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

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4211-4211
Author(s):  
He Zhu ◽  
Debra Hoppensteadt ◽  
Rakesh Wahi ◽  
Craig Paterson ◽  
Jawed Fareed
Keyword(s):  
Western Blotting ◽  
Cross Reactivity ◽  
Factor V ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Minimum Concentration ◽  
Bovine Thrombin ◽  
Coagulation Proteins ◽  
Human Thrombin

Abstract Abstract 4211 It 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.

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

2010 ◽  
Vol 16 (5) ◽  
pp. 522-528
Author(s):  
He Zhu ◽  
Debra Hoppensteadt ◽  
Michael Morris ◽  
Jawed Fareed
Keyword(s):  
Western Blotting ◽  
Cross Reactivity ◽  
Time Dependent ◽  
Protein G ◽  
Factor V ◽  
Dependent Manner ◽  
Bovine Thrombin ◽  
Time Points ◽  
Bovine Factor ◽  
Time Point

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.

Differential Effect of An Autoantibody to Thrombin on Fibrinogen Cleavage and Protein C Activation.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3652-3652
Author(s):  
Aditi Shastri ◽  
Kelty R. Baker ◽  
Perumal Thiagarajan
Keyword(s):  
Protein C ◽  
Conflicts Of Interest ◽  
Joint Disease ◽  
Left Shoulder ◽  
Factor V ◽  
Dependent Manner ◽  
Thrombin Time ◽  
Bovine Thrombin ◽  
Normal Plasma ◽  
Human Thrombin

Abstract Abstract 3652 A 76 year old male experienced an unexpected blood loss of 1300 ml during an elective left shoulder replacement for degenerative joint disease. His post-operative course was uncomplicated. Further laboratory testing revealed a prolonged thrombin time of 33 seconds (control of 17.7–20.3 seconds). The reptilase time was within normal limits. He also had a normal factor V, × and fibrinogen level. The prolonged thrombin time failed to correct upon addition of normal plasma. The patient's IgG fraction was isolated by protein G-Sepharose chromatography and tested for its effect in coagulation assays. The patient's purified IgG (2.1 mg/ml) prolonged the thrombin time in normal plasma when tested with a commercial bovine thrombin time reagent. Under similar conditions, normal IgG had no effect. These laboratory tests suggest an acquired antibody to thrombin. The antibody was further characterized by testing its effect on purified human thrombin. The antibody prolonged the clotting time of human thrombin in normal plasma in a concentration dependent manner. However, the antibody had no effect on the esterolytic activity of human or bovine thrombin as measured by the proteolysis of the chromogenic thrombin substrate, S-2238. These results suggest that the epitope for the antibody overlaps the fibrinogen binding site on thrombin but does not include thrombin's active enzymatic site. We also tested the effect of the purified antibody on thrombomodulin-dependent thrombin activation of protein C. The patient's antibody was incubated with protein C, rabbit thrombomodulin and calcium containing buffer. The activation of protein C was measured using the chromogenic protein C substrate S2366. The antibody had no effect on protein C activation by thrombin. Acquired antibodies to thrombin develop after exposure to bovine thrombin, commonly used for surgical hemostasis. These antibodies are also associated with antibodies to factor V and a low factor V level. This patient's clinical and laboratory profile is intriguing, given the lack of previous exposure to bovine or human thrombin and the presence of normal Factor V levels. This novel antibody may represent formation of a spontaneous autoantibody to thrombin due to failure of immunological surveillance mechanisms. Disclosures: No relevant conflicts of interest to declare.

Recent developments in topical thrombins

2009 ◽  
Vol 102 (07) ◽  
pp. 15-24 ◽  
Author(s):  
Thomas L. Ortel ◽  
Craig M. Kessler
Keyword(s):  
Human Plasma ◽  
Excessive Bleeding ◽  
Bovine Thrombin ◽  
Bovine Plasma ◽  
Haemostatic Agents ◽  
Coagulation Proteins ◽  
Equivalent Efficacy ◽  
Human Thrombin ◽  
Recent Developments ◽  
Surgical Bleeding

SummaryManaging 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 antibodymediated 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 bovinederived thrombin agents. Well-designed and adequately powered clinical trials should be conducted to indicate whether human thrombin products would improve the risk-benefit and costbenefit profiles for surgeries complicated by excessive bleeding.

scholarly journals Development of antibodies to thrombin and factor V with recurrent bleeding in a patient exposed to topical bovine thrombin

Blood ◽  
1990 ◽  
Vol 76 (10) ◽  
pp. 2011-2016 ◽  
Author(s):  
JL Zehnder ◽  
LL Leung
Keyword(s):  
Enzyme Linked Immunosorbent Assay ◽  
Recurrent Bleeding ◽  
Severe Bleeding ◽  
Factor V ◽  
Bleeding Events ◽  
Bovine Thrombin ◽  
Normal Plasma ◽  
Human Thrombin ◽  
Immunomodulatory Therapies ◽  
Bovine Factor

