Relative Immunologic Potential of Bovine Prothrombin and Purified Thrombins

Topical bovine thrombin preparations have been used successfully in the vast majority of patients however, isolated reports of adverse events including deranged hemostasis resulting in severe or refractory bleeding and/or thrombosis exist. It has been assumed that a severe coagulopathy following exposure to topical bovine thrombin may be attributed to the impurities in bovine thrombin preparations such as factor Va. Obtained through the activation of bovine prothrombin by thromboplastin, crude thrombin preparation was further purified using ion-exchange chromatography and membrane filtration steps yielding thrombin 4A and 4B preparations which exhibit a higher specific activity and are devoid of some of the protein contaminants seen in earlier generation products. Consequently, a purer preparation of bovine thrombin might prove to be less immunogenic. The aims of this study are

1. to evaluate the immunogenic potential of bovine prothrombin;

2. to compare the purities of crude thrombin, 4A and 4B preparations by virtue of the detection of prothrombin related antigens.

Bovine prothrombin was administered intravenously to 3 individual 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 3 rabbits were purified to obtain the immunoglobulinG (IgG) using protein G affinity columns. Utilizing western blotting, the specificity of bovine prothrombin IgG collected on each time point (day 30, 50, 105, 137 and 165) was determined by using specific human and bovine coagulation factors such as prothrombin, thrombin, factor Xa, factor VIIa and factor Va fragment. In addition, serial diluted bovine crude thrombin, 4A and 4B preparations were also probed using the prothrombin IgG from day 30 and day 165 to explore prothrombin related-antigens in these samples. Under the experimental conditions used, neither cross-reactivity with human coagulation factors nor the recognition of bovine factor Va antigen was observed with the prothrombin IgG collected on any time point. The results of Western Blotting using the prothrombin IgG collected on day 30 and day 165 revealed that the lowest amount of crude thrombin, 4A and 4B preparations which prothrombin IgG could detect was 0.25U, 10U and 20U, respectively. The rank order of the number of detectable immunoreactive bands in each preparation by prothrombin IgG was: crude thrombin > thrombin 4A > thrombin 4B. Compared with the IgG collected on day 30, the 165 day’s IgG showed a littler stronger detecting ability for the prothrombin antigens in bovine thrombin samples. The results suggest that despite of the presence of trace amounts of bovine factor Va and other coagulation factors-related antigens in bovine prothrombin preparation, these contaminants failed to elicit the generation of relevant antibodies in rabbit. The results also indicate that among the three thrombin preparations tested, thrombin 4B preparation contains the least antigens which could be found in bovine prothrombin preparation. The suggested relationship between factor Va contaminants and the development of corresponding neutralizing human antibodies that could result in a coagulopathy in humans needs further study.