FXII: A key coagulation factor in thrombosis and its therapeutic intervention

• Gene Information: Coagulation factor XII (FXII, encoded by F12) is a protein-coding gene located on chromosome 5q35.3. It encodes a circulating plasma serine protease belonging to the contact activation protease subset of the coagulation protease family.
• Protein Expression: Hepatocytes constitutively synthesize and secrete FXII zymogen into plasma. Binding to anionic surfaces during vascular injury, inflammation or foreign material contact triggers autoactivation into FXIIa.
• Signaling Pathway: FXIIa activates FXI to initiate intrinsic coagulation cascade for thrombin generation and fibrin clot formation; it also cleaves prekallikrein to drive kinin-related inflammation linked to thrombosis.
• Therapeutic Inhibition: FXII-targeted nucleic acids curb contact-pathway thrombosis with low bleeding risk; anti-FXII antibodies block excessive bradykinin to prevent HAE attacks.

Gene targeting strategy for B-hFXII mice plus. •The exons 1~12 of the mouse FXII gene were replaced by the exons 1~14 of human FXII gene in B-hFXII mice plus. The promoter, 5’UTR and 3’UTR region of the mouse gene are replaced by human counterparts.

• Mouse FXII mRNA was detectable in wild-type C57BL/6 mice.
• Human FXII mRNA was only detectable in heterozygous B-hFXII mice plus.

Strain specific analysis of FXII mRNA expression in wild-type C57BL/6 mice and B-hFXII mice plus by RT-PCR. Liver RNA were isolated from wild-type C57BL/6 mice (+/+) and heterozygous B-hFXII mice plus (H/+), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human FXII primers. Mouse FXII mRNA were detectable in wild-type C57BL/6 and heterozygous B-hFXII mice plus. Human FXII mRNA was only detectable in heterozygous B-hFXII mice plus.

• Human FXII was exclusively detectable in homozygous humanized B-hFXII mice plus but not in wild-type mice.

Strain specific FXII expression analysis in homozygous B-hFXII mice plus by ELISA. Serum were collected from wild-type mice C57BL/6 mice (+/+) (n=3, male, 6 weeks old) and homozygous B-hFXII mice plus (H/H) (n=3, male, 6 weeks old), and analyzed by ELISA with species-specific Human Factor XII ELISA Kit  (Abcam, ab192144) and Mouse FXII ELISA kit (Abcam, ab272776). (A) Mouse FXII was only detectable in wild-type mice. (B) Human FXII was exclusively detectable in homozygous B-hFXII mice plus but not in wild-type mice. Values are expressed as mean ± SEM.

• FXII expression is significantly downregulated by the human FXII-targeted nucleic acid drug.

The inhibitory efficiency of the FXII-targeted nucleic acid drugs in homozygous B-hFXII mice plus. B-hFXII mice plus were randomly divided into 4 groups (6-week-old, female). The human FXII-targeted nucleic acid drug (synthesized according to a patent) and saline were administered to the mice individually. The mice were sacrificed on day 7 or day 30. (A) The expression levels of FXI protein in serum on days -7, 7, 14, 21, 28 and 32 after administration were compared with the levels before administration. The human FXII levels in the treatment group (G4) were reduced compared to the control group (G3). (B) Activated Partial Thromboplastin Clotting Time  (aPTT) assay on Day 7 and Day 32. The APTT in the treatment group (G2) was significantly prolonged compared to the control group (G1). Values are expressed as mean ± SEM. Analyzed by 2 way-ANOVA, *P<0.05, **P<0.01, ***P<0.001.