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Identification and function probing of an antithrombin III β conformation‐specific antibody
Author(s) -
Jin Y.,
Yegneswaran S.,
Gu J.M.,
Gritzan U.,
Schönfeld D. L.,
Paz P.,
Patel C.,
Dittmer F.,
Strerath M.,
Bringmann P.,
Kauser K.,
Myles T.,
Murphy J. E.,
Hermiston T. W.
Publication year - 2016
Publication title -
journal of thrombosis and haemostasis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.947
H-Index - 178
eISSN - 1538-7836
pISSN - 1538-7933
DOI - 10.1111/jth.13198
Subject(s) - antithrombin , heparin , chemistry , hemostasis , antibody , thrombin , biochemistry , antithrombotic , gene isoform , microbiology and biotechnology , pharmacology , immunology , platelet , biology , medicine , gene
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.Summary 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 AT α, and has been shown to target antithrombotic function to the extracellular vascular endothelial injury site. Objectives To characterize a conformation‐specific antibody against AT β and begin to investigate the role of AT β 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 AT β ( AT β*H) by the use of phage display. Results SPR and binding studies showed that one of the antibodies, TPP 2009, bound specifically to AT β*H and glycosaminoglycan‐associated AT β on endothelial cells. In diluted prothrombin and activated factor X ( FX a)‐induced clotting assays, TPP 2009 dose‐dependently reduced the anticoagulant effect of heparin in non‐hemophilic and FVIII ‐deficient human plasma, with half‐maximal effective concentrations ( EC 50 ) of 10.5 n m and 4.7 n m , respectively. In AT ‐deficient human plasma, TPP 2009 dose‐dependently inhibited the effects of exogenously added AT β and heparin. In purified systems with AT β and pentasaccharide, TPP 2009 restored > 91% of FX a activity. TPP 2009 dose‐dependently reversed the effects of heparin in rabbit ( EC 50 , 25.7 n m ) and cynomolgus monkey ( EC 50 , 21.5 n m ) plasma, but not in mouse plasma. TPP 2009 was also effective in partially restoring FX a activity in rabbit and cynomolgus monkey plasma treated with FVIII function‐neutralizing antibodies. Conclusions TPP 2009 specifically targets a unique conformational epitope on AT β*H and blocks AT β‐mediated anticoagulation. It effectively promotes coagulation in plasma, indicating the importance of AT β in hemostasis.