Factor XIIa as a Novel Target for Thrombosis: Target Engagement Requirement and Efficacy in a Rabbit Model of Microembolic Signals

Abstract

Coagulation Factor XII (FXII) plays a critical role in thrombosis. What is unclear is the level of enzyme occupancy of FXIIa that is needed for efficacy and the impact of FXIIa inhibition on cerebral embolism. A selective activated FXII (FXIIa) inhibitor, recombinant human albumin-tagged mutant Infestin-4 (rHA-Mut-inf), was generated to address these questions. rHA-Mut-inf displayed potency comparable to the original wild-type HA-Infestin-4 (human FXIIa inhibition constant = 0.07 and 0.12 nM, respectively), with markedly improved selectivity against Factor Xa (FXa) and plasmin. rHA-Mut-inf binds FXIIa, but not FXII zymogen, and competitively inhibits FXIIa protease activity. Its mode of action is hence akin to typical small-molecule inhibitors. Plasma shift and aPTT studies with rHA-Mut-inf demonstrated that calculated enzyme occupancy for FXIIa in achieving a putative aPTT doubling target in human, nonhuman primate, and rabbit is more than 99.0%. The effects of rHA-Mut-inf in carotid arterial thrombosis and microembolic signal (MES) in middle cerebral artery were assessed simultaneously in rabbits. Dose-dependent inhibition was observed for both arterial thrombosis and MES. The ED of thrombus formation was 0.17 mg/kg i.v. rHA-Mut-inf for the integrated blood flow and 0.16 mg/kg for thrombus weight; the ED for MES was 0.06 mg/kg. Ex vivo aPTT tracked with efficacy. In summary, our findings demonstrated that very high enzyme occupancy will be required for FXIIa active site inhibitors, highlighting the high potency and exquisite selectivity necessary for achieving efficacy in humans. Our MES studies suggest that targeting FXIIa may offer a promising strategy for stroke prevention associated with thromboembolic events.

Plasma contact factors as therapeutic targets

“…Infestin domains inhibit several coagulation proteases, with the fourth demonstrating activity against FXIIa, and some effect on plasmin and factor Xa [178]. A recombinant protein comprised of the fourth Kazal-type domain linked to human albumin reduces thrombus formation in animal venous and arterial thrombosis models, and inhibits surface induced thrombosis [179–183]. Subsequent modifications have made it more selective for FXIIa than the native protein without loss of antithrombotic potency [178].…”

Cyclic peptide FXII inhibitor provides safe anticoagulation in a thrombosis model and in artificial lungs

“…Concordantly, the reduction of FXII expression by antisense oligonucleotides suppresses thrombosis in arterial and venous thrombosis mouse models and catheter thrombosis in rabbits 12 , 13 . The inhibition of FXII by protein-based inhibitors, such as antibodies 14 , 15 or insect- and plant-derived proteins 16 – 18 , also reduces thrombosis in mouse, rat, rabbit, and primate models of induced arterial or venous thrombosis and shows potential avenues for therapeutic anticoagulation.…”

Identification of the histidine‐rich glycoprotein domains responsible for contact pathway inhibition

“…13,14 Recent studies suggest that FXII, which is dispensable for hemostasis, is important for thrombus growth. [15][16][17][18][19][20][21][22][23] Therefore, FXIIa inhibition may attenuate thrombosis without disrupting hemostasis.…”

“…α‐FXIIa (FXIIa) propagates coagulation by activating FXI, which circulates in complex with high molecular weight kininogen (HK), and culminates in thrombin generation and fibrin formation 13,14 . Recent studies suggest that FXII, which is dispensable for hemostasis, is important for thrombus growth 15‐23 …”