Reduced Immunogenic Potential of Membrane Filtered Bovine Thrombin Preparations for Hemostatic Application
Department of Pathology, Loyola University Medical Center, Maywood, Illinois 60153, USA.Clinical and Applied Thrombosis/Hemostasis (Impact Factor: 2.39). 11/2008; 15(1):32-40. DOI: 10.1177/1076029608322550
There is concern that exposure to bovine thrombin can result in the development of antibodies, usually against factor V/Va, which can lead to hemostatic abnormalities. It is thought that purer preparations of bovine thrombin might be less immunogenic. Utilizing newer methods including a membrane filtration step, bovine crude thrombin is further purified into thrombin 4A and 4B preparations which exhibit a higher specific activity and are devoid of some of the protein contaminants. Bovine crude thrombin and its purified versions were administered intravenously to individual groups of rabbits using standard immunologic protocols. Antiserum was drawn from each rabbit and the pooled antisera were purified to obtain the IgGs using protein G affinity columns. The results suggest that the reported purification process, including filtration, resulted in the removal of most of the antigens found in crude thrombin, and that none of these preparations generated any detectable antibodies against bovine factor V related antigens.
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ABSTRACT: Using a membrane filtration step, bovine crude thrombin was purified into thrombin 4A and 4B preparations. The purpose of this study was to determine whether the improved purity of a bovine thrombin preparation can reduce its overall immunogenic potential and lower the risk of development of factor V antibodies. Bovine crude thrombin and its purified versions, thrombin 4A and 4B, were administered to individual groups of rabbits on days 0, 21, 42, 91, 123, and 151 using standard immunologic methods. Blood was drawn from each rabbit on days 30, 50, 105, 137, and 165, and the pooled antisera from individual groups were purified to obtain the Ig Gs using protein G affinity columns. Using Western blotting, the specificity of each immunoglobulin G collected at the first time point (day 30) and last time point (day 165) was determined. The results of Western blotting using the Ig Gs collected on days 30 and 165 were consistent; both demonstrating that thrombin 4B has the least immunogenic potential among the 3 thrombin preparations tested. Compared with the immunoglobulin Gs collected on day 30, the Ig Gs from day 165 did not show obvious difference regarding their ability to detect antigens in bovine thrombin samples. Neither showed cross-reactivity with human coagulation factors nor the recognition of bovine factor Va antigens. These results suggest that despite the presence of a trace amount of bovine factor Va antigen in bovine thrombin preparations, these contaminants failed to elicit the generation of antibodies against factor Va light chain in rabbit.Clinical and Applied Thrombosis/Hemostasis 02/2009; 15(1):41-9. DOI:10.1177/1076029608327863 · 2.39 Impact Factor
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ABSTRACT: The aim of this study was to compare the relative purity of bovine crude thrombin and its purified forms, namely, thrombin 4A and thrombin 4B (the products of King Pharma, Middleton, Wisconsin) by virtue of the detection of bovine prothrombin-related antigens in these preparations. Bovine prothrombin was administered intravenously to 3 individual rabbits on days 0, 21, 42, 91, 123, and 151 using standard immunologic method. Blood was drawn from each rabbit on days 30, 50, 105, 137, and 165, and the pooled antisera from 3 rabbits were purified to isolate immunoglobulin G (IgG) using protein G affinity columns. Using Western blotting method, serially diluted bovine crude thrombin, thrombin 4A, and 4B preparations were probed using the prothrombin IgGs obtained from each time point to explore prothrombin-related antigens in these preparations. The results revealed that compared with the prothrombin IgG collected on day 30, the IgGs collected on days 50 to 165 showed a time-dependent increase in their ability to detect the prothrombin-related antigens in 3 bovine thrombin preparations studied. The lowest amount of crude thrombin, thrombin 4A, and 4B preparations that prothrombin IgG could detect was 0.125, 10, and 20 U, respectively. The rank order of the number of immunoreactive bands detectable in 3 bovine thrombin preparations probed by the prothrombin IgGs collected from any given time point was always the same: crude thrombin > thrombin 4A > thrombin 4B. The results indicate that thrombin 4B preparation contains the least amount of antigens detectable by prothrombin IgG, suggesting that relatively thrombin 4B represents the most purified thrombin preparation among the 3 thrombin preparations studied.Clinical and Applied Thrombosis/Hemostasis 10/2009; 15(6):681-8. DOI:10.1177/1076029609349497 · 2.39 Impact Factor
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