Bovine Thrombin Preparations Research Articles

Acquired factor V deficiency in a patient without evidence of a classical inhibitor

Acquired factor V deficiency in a patient without evidence of a classical inhibitor

The use of rituximab therapy in patients with acquired factor v inhibitors

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Cross Reactivity of Rabbit Anti-Recothrom (Human Recombinant Thrombin) Antibodies with Molecular Variants of Human Thrombin, and Various Bovine Thrombin Preparations

AbstractAbstract 4418Bovine thrombin preparations are widely used as topical hemostatic agents. While earlier bovine thrombin preparations were reported to produce immunologic responses upon repeated exposure, the refined bovine thrombin and recombinant human thrombin preparations (Zymogenetics, Seattle, WA, USA) are claimed to be less immunogenic. It was hypothesized that recombinant human thrombin despite its reduced immunogenic nature, upon repeated administration may result in the generation of antibodies and that may cross react with bovine and human thrombin. To test this hypothesis, groups of rabbits were challenged repeatedly with human recombinant thrombin over a 6 month period. Purified IgG preparations were obtained using protein G columns.To generate specific antisera in rabbits, the Recothrom (Human recombinant thrombin) conjugated with keyhole lymphocyte hemocyanin(KLH) as a carrier were administered intravenously to individual groups of rabbits (n=3) at a dosage of 100 μ g using standard immunologic methods. Preimmune blood and antiserum were collected from each rabbit in 0,30,50,90,120,150,180 days. In order to determine their cross reactivity with 3 preparations of bovine thrombin with differing purity, namely thrombin 4A, thrombin 4B, and bovine prothrombin were evaluated by immunoblotting techniques using the rabbit IgG fractions. In addition, to determine the relative cross reactivity of anti-Recothrom antibodies with human-α and γ- thrombin, one batch of each was evaluated by immunoblotting techniques using the rabbit IgG fractions. Gel electrophoresis and Western blotting were carried out. In brief, samples were subjected to electrophoresis through 4% to 20% gradient Tris-4-(2-hydro-xyethyl)-1-piperazineethanesulfonic acid (HEPES)-sodium dodecyl sulfate (SDS) polyacrylamide mini gels and then electrotransferred onto nitrocellulose membranes. The membranes were blocked with 5% milk and probed with anti-Recothrom IgG. The membranes were then washed and incubated with horseradish peroxidase conjugated donkey anti-rabbit IgG (1:20000 dilutions).After extensively washing; immunoreactive bands were detected with SuperSignal West Pico Chemiluminescent Substrate followed by film exposure for 1- to 5 minutes. The results from these studies suggested that anti-Recothrom antibodies cross react with human thrombins (α and γ), Evithrom brand of human thrombin, and bovine thrombin in a time dependent fashion. Although the Recothrom was not immunogenic in rabbit up to 90 days, antibodies harvested on day 150 cross-reacted with human α and γ thrombin as well as Evithrom. Visible cross reactivity, immunoblots around 36 KDa with human thrombins, bovine crude thrombin, and bovine thrombin 4A and 4B were observed with anti-Recothrom IgG collected from day 180. Bovine prothrombin did not exhibit any cross reactivity with anti-recothrombin antibodies at any time period. Thus, this data suggest that it is likely that antibodies against human recombinant thrombin may also cross-react with human thrombin and bovine thrombin. Therefore, the use of Recothrom may not be recommended in patients who have been previously exposed to human thrombin and bovine thrombin preparations. Disclosures:Fareed:King Pharmaceutical: Research Funding.

Immune Response Following Exposures to Topical Bovine Thrombin Does Not Impair Hemostasis

Immune Response Following Exposures to Topical Bovine Thrombin Does Not Impair Hemostasis

Le déficit acquis en facteur V : une pathologie hémorragique rare à manifestations cliniques variables

Acquired anti-factor V (FV) antibodies are uncommon and the majority of reported cases are idiopathic or associated with pregnancy, malignancy, autoimmune diseases or the use of bovine thrombin preparations. Clinical presentation is highly variable, ranging from asymptomatic to life-threatening bleeding and the optimal treatment is not clearly established. We here report two patients with different clinical presentations. The first patient presented with an acute severe rectal bleeding related to acquired FV deficiency and recurrent colon cancer while the second patient was asymptomatic with a FV inhibitor detected during a routine blood testing. We discuss treatment modalities that are not consensual.