Abstract A 65 year old patient who was exposed to topical bovine thrombin during cardiac surgery developed markedly prolonged clotting times and a severe bleeding diathesis. Mixing studies with normal plasma failed to correct the clotting times. Platelet transfusions, immunosuppressive and immunomodulatory therapies were ineffective, but plasmapheresis was effective in decreasing clotting times and in the resolution of clinical bleeding events. The patient's purified IgG reacted with bovine thrombin by immunoblotting and enzyme-linked immunosorbent assay (ELISA). However, the IgG reacted minimally with human thrombin. In view of the severe bleeding, a coexisting inhibitor was sought. The patient's factor V activity was 1% of normal and was not corrected by mixing with normal plasma, demonstrating the presence of an inhibitor against factor V. The patient's IgG reacted with both bovine and human factor V. Immunoblotting localized the site of antibody binding to the light chain of activated bovine factor V. Detectable amounts of bovine factor V were found in commercial bovine thrombin preparations by ELISA. The data suggest that patients exposed to topical bovine thrombin may develop antibodies to thrombin and factor V. Anti-thrombin antibodies may mask coexisting factor V inhibitors responsible for clinical bleeding.

scholarly journals Cilostazol Suppresses IL-23 Production in Human Dendritic Cells via an AMPK-Dependent Pathway

2016 ◽  
Vol 40 (3-4) ◽  
pp. 499-508 ◽  
Author(s):  
Quanxing Shi ◽  
Zhao Yin ◽  
Peilin Liu ◽  
Bei Zhao ◽  
Zhong Zhang ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Dendritic Cell ◽  
Western Blotting ◽  
Human Monocyte ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Compound C ◽  
Human Dendritic Cells ◽  
Dependent Pathway

Background/Aims: Cilostazol has been previously demonstrated to inhibit IL-23 production in human synovial macrophages via a RhoA/ROCK-dependent pathway. However, whether cilostazol affects IL-23 production in human dendritic cells remains largely unknown. The present study was designed to investigate this question and elucidate the possible underlying mechanisms. Methods: Human monocyte-derived dendritic cells (mo-DCs) were pretreated with or without cilostazol and then incubated with zymosan. Enzyme-linked immunosorbent assay (ELISA) and real time PCR analyses were used to measure IL-23 protein expression and RNA levels, respectively, whereas Western blotting was used to measure the expression and phosphorylation level of AMPK. Results: Our results demonstrated that cilostazol suppressed zymosan-induced IL-23 protein production in a concentration dependent manner without affecting dendritic cell viability. In addition, it was found that cilostazol suppressed the expression of the p19 and p40 subunits of IL-23. Moreover, cilostazol mimicked the effect of the AMPK agonist A-769662, as demonstrated by the fact that IL-23 production was also inhibited by A-769662, and the effect of cilostazol on IL-23 production was blocked by the AMPK antagonist Compound C. More importantly, Western blotting demonstrated that cilostazol led to an increased phosphorylation of AMPK. Conclusion: Collectively, our data suggest that cilostazol inhibits the production of IL-23 in human mo-DCs, potentially via the activation of AMPK. This suggests that cilostazol could be an effective anti-inflammatory agent in IL-23- and dendritic cell-related diseases.

Development of antibodies to human thrombin and factor V in a pediatric patient exposed to topical bovine thrombin

2010 ◽  
Vol 55 (6) ◽  
pp. 1195-1197 ◽  
Author(s):  
Clara Y. Lo ◽  
Carol Jones ◽  
Bertil Glader ◽  
James L. Zehnder
Keyword(s):  
Pediatric Patient ◽  
Factor V ◽  
Bovine Thrombin ◽  
Human Thrombin

Clinical Significance of Antibodies to Bovine and Human Thrombin and Factor V After Surgical Use of Bovine Thrombin

1992 ◽  
Vol 97 (1) ◽  
pp. 84-91 ◽  
Author(s):  
Samuel I. Rapaport ◽  
Ariella Zivelin ◽  
Robert A. Minow ◽  
Christine S. Hunter ◽  
Kathleen Donnelly
Keyword(s):  
Clinical Significance ◽  
Factor V ◽  
Bovine Thrombin ◽  
Human Thrombin

Prevalence of Cross Reactive Epitopes to Bovine Factor Va Light Chain Fragments in Normal and Patients Plasma.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4022-4022
Author(s):  
Debra A. Hoppensteadt ◽  
Vinod Bansal ◽  
Josephine Cunanan ◽  
Kuldeep Patel ◽  
Rakesh Wahi ◽  
...  
Keyword(s):  
Western Blotting ◽  
Light Chain ◽  
Coagulation Factors ◽  
Factor V ◽  
Plasma Samples ◽  
Bovine Thrombin ◽  
Coronary Syndrome ◽  
Factor Va ◽  
V Antigen ◽  
Bovine Factor

