Isolation and characterization of an acquired antithrombin antibody

Abstract

Abstract A 68-year-old man, following mitral valve replacement, presented with a low-grade chronic consumptive coagulopathy. Laboratory analysis showed mild fibrinolysis, minimal effect of coumadin therapy, and a prolonged thrombin time (greater than 150 seconds using bovine IIa). When purified human IIa was used the thrombin time normalized to within 17 seconds of controls, suggesting a possible inhibitor of bovine IIa. An anti-IIa antibody was isolated by protein A-Sepharose (Sigma, St Louis, MO) chromatography followed by affinity chromatography using a bovine IIa-Sepharose column. The effects of this purified anti-IIa antibody on both bovine and human IIa procoagulant and anticoagulant functions were studied. The isolated immunoglobulin G (IgG) was observed to inhibit bovine IIa in all assays tested. This IgG was also able to slightly prolong fibrinogen clotting by human IIa. Using an enzyme-linked immunosorbent assay it was observed that the IgG bound to bovine IIa, bovine II, human IIa, but not to human II. Further, binding was detectable at approximately 50-fold lower concentrations to bovine IIa (1 nmol/L IgG concentration) than to human IIa (50 nmol/L IgG concentration). The effect of the antibody on the reaction between IIa and AT III/heparin was investigated. Human IIa was found to be protected from AT III/heparin neutralization in the presence of this antibody. These results suggest that this patient developed an antibody that strongly binds to and inhibits the bovine IIa in all assays tested. However, the antibody only significantly affects human IIa neutralization by AT III/heparin, and has little effect on the human IIa procoagulant activity. These data suggest that the decreased effect of AT III/heparin on this patient's IIa may have been a contributing factor in his coagulopathy. The exact cause of this antibody development is unclear, but the role of bovine topical thrombin used during cardiac valve replacement surgery is suspect.