Coagulation Factor XII (FXII) plays a critical role in thrombosis. What is unclear is the level of enzyme occupancy of FXIIa needed for efficacy, and impact of FXIIa inhibition on cerebral embolism. A selective FXIIa inhibitor, recombinant human albumin-tagged mutant Infestin4 (rHA-Mut-inf), was generated to address these questions. rHA-Mut-inf displayed potency comparable to the original wild-type HA-Infestin4 (rHA-WT-inf) (human FXIIa KI = 0.07 and 0.12 nM, respectively), with markedly improved selectivity against 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, non-human primate, and rabbit is more than 99.0%. 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 effective dose for 50% inhibition (ED50) of thrombus formation was 0.17 mg/kg rHA-Mut-inf, i.v. for the integrated blood flow and 0.16 mg/kg for thrombus weight; ED50 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.
- The American Society for Pharmacology and Experimental Therapeutics