Thrombosis and Haemostasis, Inhaltsverzeichnis

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

¹Thrombosis, Cardiometabolic Diseases, Merck Research Laboratories, Kenilworth, New Jersey, USA

,

Tian-Quan Cai

²In Vivo Pharmacology, Merck Research Laboratories, Kenilworth, New Jersey, USA

,

Gino Castriota

¹Thrombosis, Cardiometabolic Diseases, Merck Research Laboratories, Kenilworth, New Jersey, USA

,

Yuchen Zhou

²In Vivo Pharmacology, Merck Research Laboratories, Kenilworth, New Jersey, USA

,

Lizbeth Hoos

²In Vivo Pharmacology, Merck Research Laboratories, Kenilworth, New Jersey, USA

,

Nina Jochnowitz

²In Vivo Pharmacology, Merck Research Laboratories, Kenilworth, New Jersey, USA

,

Christopher Loewrigkeit

²In Vivo Pharmacology, Merck Research Laboratories, Kenilworth, New Jersey, USA

,

John A. Cook

²In Vivo Pharmacology, Merck Research Laboratories, Kenilworth, New Jersey, USA

,

Alexandra Wickham

²In Vivo Pharmacology, Merck Research Laboratories, Kenilworth, New Jersey, USA

,

Joseph M. Metzger

²In Vivo Pharmacology, Merck Research Laboratories, Kenilworth, New Jersey, USA

,

Martin L. Ogletree^*

¹Thrombosis, Cardiometabolic Diseases, Merck Research Laboratories, Kenilworth, New Jersey, USA

,

Dietmar A. Seiffert

¹Thrombosis, Cardiometabolic Diseases, Merck Research Laboratories, Kenilworth, New Jersey, USA

,

Zhu Chen

¹Thrombosis, Cardiometabolic Diseases, Merck Research Laboratories, Kenilworth, New Jersey, USA

› Institutsangaben

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 (k_on =5x10⁵ M^-1s^-1, k_off =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

Keywords

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

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