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.

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.

Relative Immunologic Potential of Bovine Prothrombin and Purified Thrombins

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5463-5463
Author(s):  
He Zhu ◽  
Debra Ann Hoppensteadt ◽  
Rodger L. Bick ◽  
Cafer Adiguzel ◽  
Rakesh Wahi ◽  
...  
Keyword(s):  
Western Blotting ◽  
Membrane Filtration ◽  
Specific Activity ◽  
Factor Viia ◽  
Rank Order ◽  
Coagulation Factors ◽  
Bovine Thrombin ◽  
Factor Va ◽  
Bovine Factor ◽  
Time Point

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.

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.

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.

EspP, An Extracellular Serine Protease From Enterohemorrhagic E. Coli, Induces Coagulopathy In Human Whole Blood and Plasma

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4416-4416
Author(s):  
Kevin H.M. Kuo ◽  
Shekeb Khan ◽  
Elena Brnjac ◽  
Emil F. Pai ◽  
Alden E. Chesney
Keyword(s):  
Serine Protease ◽  
Whole Blood ◽  
Ex Vivo ◽  
Coagulation Factor ◽  
Time Dependent ◽  
Coagulation Cascade ◽  
Factor V ◽  
Dependent Manner ◽  
E Coli ◽  
Negative Controls

Abstract Abstract 4416 EspP (E. coli secreted serine protease, large plasmid encoded) is an extracellular serine protease produced by enterohemorrhagic E. coli (EHEC) O157:H7. Brunder et al. (Mol Microbiol 1997, 24:767–78) have shown that EspP cleaves, amongst other proteins, human coagulation factor V, and the authors hypothesized that it may contribute to the mucosal hemorrhage in patients with EHEC infection. We have since shown that EspP also cleaves factor VIII. Since the mechanism by which EHEC induces diarrhea-associated Hemolytic Uremic Syndrome (D+HUS) has not been fully elucidated, and EspP has been cited as a putative virulence factor in D+HUS, we investigated the role of EspP in primary and secondary hemostasis in the pathogenesis of D+HUS. Wild type EspP (EspPwt) and EspPS263A, where the serine at the active site was mutated to an alanine thereby abolishing its proteolytic activity, were expressed in the non-pathogenic E. coli host BL21(DE3) and purified by hydrophobic interaction and size-exclusion chromatography. EspPwt at 1.0 mg/mL was incubated for 0.5, 2.0 and 4.0 hours ex vivo with citrated plasma from 6 healthy adults. EspPS263A, bovine serum albumin (BSA) and phosphate buffer saline-glycerol (PBS-G) served as negative controls. PT, aPTT and TT were found to be significantly prolonged and activity of factors V, VII, VIII and XII were reduced in a time- and concentration-dependent manner (Figures 1 and Figure 2). When citrated plasma was incubated with 1 mg/mL EspPwt at 37°C for 4 hours, PT was prolonged by 23.2 +/− 3.8 s, aPTT by 41.6 +/− 8.3 s and TT by 6.1 +/− 0.6 s, relative to the negative controls. Factor V activity decreased by 0.82 +/− 0.14 U/mL, factor VII by 0.72 +/− 0.28 U/mL, factor VIII by 0.69 +/− 0.31 U/mL and factor XII by 0.36 +/− 0.09 U/mL, relative to the negative controls. Prothrombin activity was significantly reduced (0.16 +/− 0.08 U/mL) compared to all negative controls but remained above 0.75 U/mL. Factors IX, × and XI activity, and fibrinogen concentration were not significantly different from the controls. To determine whether any cellular components in whole blood contribute to EspP's effect on the coagulation cascade, the experiment was repeated using citrated whole blood in place of plasma during the incubation phase. Plasma was then recovered and analyzed. Similar results were observed. The results suggest that EspP has proteolytic activity against specific coagulation factors at least in an ex vivo setting. In patients with EHEC infection, EspP may contribute to the hemorrhagic diarrhea by impairing the coagulation cascade. Further studies are needed to determine whether EspP is able to induce coagulopathy in vivo and if so, whether induction of such a coagulopathic state may favour the entry of Shiga toxin into systemic circulation in patients with D+HUS. Figure 1 EspP prolongs PT, aPTT and TT in a time-dependent manner. Figure 1. EspP prolongs PT, aPTT and TT in a time-dependent manner. Figure 2 EspP reduces coagulation factor activity in a time-dependent manner. Figure 2. EspP reduces coagulation factor activity in a time-dependent manner. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Genetic variation and dynamics of hepatitis C virus replicons in long-term cell culture

