title: Cleavage of prothrombin bound in immune complexes results in high thrombin enzymatic activity.
abstract: Thrombin plays a pivotal role in blood clotting as well as in the regulation of vascular remodeling and oxidative stress. Recent evidence suggests that auto-antibodies directed against prothrombin, may play an important role in the pathogenesis of atherosclerosis. It is however not clear, if prothrombin bound in an immune complex retains its clotting and regulatory properties or acts solely by increasing vascular inflammation. In order to answer this question, we used a newly developed stain for the detection of thrombin activity of such complexes. Plasma and serum samples were subjected to rocket immunoelectrophoresis in an anti-prothrombin antiserum containing agarose gel. Gel plates, covered with a nitrocellulose membrane were soaked with chromogenic thrombin substrate. The product of thrombin activity was diazotized to red azo dye bound to nitrocellulose. Activity stain revealed barely discernible rockets in plasma, but heavily stained ones in serum. Pre-incubation with trypsin enhanced activity of immunoprecipitates deriving from plasma, but not from serum. Densitometric analysis showed, that the trypsin-enhanced activity in plasma derived immune complexes was twice as high as in serum derived immunoprecipitates. Thrombin active centre is not blocked by anti-prothrombin antiserum allowing to retain thrombin activity. Moreover, prothrombin in immunoprecipitate is readily cleaved by proteolytic enzymes. This cleavage could potentially be enhanced by antibody binding, although these results need to be confirmed using different antibodies.
authors.affiliation: Department of Clinical Immunochemistry, Jagiellonian University School of Medicine, Cracow, Poland. mmpajdak@cyf-kr.edu.pl
authors.forename: W
authors.initials: W
authors.lastname: Pajdak
authors.forename: J
authors.initials: J
authors.lastname: Radwan
authors.forename: T J
authors.initials: TJ
authors.lastname: Guzik
mesh_terms: D000936:Antigen-Antibody Complex / Q000097:blood / Q000276:immunology*; D000991:Antithrombins / Q000276:immunology; D002847:Chromatography, Agarose; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D047468:Immunoprecipitation; D066298:In Vitro Techniques; D010949:Plasma / Q000276:immunology; D011516:Prothrombin / Q000276:immunology*; D044967:Serum / Q000276:immunology; D014357:Trypsin / Q000494:pharmacology
chemical_list: D000936:Antigen-Antibody Complex; D000991:Antithrombins; D007106:Immune Sera; D011516:Prothrombin; D014357:Trypsin