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Published on: November 30, 2018
Identification and function probing of an antithrombin IIIβ conformation-specific antibody
Y Jin1, S Yegneswaran2, J-M Gu2
1Global Biologics Research, Bayer HealthCare Pharmaceuticals, San Francisco, CA, USA.
Unlabelled:
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.
Objectives:
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.
