title: Discovery and characterization of an antibody directed against exosite I of thrombin.
abstract: ESSENTIALS: An IgA paraprotein with anti-thrombin activity was not associated with a severe bleeding phenotype. This observation challenges the paradigm that anticoagulant therapy necessarily increases bleeding risk. Characterization of the antibody showed that it specifically binds to thrombin exosite I. A therapeutic drug with the properties of this antibody might be an antithrombotic that doesn't cause bleeding.
  
  
  BACKGROUND
  We report the case of a 54-year-old female who presented with a traumatic subdural hemorrhage. Coagulation tests were markedly prolonged due to the presence of an anti-thrombin IgA paraprotein at 3 g L(-1) . The patient made a complete recovery and has had no abnormal bleeding during a 7-year follow-up, despite the persistence of the paraprotein.
  
  
  OBJECTIVE
  To determine how the paraprotein prolonged clotting tests by defining its target and its epitope.
  
  
  METHODS
  The paraprotein was purified and added to normal pooled plasma for in vitro clotting assays. Binding studies were conducted to determine the affinity of the IgA for thrombin. The Fab was isolated and crystallized with thrombin.
  
  
  RESULTS
  The purified IgA was sufficient to confer the patient's in vitro coagulation profile in normal pooled plasma, and was found to bind specifically and with high affinity to thrombin. A crystal structure of the Fab fragment in complex with thrombin revealed an exosite I interaction involving CDRH3 of the antibody.
  
  
  CONCLUSIONS
  Although the patient originally presented with a subdural bleed, the hematoma resolved without intervention, and no other bleeding event occurred during the subsequent 7 years. During this period, the patient's IgA paraprotein levels have remained constant at 3 g L(-1) , suggesting that the presence of a high-affinity, exosite I-directed antibody is consistent with normal hemostasis. A therapeutic derivative of this antibody might therefore permit antithrombotic dose escalation without an associated increase in the risk of bleeding.
authors.affiliation: Department of Haematology, Addenbrooke's Hospital, Cambridge University Hospitals NHS Trust, Cambridge, UK.
authors.forename: T P
authors.initials: TP
authors.lastname: Baglin
authors.affiliation: Department of Haematology, Addenbrooke's Hospital, Cambridge University Hospitals NHS Trust, Cambridge, UK.
authors.forename: J
authors.initials: J
authors.lastname: Langdown
authors.affiliation: Department of Haematology, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK.
authors.forename: R
authors.initials: R
authors.lastname: Frasson
authors.affiliation: Department of Haematology, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK.
authors.forename: J A
authors.initials: JA
authors.lastname: Huntington
mesh_terms: D000925:Anticoagulants / Q000737:chemistry; D000991:Antithrombins / Q000737:chemistry / Q000276:immunology*; D001777:Blood Coagulation; D001780:Blood Coagulation Tests; D000939:Epitopes / Q000737:chemistry; D005260:Female; D005338:Fibrin Fibrinogen Degradation Products / Q000737:chemistry; D006408:Hematoma, Subdural / Q000276:immunology; D006470:Hemorrhage / Q000276:immunology*; D006487:Hemostasis / Q000276:immunology; D006801:Humans; D007070:Immunoglobulin A / Q000737:chemistry / Q000276:immunology*; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry; D008875:Middle Aged; D010641:Phenotype; D013917:Thrombin / Q000737:chemistry* / Q000276:immunology
chemical_list: D000925:Anticoagulants; D000991:Antithrombins; D000939:Epitopes; D005338:Fibrin Fibrinogen Degradation Products; D007070:Immunoglobulin A; D007140:Immunoglobulin Fab Fragments; C036309:fibrin fragment D; D013917:Thrombin
keywords: anticoagulants; antithrombins; blood coagulation; crystallography; paraproteins; thrombin