Cross-Reactivity of Rabbit Anti-Bovine Thrombin IgGs With Human α-Thrombin and a Recombinant Version of Human Thrombin (Recothrom)

It has been reported that patients exposed to topical bovine thrombin preparations may develop antibodies against bovine thrombin, factr V or various other proteins found in these preparations. Such antibodies can cross-react with human endogenous coagulation proteins and may lead to alterations in the coagulation laboratory parameters, hypersensitivity reactions, and severe bleeding or thrombosis. The underlying mechanisms for the coagulopathy are not fully understood yet. To better understand the cross-reactivity of anti-bovine thrombin antibodies with human corresponding coagulation proteins, bovine crude thrombin, and its purified versions, thrombin 4A (the old version of Thrombin-JMI before year 2008) and thrombin 4B (the current version of Thrombin-JMI on market), were used to generate relevant anti-bovine thrombin immunoglobulin G (IgGs) in rabbits. Using Western blotting, the cross-reactivity of each IgG with human alpha-thrombin and a recombinant version of human thrombin (Recothrom) was investigated. The results indicated that no cross-reactivity with either human alpha-thrombin or Recothrom was observed with both anti-bovine crude thrombin IgGs and thrombin 4B IgGs. However, anti-bovine thrombin 4A IgGs showed apparent cross-reactivity with human alpha-thrombin and Recothrom in a protein amount-dependent manner. Furthermore, the results revealed that the cross-reactivity of anti-bovine thrombin 4A IgGs with human alpha-thrombin and Recothrom was immunization time-dependent. The minimum concentration of 4A IgG required to exhibit cross-reactivity with human alpha-thrombin and Recothrom varied considerably among individual rabbits. These results indicate that rabbit anti-bovine thrombin IgGs can cross-react with human alpha-thrombin and Recothrom, suggesting that human antibodies against bovine thrombin may also cross-react with human recombinant thrombin. Thus, the patients who were previously exposed to bovine thrombin may also develop antibodies which can cross-react with human recombinant thrombin.

Development of Factor V and Thrombin Inhibitors in Children Following Bovine Thrombin Exposure during Cardiac Surgery: A Report of Three Cases

Factor V and thrombin inhibitors may develop following exposure to bovine thrombin preparations. In patient populations where exposure to bovine thrombin is common, such as children undergoing cardiovascular surgery, the development of such inhibitors should be considered in the evaluation of prolonged prothrombin times. We present three cases of children developing factor V and thrombin inhibitors following repeated exposure during cardiac surgical procedures.

Cross‐Reactivity of Rabbit Anti‐Bovine Thrombin IgGs with a Human Plasma Derived Thrombin Preparation (Evithrom™)

Studies have shown that rabbit anti‐bovine thrombin IgGs cross‐react with human alpha‐thrombin and a recombinant version of human thrombin (Recothrom™). This study characterized the cross‐reactivity of anti‐bovine thrombin antibodies with a human plasma‐derived topical thrombin preparation (Evithrom™). Gel electrophoresis revealed that in addition to a human α‐thrombin protein band, Evithrom™ also contained a large amount of human albumin and a few faint high‐molecular weight bands which were absent in bovine thrombin, human α‐thrombin, human γ‐thrombin and Recothrom™. Western blotting results demonstrated no cross‐reactivity with Evithrom™ when anti‐bovine crude thrombin IgGs and thrombin 4B IgGs were used. However, Evithrom™ was recognized by rabbit anti‐bovine thrombin 4A IgG. Furthermore, the results revealed the cross‐reactivity of anti‐bovine thrombin 4A IgG with Evithrom™ was time‐dependent. These results indicate that besides human α‐thrombin and Recothrom™, rabbit anti‐bovine thrombin IgGs can also cross‐react with Evithrom™ suggesting that human antibodies against bovine thrombin may also cross‐react with human thrombin as well as human recombinant thrombin. Thus, the use of Evithrom™ and RecothromTM may not be recommended in patients who have been previously treated with/exposed to bovine thrombin preparations.

