title: 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 ED50of 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 ED50for 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.
authors.affiliation: In Vitro Pharmacology (C.M.B., A.M.O., K.O., D.C.) and Cardiometabolic Diseases (X.W., W.W., X.Z., G.C., D.A.S., D.E.G., Z.C.), Merck & Co., Inc., Kenilworth, New Jersey.
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Barbieri
authors.affiliation: In Vitro Pharmacology (C.M.B., A.M.O., K.O., D.C.) and Cardiometabolic Diseases (X.W., W.W., X.Z., G.C., D.A.S., D.E.G., Z.C.), Merck & Co., Inc., Kenilworth, New Jersey.
authors.forename: Xinkang
authors.initials: X
authors.lastname: Wang
authors.affiliation: In Vitro Pharmacology (C.M.B., A.M.O., K.O., D.C.) and Cardiometabolic Diseases (X.W., W.W., X.Z., G.C., D.A.S., D.E.G., Z.C.), Merck & Co., Inc., Kenilworth, New Jersey.
authors.forename: Weizhen
authors.initials: W
authors.lastname: Wu
authors.affiliation: In Vitro Pharmacology (C.M.B., A.M.O., K.O., D.C.) and Cardiometabolic Diseases (X.W., W.W., X.Z., G.C., D.A.S., D.E.G., Z.C.), Merck & Co., Inc., Kenilworth, New Jersey.
authors.forename: Xueping
authors.initials: X
authors.lastname: Zhou
authors.affiliation: In Vitro Pharmacology (C.M.B., A.M.O., K.O., D.C.) and Cardiometabolic Diseases (X.W., W.W., X.Z., G.C., D.A.S., D.E.G., Z.C.), Merck & Co., Inc., Kenilworth, New Jersey.
authors.forename: Aimie M
authors.initials: AM
authors.lastname: Ogawa
authors.affiliation: In Vitro Pharmacology (C.M.B., A.M.O., K.O., D.C.) and Cardiometabolic Diseases (X.W., W.W., X.Z., G.C., D.A.S., D.E.G., Z.C.), Merck & Co., Inc., Kenilworth, New Jersey.
authors.forename: Kim
authors.initials: K
authors.lastname: O'Neill
authors.affiliation: In Vitro Pharmacology (C.M.B., A.M.O., K.O., D.C.) and Cardiometabolic Diseases (X.W., W.W., X.Z., G.C., D.A.S., D.E.G., Z.C.), Merck & Co., Inc., Kenilworth, New Jersey.
authors.forename: Donald
authors.initials: D
authors.lastname: Chu
authors.affiliation: In Vitro Pharmacology (C.M.B., A.M.O., K.O., D.C.) and Cardiometabolic Diseases (X.W., W.W., X.Z., G.C., D.A.S., D.E.G., Z.C.), Merck & Co., Inc., Kenilworth, New Jersey.
authors.forename: Gino
authors.initials: G
authors.lastname: Castriota
authors.affiliation: In Vitro Pharmacology (C.M.B., A.M.O., K.O., D.C.) and Cardiometabolic Diseases (X.W., W.W., X.Z., G.C., D.A.S., D.E.G., Z.C.), Merck & Co., Inc., Kenilworth, New Jersey.
authors.forename: Dietmar A
authors.initials: DA
authors.lastname: Seiffert
authors.affiliation: In Vitro Pharmacology (C.M.B., A.M.O., K.O., D.C.) and Cardiometabolic Diseases (X.W., W.W., X.Z., G.C., D.A.S., D.E.G., Z.C.), Merck & Co., Inc., Kenilworth, New Jersey.
authors.forename: David E
authors.initials: DE
authors.lastname: Gutstein
authors.affiliation: In Vitro Pharmacology (C.M.B., A.M.O., K.O., D.C.) and Cardiometabolic Diseases (X.W., W.W., X.Z., G.C., D.A.S., D.E.G., Z.C.), Merck & Co., Inc., Kenilworth, New Jersey zchen17@hotmail.com.
authors.forename: Zhu
authors.initials: Z
authors.lastname: Chen
mesh_terms: D000818:Animals; D000925:Anticoagulants / Q000494:pharmacology; D001777:Blood Coagulation / Q000187:drug effects / Q000502:physiology; D015956:Factor XIIa / Q000037:antagonists & inhibitors*; D005343:Fibrinolytic Agents / Q000494:pharmacology; D019476:Insect Proteins / Q000494:pharmacology*; D020766:Intracranial Embolism / Q000097:blood / Q000188:drug therapy; D020767:Intracranial Thrombosis / Q000097:blood / Q000188:drug therapy; D023421:Models, Animal; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000494:pharmacology*; D012709:Serum Albumin / Q000494:pharmacology*; D000075462:Serum Albumin, Human
chemical_list: D000925:Anticoagulants; D005343:Fibrinolytic Agents; D019476:Insect Proteins; D011993:Recombinant Fusion Proteins; D012709:Serum Albumin; C000609004:rHA-infestin-4 protein; D015956:Factor XIIa; D000075462:Serum Albumin, Human