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

• Gene Information: Coagulation factor XI (FXI, encoded by F11) is a protein-coding gene located on chromosome 4q35.2. It encodes a circulating plasma serine protease that is a member of the coagulation protease family.
• Protein Expression: FXI zymogen is predominantly produced by hepatic hepatocytes and secreted into blood plasma under basal status; it is proteolytically cleaved into activated FXIa upon contact pathway stimulation during thrombotic or inflammatory injury.
• Signaling Pathway: Activated FXIa specifically cleaves and activates FIX to assemble tenase complex, which drives FX activation, amplifies prothrombin conversion into thrombin and augments overall clot formation via intrinsic coagulation cascade.
• Therapeutic Inhibition: Anti-FXI mAbs, FXI targeted nucleic acid drugs block hFXI synthesis or FXIa catalytic activity to restrain contact pathway-mediated thrombosis, with lower bleeding risk than classic vitamin K antagonists and DOACs.

The exons 1~15 of the mouse FXI gene that encode full-length molecule (ATG to STOP codon), including 3’UTR were replaced by human counterparts in B-hFXI mice. The human FXI expression is driven by the human FXI promoter and 5’UTR, while mouse FXI gene transcription and translation will be disrupted.

• Mouse FXI mRNA was detectable in wild-type C57BL/6 mice.
• Human FXI mRNA was only detectable in homozygous B-hFXI mice.

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

• Human FXI was exclusively detectable in homozygous humanized B-hFXI mice but not in wild-type mice.

Strain specific FXI expression analysis in wild-type C57BL/6JNifdc mice and homozygous humanized B-hFXI mice by ELISA. Serum were collected from wild-type mice C57BL/6JNifdc mice (+/+) (n=2, male, 7-week-old) and homozygous B-hFXI mice (H/H) (n=3, male, 7-week-old; n=3, female, 7-week-old), and analyzed by ELISA with species-specific FXI ELISA kit (Human Coagulation Factor XI ELISA Kit, Invitrogen, EH118RB). Human FXI was exclusively detectable in homozygous humanized B-hFXI mice but not in wild-type mice. Values are expressed as mean ± SEM.

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

The inhibitory efficiency of the FXI-targeted nucleic acid drugs in homozygous B-hFXI mice. B-hFXI mice were randomly divided into 2 groups (8-week-old, female). The human FXI-targeted nucleic acid drug (from a client) and saline were administered to the mice individually. The mice were sacrificed on day 30. (A) The expression levels of FXI protein in serum on days -7, 7, 14, and 30 after administration were compared to the levels before administration. The human FXI levels in the treatment group were reduced compared to the control group (G1). (B) Activated Partial Thromboplastin Clotting Time  (aPTT) assay on Day 30.

Values are expressed as mean ± SEM. Analyzed by 2 way-ANOVA, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

• Abelacimab analog markedly prolonged APTT relative to the control group.

APTT of anti-human FXI antibody in B-hFXI mice. B-hFXI mice were randomly divided into 6 groups (6-week-old, male). The anti-human FXI antibody abelacimab analog and PBS were administered to the mice individually. The mice were sacrificed on day 1 or day 7. (A) Activated Partial Thromboplastin Clotting Time  (aPTT) assay on Day 1. (B) Activated Partial Thromboplastin Clotting Time  (aPTT) assay on Day 7. The APTT in the treatment group were increased compared to the control group. Values are expressed as mean ± SEM. Analyzed by 2 way-ANOVA, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.