Decreased Immunogenicity of Purified Topical Bovine Thrombin Preparations.

Abstract 4209It 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. Disclosures:No relevant conflicts of interest to declare.

Cross-Reactivity of Rabbit Anti-Bovine Thrombin IgGs with Human α-Thrombin and a Recombinant Version of Human Thrombin (Recothrom™).

Abstract 4211It has been reported that patients exposed to topical bovine thrombin preparations may develop antibodies to bovine thrombin, factor V or various other proteins. In some cases these antibodies can cross-react with the corresponding human coagulation proteins. The present study was undertaken to determine if the antibodies induced by bovine thrombin antigens in rabbits could also cross-react with human a-thrombin and RecothromTM. Bovine crude thrombin and its purified versions, thrombin 4A and 4B, were administered to individual groups of rabbits on 0, 21, 42, 91, 123 and 151 days. Blood was drawn from each rabbit on days 30, 50, 105, 137 and 165, respectively. The antiserum from each rabbit and the pooled antisera from individual groups were purified to obtain the IgGs. Utilizing western blotting method, the possible cross-reactivity of each IgG with human thrombin and RecothromTM was explored using serial diluted human α-thrombin (20μg, 10μg, and 5μg) and RecothromTM (10U, 5U, and 2.5U). No cross-reactivity with either human α-thrombin or RecothromTM was observed with both anti-bovine crude thrombin IgGs and thrombin 4B IgGs collected on day 30 and day 165. However, anti-bovine thrombin 4A IgGs showed cross-reactivity with both human α-thrombin and RecothromTM. Cross-reactivity of anti-bovine thrombin 4A IgGs with human α-thrombin and RecothromTM was augmented with time. The minimum concentration of 4A IgG required to exhibit cross-reactivity with human α-thrombin and RecothromTM varied considerably among individual rabbits. These results demonstrate that rabbit anti-bovine thrombin 4A IgG could cross-react with both human a-thrombin and RecothromTM in a time/concentration-dependent manner. Disclosures:Paterson:King Pharmaceuticals: Employment.

Relative Purity of Different Bovine Thrombin Preparations

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.

Cross Reactivity of Human Recombinant thrombin (Recothrombin) to Antibovine Thrombin Antibodies. Clinical Implications

Bovine thrombin preparations are widely used topical hemostatic agents. Whereas older bovine thrombin preparations were reported to produce immunologic responses upon repeated exposure, the newly introduced recombinant human thrombin preparation (Zymogenetics, Seattle, WA, USA) and bovine thrombin preparations purified by membrane filtration are claimed to be less immunogenic. Despite their reduced immunogenic nature, repeated administration of purified bovine thrombin preparations can result in the generation of antibovine thrombin antibodies. In this study, groups of rabbits were challenged repeatedly with bovine thrombin over a 6 months period and purified IgG preparations were obtained using protein G columns. In order to determine their cross reactivity with human recombinant thrombin (Recothrombin), two batches of this agent were evaluated by immunoblotting techniques using the rabbit IgG fractions. Recothrombin preparations cross reacted with the rabbit antibovine thrombin IgG. The cross reactivity of Recothrombin with antibodies was time dependent and a progressive increase in the immunoreactivity was evident. These results demonstrate that human recombinant thrombin can cross react with antibodies to bovine thrombin. Thus, the use of Recothrombin may not be recommended in patients previously treated/exposed to bovine thrombin.

