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Thromb Haemost 2014; 111(04): 694-704
DOI: 10.1160/TH13-08-0668
Blood Coagulation, Fibrinolysis and Cellular Haemostasis
Schattauer GmbH

Factor XIIa inhibition by Infestin-4: in vitro mode of action and in vivo antithrombotic benefit

Yiming Xu
1   Thrombosis, Cardiometabolic Diseases, Merck Research Laboratories, Kenilworth, New Jersey, USA
,
Tian-Quan Cai
2   In Vivo Pharmacology, Merck Research Laboratories, Kenilworth, New Jersey, USA
,
Gino Castriota
1   Thrombosis, Cardiometabolic Diseases, Merck Research Laboratories, Kenilworth, New Jersey, USA
,
Yuchen Zhou
2   In Vivo Pharmacology, Merck Research Laboratories, Kenilworth, New Jersey, USA
,
Lizbeth Hoos
2   In Vivo Pharmacology, Merck Research Laboratories, Kenilworth, New Jersey, USA
,
Nina Jochnowitz
2   In Vivo Pharmacology, Merck Research Laboratories, Kenilworth, New Jersey, USA
,
Christopher Loewrigkeit
2   In Vivo Pharmacology, Merck Research Laboratories, Kenilworth, New Jersey, USA
,
John A. Cook
2   In Vivo Pharmacology, Merck Research Laboratories, Kenilworth, New Jersey, USA
,
Alexandra Wickham
2   In Vivo Pharmacology, Merck Research Laboratories, Kenilworth, New Jersey, USA
,
Joseph M. Metzger
2   In Vivo Pharmacology, Merck Research Laboratories, Kenilworth, New Jersey, USA
,
Martin L. Ogletree   *
1   Thrombosis, Cardiometabolic Diseases, Merck Research Laboratories, Kenilworth, New Jersey, USA
,
Dietmar A. Seiffert
1   Thrombosis, Cardiometabolic Diseases, Merck Research Laboratories, Kenilworth, New Jersey, USA
,
Zhu Chen
1   Thrombosis, Cardiometabolic Diseases, Merck Research Laboratories, Kenilworth, New Jersey, USA
› Author Affiliations
Further Information

Publication History

Received: 14 August 2013

Accepted after major revision: 06 November 2013

Publication Date:
29 November 2017 (online)

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Summary

Coagulation factor XII (FXII) plays a central role in initiating the intrinsic cascade of blood coagulation. Purified recombinant Human Albumin- tagged Infestin-4 (rHA-Infestin-4) is a recently described FXIIa inhibitor that displayed strong anticoagulant activity without compromising haemostasis in several animal models. We pursued detailed in vitro characterisation of rHA-Infestin-4 and demonstrated that it is a competitive inhibitor of FXIIa with slow on and off rate constants for binding (kon =5x105 M-1s-1, koff =6x10–4 s-1), it can block FXIIa activation of its physiological substrates (plasma prekallikrein and FXI), and it can inhibit ellagic acid-triggered thrombin generation in plasma. Potency and selectivity profiling in enzyme assays suggest that rHAInfestin- 4 is indeed highly potent on FXIIa (IC50=0.3 ± 0.06, 1.5 ± 0.06, 1.2 ± 0.09 nM, for human, rat, and rabbit FXIIa, respectively) with at least >100-fold selectivity against factors IIa, Xa, IXa, XIa, VIIa, and plasma kallikrein in all three species. rHA-Infestin-4 dose-dependently and markedly reduced clot weight in the arteriovenous shunt thrombosis model in rats and rabbits, accompanied with minimal increase in cuticle bleeding times in either species. rHA-Infestin-4 treatment at 5 mg/kg in rabbit resulted in a 13% reduction in ex vivo FXa activity, demonstrating a modest off-target effect. In summary, our findings confirmed and extended previous reports that inhibition of FXIIa by rHA-Infestin-4 can produce strong antithrombotic efficacy while preserving haemostasis. Our comprehensive selectivity profiling, mode of action, and kinetic studies of rHA-Infestin-4 reveal limitations of this molecule and offer new perspectives on any potential effort of discovering novel FXIIa inhibitors.

Keywords

Factor XIIa - Infestin-4 - coagulation - thrombosis - bleeding

* Current affiliation: Department of Pharmacology, Vanderbilt University, Nash ville, TN, USA.


 

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