Prothrombin-Sepharose-Purified Factor V and Its Role in Prothrombin Conversion

Bovine Factor V isolated by the method of Esnouf and Jobin (1967) has been further purified by affiniy chromotgraphy through prothrombin-sepharose. Factor V bound quantitative to the prothrombin-sepharose column. There was a 2-fold increase in the average specific activity (260.000 units/mg protein) of the Factor V recovered. Recovery of total Factor V activity and total protein was about 95% and 90% respectively.

Cross-Reactivity of Rabbit Anti-Bovine Prothrombin/Thrombin IgGs With Bovine Factor V/Va-Related Antigens

The purpose of this study was to determine whether rabbit anti-bovine prothrombin/thrombin immunoglobulin Gs (IgGs) would cross-react with bovine factor V/Va-related antigens. Bovine prothrombin, crude thrombin, as well as 2 purified versions of thrombin, that is, thrombin 4A (the previous version of Thrombin-JMI marketed prior to 2008) and 4B (the currently marketed version of Thrombin-JMI), were administrated 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 IgGs using protein G affinity columns. By probing bovine factor V/Va samples, the possible cross-reactivity of each IgG collected at different time points (from day 30 to day 165) was explored using Western blotting techniques. The results indicated that rabbit anti-bovine prothrombin and crude thrombin IgGs could cross-react strongly with bovine factor V/Va in an immunization time-dependent manner. However, antibodies generated in thrombin 4A-treated rabbits presented much weaker cross-reactivity with bovine factor V/Va. Furthermore, no cross-reactivity with bovine factor V/Va-related antigens was observed when the anti-bovine thrombin 4B IgG collected at any time point was used. The results suggest that thrombin 4B preparation contains the least bovine factor V/Va contaminants among the bovine prothrombin/thrombin preparations studied and the amount of bovine factor V/Va contaminants in bovine thrombin 4B is too small to elicit the generation of antibodies against bovine factor V/Va in rabbits.

Differences in prethrombin-1 activation with human or bovine factor Va can be attributed to the heavy chain

SummaryHuman and bovine factor Va (FVa) function similarly in the activation of prothrombin but differently in the activation of prethrombin-1 (Pre-1). Pre-1 activation with human FVa proceeds at about 22 percent of the rate with bovine FVa. The dependencies of initial rates on the FVa and Pre-1 concentrations indicate that the differential activity is expressed in kcat differences, rather than differences in the assembly of prothrombinase or the Km value of the substrate. The heavy and light chains of both species of FVa were separated and interspecies hybrids were constructed in the presence of Ca++. Studies of the activation of Pre-1 with these hybrids indicate that the species difference can be attributed specifically to the heavy chain of FVa. Analyses of the reactions by SDS-PAGE indicated that cleavage at Arg271 occurs at about the same rate with both species of FVa, but cleavage at Arg320 with human FVa is specifically retarded. A major difference in primary structure between the human and bovine FVa heavy chains comprises 10 residues at COOH-terminus, adjacent to the negatively charged hirudin-like DYDYQ sequence. These residues have pI values of 12.5 and 4.26 in human and bovine FVa, respectively. The lower value would complement the negatively charged DYDYQ sequence but the higher value would counteract it. Thus, we suggest that the differences in the COOH-terminus of the heavy chain are responsible for the differences in Pre-1 activation, and that it specifically influences cleavage at Arg320 in Pre-1.

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 to evaluate the immunogenic potential of bovine prothrombin; 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.

Further Removal of Factor V Related Antigen From Bovine Thrombin by Utilizing a Membrane-Filtration Step

Topical bovine thrombin is commonly used during surgery to maintain hemostasis and is rarely associated with abnormalities in hemostasis, including coagulopathies and bleeding. Coagulopathies may be related to the formation of cross-reactive antibodies to bovine factor V. Effectiveness of a new filtration step to remove factor V/Va from bovine thrombin was evaluated. A highly sensitive and specific Western blot capable of detecting minute amounts of factor V/Va and/or its fragments was developed. Samples were evaluated for bovine factor V related antigens using the Western blot method and a competitive enzyme-linked immunosorbent assay. Factor Va light chain fragment levels were detectable in crude thrombin and chromatographically purified thrombin but not in chromatographically purified and virally filtered preparations. Therefore, inclusion of the viral-filtration step during purification of thrombin is effective in reducing factor V or its fragments to undetectable levels, thus enhancing product purity.