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.

Factor XII full and partial null in rat confers robust antithrombotic efficacy with no bleeding

“…Rat arteriovenous shunt thrombosis and cuticle bleeding models Arteriovenous shunt thrombosis and cuticle bleeding studies were performed in Inactin (100 mg/kg, intraperitoneal) anesthetized animals based on published procedures [16,18]. Briefly, an extracorporeal shunt (Tygon tubing containing a silk thread of known weight) was used to connect the carotid artery and jugular vein cannulas.…”

“…Ex-vivo activated partial thromboplastin time, prothrombin time , thrombin generation assay Ex-vivo activated partial thromboplastin time (aPTT) and prothrombin time (PT) were measured as described before [16].…”

“…Ex-vivo thrombin generation assay (TGA) was performed as previously described [14,16]. Ellagic acid (100 and 0.5 mmol/l) and tissue factor (20 and 1 pmol/l) were included as different trigger conditions.…”

“…They include monoclonal antibodies [9,10], natural peptide or protein inhibitors [11,12], small molecule inhibitors [6,13], RNA aptamer [14], and antisense oligonucleotide (ASO) [15]. Although results with these various tool molecules in aggregate clearly strengthen the notion that targeting FXII may provide strong antithrombotic efficacy without conferring bleeding diathesis, some of the reagents bear the limitation of insufficient selectivity [13,16]. With respect to exposure-response relationships, it is known that FXII heterozygous mice (i.e.…”

Future prospects for contact factors as therapeutic targets

“…At higher doses of rHA-Infestin-4, the cuticle bleeding time in rats and rabbits was prolonged <2-fold, possibly because of a modest off-target effect on factor Xa. 33 Despite this, infestin-4 can serve as a model for the development of even more specific fXIIa inhibitors.…”