2005 ◽  
Vol 86 (3) ◽  
pp. 645-656 ◽  
Author(s):  
Nobuyuki Kato ◽  
Takashi Nakamura ◽  
Hiromichi Dansako ◽  
Katsuyuki Namba ◽  
Ken-ichi Abe ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Cell Culture ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Colony Formation ◽  
Time Dependent ◽  
Genetic Mutations ◽  
Dependent Manner ◽  
Time Points

Hepatitis C virus (HCV) genomic sequences are known to vary widely among HCV strains, but to date there have been few reports on the genetic variations and dynamics of HCV in an experimental system of HCV replication. In this study, a genetic analysis of HCV replicons obtained in long-term culture of two HCV replicon cells (50-1 and 1B-2R1), which were established from two HCV strains, 1B-1 and 1B-2, respectively, was performed. One person cultured 50-1 cells for 18 months, and two people independently cultured 50-1 cells for 12 months. 1B-2R1 cells were also cultured for 12 months. The whole nucleotide sequences of the three independent replicon RNA clones obtained at several time points were determined. It was observed that genetic mutations in both replicons accumulated in a time-dependent manner, and that the mutation rates of both replicons were approximately 3·0×10−3 base substitutions/site/year. The genetic diversity of both replicons was also enlarged in a time-dependent manner. The colony formation assay by transfection of total RNAs isolated from both replicon cells at different time points into naïve HuH-7 cells revealed that the genetic mutations accumulating with time in both replicons apparently improved colony formation efficiency. Taken together, these results suggest that the HCV replicon system is useful for the analysis of evolutionary dynamics and variations of HCV. Using this replicon cell culture system, it was demonstrated further that neither ribavirin nor its derivative mizoribine accelerated the mutation rate or the increase in the genetic diversity of HCV replicon.

scholarly journals Involvement of GluA1-AMPAR-mediated LTP in time-dependent decline of cognitive function in rats with temporal lobe epilepsy

2021 ◽  
Vol 3 (1) ◽  
Author(s):  
Xiaoqing Luo ◽  
Xiaoli Yu ◽  
Jufang Liang ◽  
Ruidi Sun ◽  
Cheng Li ◽  
...  
Keyword(s):  
Cognitive Impairment ◽  
Cognitive Function ◽  
Temporal Lobe Epilepsy ◽  
Learning And Memory ◽  
Western Blotting ◽  
Temporal Lobe ◽  
Long Term Potentiation ◽  
Time Dependent ◽  
Dependent Manner ◽  
Behavioral Tests

