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

Blood Coagul Fibrinolysis. 2015 Dec;26(8):893-902. doi: 10.1097/MBC.0000000000000337.

Abstract

This report aims at exploring quantitatively the relationship between FXII inhibition and thromboprotection. FXII full and partial null in rats were established via zinc finger nuclease-mediated knockout and siRNA-mediated knockdown, respectively. The rats were subsequently characterized in thrombosis and hemostasis models. Knockout rats exhibited complete thromboprotection in both the arteriovenous shunt model (∼100% clot weight reduction) and the FeCl3-induced arterial thrombosis model (no reduction in blood flow), without any increase in cuticle bleeding time compared with wild-type control rats. Ex-vivo aPTT and the ellagic acid-triggered thrombin generation assay (TGA) exhibited anticoagulant changes. In contrast, ex-vivo PT or high tissue factor-triggered TGA was indistinguishable from control. Rats receiving single doses (0, 0.01, 0.03, 0.1, 0.3, 1 mg/kg) of FXII siRNA exhibited dose-dependent knockdown in liver FXII mRNA and plasma FXII protein (95 and 99%, respectively, at 1 mg/kg) at day 7 post dosing. FXII knockdown was associated with dose-dependent thromboprotection (maximal efficacy achieved with 1 mg/kg in both models) and negligible change in cuticle bleeding times. Ex-vivo TGA triggered with low-level (0.5 μmol/l) ellagic acid tracked best with the knockdown levels and efficacy. Our findings confirm and extend literature reports of an attractive benefit-to-risk profile of targeting FXII for antithrombotic therapies. Titrating of FXII is instructive for its pharmacological inhibition. The knockout rat is valuable for evaluating both mechanism-based safety concerns and off-target effects of FXII(a) inhibitors. Detailed TGA analyses will inform on optimal trigger conditions in studying pharmacodynamic effects of FXII(a) inhibition.

MeSH terms

  • Animals
  • Arteriovenous Shunt, Surgical
  • Chlorides / pharmacology
  • Disease Models, Animal
  • Dose-Response Relationship, Drug
  • Ellagic Acid / pharmacology
  • Endodeoxyribonucleases / genetics*
  • Endodeoxyribonucleases / metabolism
  • Factor XII / antagonists & inhibitors*
  • Factor XII / genetics
  • Factor XII / metabolism
  • Ferric Compounds / pharmacology
  • Gene Knockout Techniques
  • Hemorrhage / prevention & control
  • Humans
  • Liver / metabolism
  • Male
  • Partial Thromboplastin Time
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • RNA, Small Interfering / administration & dosage*
  • RNA, Small Interfering / genetics
  • RNA, Small Interfering / metabolism
  • Rats
  • Rats, Sprague-Dawley
  • Thrombin / metabolism
  • Thrombolytic Therapy / methods*
  • Thrombosis / chemically induced
  • Thrombosis / genetics
  • Thrombosis / pathology
  • Thrombosis / therapy*
  • Zinc Fingers / genetics

Substances

  • Chlorides
  • Ferric Compounds
  • RNA, Messenger
  • RNA, Small Interfering
  • Ellagic Acid
  • Factor XII
  • Endodeoxyribonucleases
  • Thrombin
  • ferric chloride