Recent developments in topical thrombins

Managing blood loss is part of the surgeon's responsibility during surgical procedures, and a variety of therapeutic strategies are available to help accomplish this. Topical haemostatic agents are among the agents used to control surgical bleeding and locally arrest blood flow. Bovine thrombin is a commonly used topical haemostatic agent; however, its use has been associated with potential risks, including well-documented cases of antibody-mediated coagulopathy. This coagulopathy develops as a consequence of antibody formation directed against bovine thrombin, other bovine coagulation proteins, and their human orthologs. The fact that a coagulopathy can result in association with the use of bovine plasma-derived thrombin preparations prompted the FDA to require pharmaceutical companies to place a black-box warning in their prescribing information for products containing bovine plasma-derived thrombin. Recently, human plasma-derived thrombin and recombinant human thrombin have been approved by the FDA with the expectation that they will be less immunogenic than the bovine-derived product. In clinical studies, purified human plasma-derived thrombin and recombinant thrombin have demonstrated equivalent efficacy and safety, with improved immunogenicity profiles compared with bovine-derived thrombin agents. Well-designed and adequately powered clinical trials should be conducted to indicate whether human thrombin products would improve the risk-benefit and cost-benefit profiles for surgeries complicated by excessive bleeding.

Relative Immunologic Potential of Bovine Prothrombin and Purified Thrombins

Abstract 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.

Favorable Immunologic Response to Recombinant Thrombin Application in Surgery among Subjects with Pre-Existing Antibodies to Bovine Thrombin.

Recombinant thrombin (rThrombin) is an active topical hemostat that is approved by the FDA as an aid to hemostasis. The safety, immunogenicity, and efficacy of rThrombin have been demonstrated in 1 Phase 1 study, 5 Phase 2 studies, and 1 randomized, double-blind Phase 3 study. No safety observations have been deemed clearly causally related to exposure to rThrombin; favorable immunologic response to rThrombin was observed in these trials. Treatment with thrombin derived from bovine sources has occasionally been associated with adverse events which appear to be related to the formation of antibodies against bovine thrombin and/or Factor V. A boxed product warning indicates that patients with antibodies to bovine thrombin preparations should not be re-exposed to these products. Secondary immune responses to bovine thrombin re-exposure may occur in patients with antibodies to bovine thrombin product. This Phase 3b open-label, single-group, multisite study was designed to evaluate the immunogenicity and safety of rThrombin among subjects at increased risk for having anti-bovine thrombin product antibodies as a result of prior surgery with a high likelihood of bovine thrombin exposure (N=209). Topical rThrombin was applied during a single spinal or vascular surgical procedure. Immunogenicity was evaluated by enzyme-linked immunosorbent assay at baseline and Day 29; safety measures were evaluated through Day 29. Subjects were eligible for participation if they had definite or highly likely prior exposure to bovine thrombin within the previous 3 years. Preliminary results from 162 subjects enrolled and evaluated as of the cut-off date (28 July 2008) are presented herein; complete study results will be available at the meeting. Of the subjects evaluated to date, 78 (48%) had definite prior exposure to bovine thrombin; prior exposure was highly likely for 85 subjects (52%) and unknown for 1 subject. At baseline, 139 subjects (86%) were seronegative and 23 (14%) were seropositive for anti-bovine thrombin product antibodies. Anti-rThrombin product antibodies did not develop through Day 29 post-treatment in any subject; thus, the rate of antibody formation to rThrombin was the same regardless of anti-bovine thrombin product antibody status. Of the 4 subjects who had anti-rThrombin product antibodies at baseline, none had a change in titer ≥1.0 unit at Day 29. Common adverse events (AEs; reported by ≥10% of subjects) were consistent with the post-surgical setting and with prior studies of rThrombin in these surgical populations. Common AEs included incision site pain, procedural pain, nausea, constipation, anemia, muscle spasms, hypotension, and pyrexia. Topical application of rThrombin was well tolerated, and there was no difference in the formation of antibodies to rThrombin at Day 29 in subjects with or without pre-existing antibodies to bovine thrombin product. The results from this study provide additional information about the immunologic safety of rThrombin and suggest that rThrombin can be safely applied as an aid to hemostasis in patients with pre-existing antibodies to bovine thrombin.

Comparison of Immunogenic Potentials of Bovine Thrombin Preparations

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.