Abstract Background Cognitive impairment is one of the common comorbidities in patients with temporal lobe epilepsy (TLE), but the underlying mechanisms remain largely unknown. Previous studies have found significant decay of hippocampal long-term potentiation (LTP) in TLE rats with cognitive impairment. As the activation of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs) is responsible for LTP formation and learning and memory, we investigated whether AMPARs are involved in the LTP inhibition and the TLE-associated cognitive impairments. Methods TLE rat model was established by intraperitoneal injection of lithium chloride-pilocarpine on postnatal day 21 (P21). Learning and memory performance, hippocampal expression of membrane GluA1-AMPARs, and hippocampal LTP were tested by behavioral tests, western blotting, and field potential recording, respectively, at 1, 5 and 13 weeks after induction of status epilepticu (SE). Finally, the effects of (S)-AMPA, an agonist of AMPARs, on LTP and cognitive function were tested. Results Results of behavioral tests revealed an time-dependent decline in the learning and memory of TLE rats when compared to the age-matched controls at week 5 and 13, rather than at week 1 after the induction of SE. Western blotting showed that the hippocampal expression of membrane GluA1 was significantly decreased in a time-dependent manner in the TLE rats when compared to the age-matched controls at weeks 5 and 13, rather than at week 1 after the induction of SE. Similarly, the hippocampal LTP was inhibited in a time-dependent manner in TLE rats at weeks 5 and 13, rather than at week 1 after the induction of SE. Moreover, intra-hippocampal injection of (S)-AMPA ameliorated the deficits in learning as well as spatial and emotional memory in a dose-dependent manner, and partially reversed the inhibition of CA1 LTP in the TLE rats at week 13 after the induction of SE. Conclusions The reduced expression of hippocampal membrane GluA1 may be involved in LTP decay in CA1 and cognition impairment in TLE rats.

Endotoxin transiently inhibits protein synthesis through Akt and MAPK mediating pathways in C2C12 myotubes

2011 ◽  
Vol 301 (4) ◽  
pp. C895-C902 ◽  
Author(s):  
R. Tarabees ◽  
D. Hill ◽  
C. Rauch ◽  
P. A. Barrow ◽  
P. T. Loughna
Keyword(s):  
Protein Synthesis ◽  
Intracellular Signaling ◽  
Kinase Inhibitor ◽  
Mammalian Target Of Rapamycin ◽  
Polymyxin B ◽  
C2c12 Myotubes ◽  
Dependent Manner ◽  
Time Points ◽  
Dose Dependent ◽  
Time Point

In this study, the effect of lipopolysaccharide (LPS) on protein synthesis (PS) and intracellular signaling factors that regulate it have been investigated in C2C12 murine-derived myotubes. In particular, the role of Akt/mammalian target of rapamycin (mTOR) and the mitogen-activated protein kinases (MAPKs) [p38 and extracelluar regulated protein kinase (ERK1/2)] have been examined. The direct effect of LPS on PS was measured at 3 and 18 h. LPS significantly decreased PS at 3 h but not at the 18-h time point. This effect was preceded by decreased Akt phosphorylation at 5 and 30 min after LPS administration. The mTOR phosphorylation exhibited a long time dose-dependent increase at all the time points. Similarly, the activity-related phosphorylation of p38 and ERK1/2 significantly increased in a time- and dose-dependent manner at all the time points. Polymyxin B abolished the LPS-induced decrease in PS rate. The phosphatidylinositol 3-kinase inhibitor LY-0294002 in combination with LPS significantly decreased the rate of PS by 81% and alone by 66%, respectively, for the 3- and 18-h time points, whereas p38 and ERK inhibitors in combination with LPS significantly decreased the rate PS rate at the 18-h time point by 41% and 59%, respectively, compared with control cells. In conclusion, LPS alone transiently decreased the rate of PS by 50% at 3 h; this effect is most likely mediated via the Toll-like receptor 4 (TLR4)-Akt/mTOR pathway, and both p38 and ERK when inhibited in the presence of LPS at 3 h have a similar effect in preventing the LPS-induced reduction in PS.

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

2008 ◽  
Vol 14 (2) ◽  
pp. 135-140 ◽  
Author(s):  
Abdel Terrab ◽  
Dan Pawlak ◽  
Pat Spaay ◽  
Debra Hoppensteadt ◽  
Jawed Fareed
Keyword(s):  
Western Blot ◽  
Membrane Filtration ◽  
Enzyme Linked Immunosorbent Assay ◽  
Factor V ◽  
Bovine Thrombin ◽  
Western Blot Method ◽  
Factor Va ◽  
Highly Sensitive ◽  
Filtration Step ◽  
Bovine Factor

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.

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