title: Autoantibodies to thrombin directed against both of its cryptic exosites.
abstract: Immunoglobulin G (IgG) anti-thrombin autoantibodies (ATA) were purified from the plasma of a patient referred for haematological investigation because of bleeding for 24 h following a dental extraction. The ATA did not inhibit the catalytic activity of thrombin against a chromogenic substrate, suggesting that they did not interact with the active site of thrombin. The ATA did, however, prolong the time required to generate thrombin in plasma, suggesting that they inhibited factor V and factor VIII activation. Surface plasmon resonance (SPR) was used to demonstrate that ATA bound to thrombin with high affinity. Competition of thrombin-ATA binding, using known thrombin exosite I and II ligands (hirudin, thrombomodulin and heparin), demonstrated that ATA bound to both thrombin exosites. Thrombin residues that are important for ATA binding were identified using a library of 53 recombinant thrombin variants encompassing alanine substitutions of 78 surface-exposed residues. They were H66, R68, R70 and Y71 in exosite I, and R89, R93, E94, R98, R245 and K248 in exosite II. ATA bound predominantly to exosite II. They did not bind to prothrombin, illustrating the cryptic nature of both exosites exposed and presented as potential antigens following prothrombin conversion to thrombin.
authors.affiliation: Department of Haematology, Faculty of Medicine, Imperial College London, Hammersmith Hospital Campus, London, UK. l.mollica@imperial.ac.uk
authors.forename: Luigina
authors.initials: L
authors.lastname: Mollica
authors.forename: Roger J S
authors.initials: RJ
authors.lastname: Preston
authors.forename: Alain Chan Kwo
authors.initials: AC
authors.lastname: Chion
authors.forename: Steve J
authors.initials: SJ
authors.lastname: Lees
authors.forename: Peter
authors.initials: P
authors.lastname: Collins
authors.forename: Sarah
authors.initials: S
authors.lastname: Lewis
authors.forename: David A
authors.initials: DA
authors.lastname: Lane
mesh_terms: D000940:Antigenic Variation; D000991:Antithrombins / Q000032:analysis* / Q000378:metabolism; D001323:Autoantibodies / Q000032:analysis* / Q000378:metabolism; D001665:Binding Sites; D001667:Binding, Competitive; D005337:Fibrin / Q000378:metabolism; D006493:Heparin / Q000378:metabolism; D006629:Hirudins / Q000378:metabolism; D006801:Humans; D007074:Immunoglobulin G / Q000032:analysis*; D008297:Male; D008875:Middle Aged; D011516:Prothrombin / Q000378:metabolism; D020349:Surface Plasmon Resonance; D013917:Thrombin / Q000276:immunology* / Q000378:metabolism; D018180:Thrombomodulin / Q000378:metabolism
chemical_list: D000991:Antithrombins; D001323:Autoantibodies; D006629:Hirudins; D007074:Immunoglobulin G; D018180:Thrombomodulin; D011516:Prothrombin; D005337:Fibrin; D006493:Heparin; D013917:Thrombin