title: Factor XIIa inhibition by Infestin-4: in vitro mode of action and in vivo antithrombotic benefit.
abstract: 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 rHA-Infestin-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.
authors: Yiming
authors: Y
authors: Xu
authors: Tian-Quan
authors: TQ
authors: Cai
authors: Gino
authors: G
authors: Castriota
authors: Yuchen
authors: Y
authors: Zhou
authors: Lizbeth
authors: L
authors: Hoos
authors: Nina
authors: N
authors: Jochnowitz
authors: Christopher
authors: C
authors: Loewrigkeit
authors: John A
authors: JA
authors: Cook
authors: Alexandra
authors: A
authors: Wickham
authors: Joseph M
authors: JM
authors: Metzger
authors: Martin L
authors: ML
authors: Ogletree
authors: Dietmar A
authors: DA
authors: Seiffert
authors: Zhu Chen, PhD, Cardiometabolic Diseases, Merck Research Laboratories, K15-3A-309, Galloping Hill Road, Kenilworth, NJ 07033, USA, Tel.: +1 908 740 0642, E-mail: zhu_chen@merck.com.
authors: Zhu
authors: Z
authors: Chen
mesh_terms: D000818:Animals; D001777:Blood Coagulation / Q000187:drug effects; D004195:Disease Models, Animal; D015956:Factor XIIa / Q000037:antagonists & inhibitors*; D015951:Factor Xa / Q000378:metabolism; D005343:Fibrinolytic Agents / Q000008:administration & dosage* / Q000009:adverse effects; D006470:Hemorrhage / Q000209:etiology; D006487:Hemostasis / Q000187:drug effects; D006801:Humans; D019476:Insect Proteins / Q000008:administration & dosage* / Q000009:adverse effects / Q000494:pharmacology; D007610:Kallikreins / Q000097:blood; D008297:Male; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D013917:Thrombin / Q000378:metabolism*; D013927:Thrombosis / Q000097:blood / Q000188:drug therapy*
chemical_list: D005343:Fibrinolytic Agents; D019476:Insect Proteins; C467902:infestin protein, Triatoma infestans; D007610:Kallikreins; D015956:Factor XIIa; D013917:Thrombin; D015951:Factor Xa
keywords: Factor XIIa; Infestin-4; bleeding; coagulation; thrombosis