Cross-Reactivity of Various Thrombin Products With Anti-Rabbit Antibodies to Bovine, Human, and Recombinant Thrombin

Abstract

JMI-thrombin is used as topical hemostatic agent. While earlier clinically available JMI were reported to produce immunologic responses upon repeated exposure, the improved JMI, Recothrom?, and Evithrom? are claimed to be less immunogenic. Recothrom, despite its reduced immunogenic nature, upon repeated administration may result in the generation of antibodies (Abs) and that may cross react with bovine and human thrombin. Therefore, groups of rabbits were challenged repeatedly with Recothrom, Evithrom, and JMI over a 9-month period. Pre-immune blood and antiserum were collected from each rabbit on different time point. To determine their relative cross reactivity, JMI, Recothrom, and Evithrom were evaluated by western blotting using the rabbit IgG fractions. The results suggest that anti-Recothrom Abs cross-react with Evithrom and JMI in a time dependent fashion. Anti-JMI Abs did not cross-react with Recothrom, and Evithrom. Also, anti-Evithrom did not show any cross-reactivity with Recothrom and JMI at any time.

Compositional Differences in Commercially Available Prothrombin Complex Concentrates

“…Preimmune blood and antiserum were collected from each rabbit in serial order as shown previously. 20 The antiserum from each rabbit and the pooled antisera obtained from 3 individual rabbits in each treatment group were stored at −80ºC prior to IgG isolation.…”

“…Gel electrophoresis and Western blotting were carried out as described previously. 20 In brief, samples were denatured and subjected to electrophoresis through 4% to 20% gradient Tris-N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid–sodium dodecyl sulfate (SDS) polyacrylamide mini gels (Pierce Biotechnology, Rockford, Illinois) and then electrotransferred onto nitrocellulose membrane overnight at 4°C. Precision Dual Color Plus protein standards (Bio-Rad Laboratories) were included in each gel.…”

Recombinant Factor VIIa–Mediated Activation of Prothrombin Complex Concentrates

“…The 36-kDa band in the presence of generation of thrombin from prothrombin has been previously reported in a study to represent thrombin on the immunoblotting human and bovine thrombin. 27 In order to further investigate the effect of AryoSeven and NovoSeven preparations with final concentration of 0.25 and 0.5 mg/mL in 30-minute incubation time in generation of thrombin upon supplementation in activated Profilnine, immunoblotting studies were carried out. This study revealed a strong band at 36 kDa and a very faint band at 50 kDa, which were comparable in all products.…”

Tissue Factor-Mediated Activation of the Prothrombin Complex Concentrate (PCC) is Differently Inhibited by Dabigatran, Rivaroxaban, and Apixaban

“…Preimmune blood and antiserum were collected from each rabbit in serial order as shown previously. 27 The antiserum from each rabbit and the pooled antisera obtained from 3 individual rabbits in each treatment group were stored at −80°C prior to IgG isolation.…”

“…Gel electrophoresis and Western blotting were carried out as described previously. 27 In brief, samples were denatured and subjected to electrophoresis through 4% to 20% gradient Tris-N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid–sodium dodecyl sulfate (SDS) polyacrylamide mini gels (Pierce Biotechnology, Rockford, Illinois) and then electrotransferred onto nitrocellulose membrane overnight at 4°C. Precision Plus Protein Dual Color standards (Bio-Rad Laboratories) were included in each gel.…”