title: Identification and function probing of an antithrombin IIIβ conformation-specific antibody.
abstract: ESSENTIALS: Antithrombin III (AT)β binds heparin with higher affinity than ATα. A conformation-specific antibody against ATβ, TPP2009, was made to investigate ATβ in hemostasis. TPP2009 bound specifically to heparin-ATβ and greatly reduced the anticoagulant effect of AT. This antibody was effective in elucidating the importance of ATβ in hemostasis.
  
  
  BACKGROUND
  Antithrombin III (AT)β is an isoform of AT that lacks the post-translational carbohydrate modification at Asn135. This isoform binds heparin with greater affinity than ATα, and has been shown to target antithrombotic function to the extracellular vascular endothelial injury site.
  
  
  OBJECTIVE
  To characterize a conformation-specific antibody against ATβ and begin to investigate the role of ATβ in maintaining hemostasis.
  
  
  METHODS
  Surface plasmon resonance (SPR), antigen binding and functional assays were conducted to characterize the mode of action of antibodies generated against heparin-bound ATβ (ATβ*H) by the use of phage display.
  
  
  RESULTS
  SPR and binding studies showed that one of the antibodies, TPP2009, bound specifically to ATβ*H and glycosaminoglycan-associated ATβ on endothelial cells. In diluted prothrombin and activated factor X (FXa)-induced clotting assays, TPP2009 dose-dependently reduced the anticoagulant effect of heparin in non-hemophilic and FVIII-deficient human plasma, with half-maximal effective concentrations (EC50 ) of 10.5 nm and 4.7 nm, respectively. In AT-deficient human plasma, TPP2009 dose-dependently inhibited the effects of exogenously added ATβ and heparin. In purified systems with ATβ and pentasaccharide, TPP2009 restored > 91% of FXa activity. TPP2009 dose-dependently reversed the effects of heparin in rabbit (EC50 , 25.7 nm) and cynomolgus monkey (EC50 , 21.5 nm) plasma, but not in mouse plasma. TPP2009 was also effective in partially restoring FXa activity in rabbit and cynomolgus monkey plasma treated with FVIII function-neutralizing antibodies.
  
  
  CONCLUSIONS
  TPP2009 specifically targets a unique conformational epitope on ATβ*H and blocks ATβ-mediated anticoagulation. It effectively promotes coagulation in plasma, indicating the importance of ATβ in hemostasis.
authors.affiliation: Global Biologics Research, Bayer HealthCare Pharmaceuticals, San Francisco, CA, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Jin
authors.affiliation: Hematology Research, Bayer HealthCare Pharmaceuticals, San Francisco, CA, USA.
authors.forename: S
authors.initials: S
authors.lastname: Yegneswaran
authors.affiliation: Hematology Research, Bayer HealthCare Pharmaceuticals, San Francisco, CA, USA.
authors.forename: J-M
authors.initials: JM
authors.lastname: Gu
authors.affiliation: Global Biologics Research, Bayer HealthCare Pharmaceuticals, Cologne, Germany.
authors.forename: U
authors.initials: U
authors.lastname: Gritzan
authors.affiliation: Global Biologics Research, Bayer HealthCare Pharmaceuticals, Wuppertal, Germany.
authors.forename: D L
authors.initials: DL
authors.lastname: Schönfeld
authors.affiliation: Global Biologics Research, Bayer HealthCare Pharmaceuticals, San Francisco, CA, USA.
authors.forename: P
authors.initials: P
authors.lastname: Paz
authors.affiliation: Hematology Research, Bayer HealthCare Pharmaceuticals, San Francisco, CA, USA.
authors.forename: C
authors.initials: C
authors.lastname: Patel
authors.affiliation: Global Biologics Research, Bayer HealthCare Pharmaceuticals, Cologne, Germany.
authors.forename: F
authors.initials: F
authors.lastname: Dittmer
authors.affiliation: Global Biologics Research, Bayer HealthCare Pharmaceuticals, Wuppertal, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Strerath
authors.affiliation: Global Biologics Research, Bayer HealthCare Pharmaceuticals, San Francisco, CA, USA.
authors.forename: P
authors.initials: P
authors.lastname: Bringmann
authors.affiliation: Hematology Research, Bayer HealthCare Pharmaceuticals, San Francisco, CA, USA.
authors.forename: K
authors.initials: K
authors.lastname: Kauser
authors.affiliation: Hematology Research, Bayer HealthCare Pharmaceuticals, San Francisco, CA, USA.
authors.forename: T
authors.initials: T
authors.lastname: Myles
authors.affiliation: Global Biologics Research, Bayer HealthCare Pharmaceuticals, San Francisco, CA, USA.
authors.forename: J E
authors.initials: JE
authors.lastname: Murphy
authors.affiliation: Global Biologics Research, Bayer HealthCare Pharmaceuticals, San Francisco, CA, USA.
authors.forename: T W
authors.initials: TW
authors.lastname: Hermiston
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000378:metabolism / Q000494:pharmacology*; D000918:Antibody Specificity; D000990:Antithrombin III / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D001666:Binding Sites, Antibody; D001777:Blood Coagulation / Q000187:drug effects*; D001780:Blood Coagulation Tests; D002460:Cell Line; D061505:Cell Surface Display Techniques; D003029:Coagulants / Q000276:immunology / Q000378:metabolism / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism; D018604:Epitope Mapping; D006801:Humans; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011817:Rabbits; D013329:Structure-Activity Relationship; D020349:Surface Plasmon Resonance; D013997:Time Factors
chemical_list: D000906:Antibodies; D003029:Coagulants; C542513:SERPINC1 protein, human; D000990:Antithrombin III
keywords: antithrombin III; blood coagulation; endothelium; heparin; neutralizing antibody