Reduced Immunogenic Potential of Membrane Filtered Bovine Thrombin Preparations for Hemostatic Application

Abstract

on the reduction of trace contaminants such as the bovine factor Va/Va light chain fragments to an undetectable level in thrombin 4B. In order to compare the relative immunogenic potential, bovine crude thrombin and its purified versions, 4A and 4B preparations, were administered intravenously to individual groups of rabbits on day 0 and day 21 using standard immunologic protocols. Antiserum was drawn from each rabbit on day 30 and the pooled antisera were purified to obtain the IgGs using protein G affinity columns. The antibody profile for each of these IgGs was determined by using specific human and bovine coagulation factors such as prothrombin, thrombin, factor Xa, factor VIIa, and factor Va fragment. In addition, bovine crude thrombin as well as 4A and 4B preparations were also profiled in cross immunoblotting studies. Western blotting results suggest thrombin 4B IgG has the most selective binding with thrombin antigen, and thrombin 4B has the least immunogenic potential among the 3 thrombin preparations tested. Furthermore, neither cross-reactivity A lthough topical bovine thrombin preparations have been widely used for hemostatic purposes in clinical practice, 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 coagulopathy following exposure to topical thrombin may be attributed to the impurities in bovine thrombin preparations and 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. A previous study has reported 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.

Comparison of Immunogenic Potentials of Bovine Thrombin Preparations

“…The data from our initial study had shown that preimmune IgG obtained from the rabbits did not demonstrate any recognition of epitopes in bovine crude thrombin, thrombin 4A, thrombin 4B, and factor Va light chain. 27 The results from this study clearly demonstrated that factor V/Va contaminants in bovine thrombin preparations do not appear to generate an immune response in a naive rabbit model even after repeated exposure. Therefore, this observation points to a possibility that the reported presence of anti-bovine factor Va antibodies in some patients exposed to bovine crude thrombin may be caused by an immune response to the preexisting bovine immunogens in these patients.…”

“…27 This finding in rabbits is inconsistent with the data obtained from human patients following exposure to topical bovine thrombin during surgery. [4][5][6][7][8] It was thought that the development of anti-bovine factor Va antibodies might have slower onset as compared to antithrombin IgGs in rabbit and therefore, a longer immunization time may be needed to generate anti-bovine factor Va antibodies in rabbits than in humans.…”

“…Lastly, the specificity of each IgG collected on day 165 was also determined using specific human and bovine coagulation factors. 27 Serial amounts of a bovine factor Va light chain standard were probed using the IgGs from day 165 to investigate whether there is any anti-bovine factor Va antibody in the anti-bovine thrombin IgGs. As shown in Figure 5, no immunoreactive band was detected from bovine factor Va light chain standard by any of IgGs collected from day 165.…”

“…All other human and bovine coagulation factors used in this study were obtained commercially from Enzyme Research Laboratories (South Bend, IN) and American Diagnostica (Stamford, CN) and diluted with saline accordingly before loading. 27 Molecular weight standards ranging from 10.0 to 250 kd were purchased from Pierce (Rockford, IL). These standards were used to crossreference the molecular weight of various bands in the electrophoresis and Western blotting methods.…”

“…26 To determine whether the improved purity of bovine thrombin can reduce its overall immunogenic potential and lower the risk of development of factor V antibodies following exposure, we performed a pilot experiment to compare the acute immunogenic potential of crude thrombin and its purified forms, thrombin 4A and thrombin 4B. 27 Our preliminary data obtained using the anti-bovine thrombin immunoglobulin Gs (IgGs) collected on day 30 suggested that the reported purification process resulted in the removal of most of the antigens found in crude thrombin, and that none of the preparations tested generated any detectable anti-bovine factor Va antibodies in rabbits. 27 In this study, we repeated the Western blotting experiments using anti-bovine thrombin IgGs collected on day 165.…”