Abstract Several reports have described the presence of antibodies to bovine coagulation factors, such as factor V, prothrombin and factor X in plasma samples obtained from patients exposed to topical bovine thrombin. Other reports have also demonstrated the presence of anti-bovine coagulation factors in patients who have not been exposed to bovine thrombins, suggesting that anti-bovine protein antibodies can be generated in normal individuals. It has been suggested that surgical patients treated with topical bovine thrombin develop specific antibodies to bovine factor V which may be responsible for the bleeding and thrombotic complications. However, there is no definitive clinical study demonstrating a relationship between the apparent hemostatic defects and the presence of bovine factor Va antibodies. It was hypothesized that bovine factor Va antibodies are usually present in patients plasma because of the exposure to dietary bovine products. To test this hypothesis plasma samples from patients with end state renal disease (ESRD)(n=80), acute coronary syndrome (ACS)(n=160), burns (n=40) and healthy normal volunteers (n=140) were profiled for the presence of human factor V antigen (HFVA), bovine FVa antigen as measured by using a modified Elisa method and western blotting methods where bovine factor Va light chain fragment is used as a probe. In contrast to the normals (89±12%), the factor V antigen levels were found to be increased in the ESRD (148±30%), ACS (164±41%) and burn (145±27%) patients. Thus, there appears to be an up regulation of factor V antigen in these patients. All of the groups tested for the presence of immunoreactive material to the bovine factor Va light chain exhibited 2–3 ug/ml levels which were not significantly different. However, in the western blotting studies all groups exhibited cross reactivity with the factor Va light epitopes. There were bands present in the molecular weight range of 22, 36, 45 and 97 Kda in both the ESRD and burn patients. In the ACS patients there was an additional band observed at 166 Kda. These observations underscore the notion that bovine antifactor Va antibodies are non-specific and highly prevalent in both the surgical/interventional patients and normal population. A possible explanation for the presence of these antibodies is that most normal individual and patients problem are exposed to bovine proteins. Moreover, the higher prevalence of these antibodies in the ESRD and ACS patients may be due to additional exposure to heparin and aprotonin.

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

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4418-4418
Author(s):  
Nasir Sadeghi ◽  
Josephine Cunanan ◽  
Debra Hoppensteadt ◽  
Indermohan Thethi ◽  
Rakesh Wahi ◽  
...  
Keyword(s):  
Sodium Dodecyl ◽  
Pharmaceutical Research ◽  
Repeated Administration ◽  
Cross Reactivity ◽  
Bovine Thrombin ◽  
Time Period ◽  
Molecular Variants ◽  
Rabbit Igg ◽  
Human Thrombin ◽  
Piperazineethanesulfonic Acid

Abstract Abstract 4418 Bovine 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.

scholarly journals Application of Electric Cell-Substrate Impedance Sensing to Investigate the Cytotoxic Effects of Andrographolide on U-87 MG Glioblastoma Cell Migration and Apoptosis

Sensors ◽  
2019 ◽  
Vol 19 (10) ◽  
pp. 2275 ◽  
Author(s):  
Sheng-Po Chiu ◽  
Buyandelger Batsaikhan ◽  
Huei-Mei Huang ◽  
Jia-Yi Wang
Keyword(s):  
Cell Migration ◽  
Western Blotting ◽  
Primary Brain Tumor ◽  
Dependent Manner ◽  
Inhibitory Effects ◽  
Concentration Dependent Manner ◽  
Cytotoxic Effects ◽  
Impedance Sensing ◽  
Cell Substrate ◽  
Related Proteins

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor in adults. In recent studies, the efficacy of suberoylanilide hydroxamic acid (SAHA) has been investigated for GBM. We explored the effects of two exploratory compounds, the histone deacetylase SAHA and the natural product andrographolide, on Uppsala 87 Malignant Glioma (U-87 MG) cell migration and viability in comparison with the clinically used therapeutic agent temozolomide (TMZ). We used the electric cell–substrate impedance sensing (ECIS) system to monitor the migration of U-87 MG cells after treatment with various concentrations of these compounds. Moreover, we used the Alamar blue assay and western blotting to observe the concentration-dependent changes in the viability and apoptosis of U-87 MG cells. Our results demonstrated that both SAHA and andrographolide (10–300 μM) significantly inhibited GBM cell migration in a concentration-dependent manner, and 10 μM SAHA and 56 μM andrographolide demonstrated remarkable inhibitory effects on U-87 MG migration. Western blotting indicated that compared with TMZ, both SAHA and andrographolide induced higher expression levels of apoptosis-related proteins, such as caspase-3, BAX, and PARP in U-87 MG cells. Furthermore, all three drugs downregulated the expression of the antiapoptotic protein Bcl-2. In conclusion, SAHA and andrographolide showed exceptional results in inhibiting cell migration and motility. The ECIS wound healing assay is a powerful technique to identify and screen potential therapeutic agents that can inhibit cancer cell migration.

Sign in / Sign up

Export Citation Format

Share Document