immunogenic form among three thrombin preparations studied.