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

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.

Recombinant Thrombin Has a Superior Immunogenicity Profile Compared with Bovine Thrombin in a Well-Controlled, Randomized, Double-Blind, Phase 3 Study of Topically Applied Thrombins in Four Surgical Settings.

Topical hemostatic agents have been used for more than 50 years. Currently, bovine thrombin is isolated from bovine plasma and has an inherent risk of transmitting blood-borne pathogens. Approximately 20% or more patients who receive various bovine thrombin preparations have been reported to develop antibodies to bovine thrombin product and impurities. In rare cases, these antibodies can cross-react with human endogenous coagulation proteins and may lead to alterations in coagulation laboratory parameters, hypersensitivity reactions, or severe bleeding or thrombosis. ZymoGenetics, Inc. has developed recombinant Thrombin (rThrombin) as an alternative to bovine and human plasma-derived thrombin preparations. The potential immunogenicity of rThrombin (n=198) and bThrombin (n=200) were compared in a well-controlled, randomized, double-blind Phase 3 study. Two enzyme-linked immunosorbent assays (ELISAs) were developed and validated under the principles of FDA Guidance Documents (FDA Guidance for Industry: BioAnalytical Method Validation, May 2001) to detect antibodies binding to either rThrombin or bThrombin product. A time-to-clot based assay was developed and validated to characterize whether antibodies to rThrombin had neutralizing activity for plasma-derived thrombin. Study results demonstrated rThrombin's superior immunogenicity profile compared with bThrombin (1.5% versus 22%, respectively, p < 0.0001; antibodies developed to the products, as defined by seroconversion or significant change in titer units). Characterization of the binding antibodies demonstrated none of the anti-rThrombin antibodies neutralized human plasma thrombin. No subjects in either group had evidence of antibody-mediated coagulopathy. These results demonstrate that rThrombin has a superior immunogenicity profile when compared with bThrombin.

165. Topical Recombinant Human Thrombin for Surgical Hemostasis in Subjects Undergoing Spine Surgery

BACKGROUND CONTEXT: Patients treated with topical bovine thrombin preparations may develop antibodies directed against bovine coagulation factors that can cross-react with human endogenous coagulation proteins and lead to alterations in coagulation laboratory parameters or in some cases to severe bleeding or thrombosis.

Unraveling thrombin's true microglia‐activating potential: markedly disparate profiles of pharmaceutical‐grade and commercial‐grade thrombin preparations

Microglia are the resident immune cells of the CNS. Brain injury triggers microglial activation, leading to proliferation, changes in antigenic profile, NO production and cytokine release. It is widely believed that serum factors inundating the injured tissue can prompt this activation, leading to long-term phenotypic changes. We and others have recently reported that commercial-grade preparations of thrombin, a serine protease known for its central function in blood coagulation, activate microglial cells. Recent findings, however, have called into question the involvement of thrombin itself in the induction of microglial cytokine release and led us to systematically re-investigate the ability of the protease to induce a broad spectrum of microglial activation parameters. We used a pharmaceutical-grade recombinant human thrombin (rh-thr) and compared it with a commercial-grade plasma-derived bovine thrombin (pb-thr) preparation that has been used extensively in the literature, including in our own earlier report. We investigated the effect of these two thrombin preparations on proliferation, NO production, interleukin-6 and tumour necrosis factor-alpha release, intracellular calcium signaling and cell surface expression of CD95 (Fas) and CD40. Pb-thr induced robust responses in all variables tested. In contrast, rh-thr triggered calcium signals and induced small but significant changes in the expression of cell surface antigens, but had no effect on proliferation, NO production or cytokine release. Control studies assured equivalent thrombin potencies and excluded both species-specific effects and endotoxin (lipopolysaccharide) contamination as possible causes of the disparity. Our results indicate a substantially more restricted role for thrombin itself in microglial activation than previously appreciated, but point to several potentially important co-stimulatory effects. In addition, these results suggest that previous studies examining thrombin's activation of microglia should be cautiously re-interpreted.