Every year on April 17th, we observe World Hemophilia Day, focusing on this rare inherited bleeding disorder, often referred to as the "glass man" disease. In people with hemophilia, the synthesis or function of key clotting factors is impaired, interrupting the coagulation cascade at a critical node in the intrinsic pathway. This prevents the completion of the final "thrombin burst," leading to failure of the physiological hemostatic mechanism. Consequently, even minor trauma can trigger uncontrolled bleeding. Today, let's delve deeper into the pathogenesis of hemophilia and the mouse models used in related research.
 

What is Hemophilia?
 

Hemophilia is an X-linked recessive bleeding disorder. It is caused by pathogenic variants in either the F8gene on Xq28 or the F9gene on Xq27, leading to impaired synthesis or function of coagulation factor VIII (FVIII) or factor IX (FIX), respectively. Clinically, it manifests as spontaneous or trauma-induced bleeding due to impaired thrombin generation. The global estimated prevalence is approximately 17.1 per 100,000 male residents.
 

Hemophilia A (HA), caused by FVIII deficiency, accounts for 80%-85% of cases, while Hemophilia B (HB), caused by FIX deficiency, accounts for 15%-20%. Based on plasma coagulation factor activity levels (FVIII:C/FIX:C), it is classified as severe (<1 IU/dL), moderate (1-5 IU/dL), or mild (5-40 IU/dL).

Image source: Insights into the Molecular Genetic of Hemophilia A and Hemophilia B: The Relevance of Genetic Testing in Routine Clinical Practice
 

Pathogenesis
 

The core pathological mechanism of hemophilia is the interruption of the coagulation cascade's intrinsic pathway at the stage of the thrombin burst. In normal hemostasis, activated factor IXa (FIXa) requires its cofactor FVIIIa, Ca²⁺ ions, and a phospholipid surface (usually platelets) to form the intrinsic tenase complex. This complex catalyzes the activation of factor X (FX) to FXa with extremely high efficiency. FXa then drives the assembly of the prothrombinase complex, enabling explosive generation of thrombin, ultimately leading to the formation of a stable cross-linked fibrin clot.

Image source: MSD Manuals
 

Hemophilia A​ is primarily caused by mutations or deletions in the F8gene leading to coagulation factor VIII deficiency. FVIII is a glycoprotein with a molecular weight of approximately 330 kDa, mainly synthesized by hepatocytes. In the coagulation process, FVIII acts as a cofactor for FIXa, activating factor X in the presence of Ca²⁺ ions and phospholipids, allowing the coagulation cascade to proceed. When FVIII activity is reduced, thrombin generation is delayed, fibrin formation is delayed, leading to prolonged bleeding.
 

Hemophilia B​ is mainly caused by mutations in the F9gene leading to coagulation factor IX deficiency. FIX is a vitamin K-dependent serine protease with a molecular weight of about 56 kDa, synthesized in the liver. In the coagulation cascade, FIX, with Ca²⁺ ions, membrane phospholipids, and FVIII, activates factor X. When FIX is deficient, the intrinsic pathway is blocked, and the activated partial thromboplastin time (aPTT) is significantly prolonged.
 

Normal coagulation requires factor VIII and IX levels >30% of normal. Due to the deficiency of these key factors, people with hemophilia have impaired intrinsic pathway thromboplastin generation, leading to prolonged clotting time. When factor activity is below 1%, spontaneous bleeding occurs, especially in joints and muscles.
 

Image source: Based on a population PK model to evaluate the relationship between FVIII activity levels and bleeds
 

Mouse Models
 

F8 KO Mice:​ Knockout of the coagulation factor VIII(F8) gene (e.g., exon 17), leading to loss of FVIII function. Mice exhibit prolonged clotting time and internal bleeding. Used to evaluate the efficacy of therapeutic strategies and technologies for FVIII deficiency.
 

F9 KO Mice:​ Knockout of the coagulation factor IX(F9) gene, leading to loss of FIX function. Symptoms are generally milder compared to hemophilia A mice.
 

F11 KO Mice:​ Knockout of the coagulation factor XI(F11) gene, leading to FXI deficiency. Bleeding symptoms are even milder compared to hemophilia A and B.
 

R333Q-hFIX Mice:​ Generated by targeted knock-in of a human F9gene carrying the R333Q missense mutation. They express an antigenic but inactive FIX protein (CRM⁺), used to model a "low immunogenicity" phenotype with a low risk of inhibitor development.
 

R29X-hFIX Mice:​ Carry the R29X nonsense mutation, leading to complete absence of FIX protein (CRM⁻). This is a key model for evaluating treatment safety in high-risk genotypes.
 

Supporting Gene Therapy
 

Gene therapy offers hope for rare diseases, but its development and validation rely heavily on animal model support. MingCeler Biotech, leveraging its self-developed TurboMice™ technology, has developed multiple rare disease mouse models. TurboMice™ technology overcomes the technical challenges of long mouse model generation cycles and low success rates for complex models, enabling editing at almost any target gene locus. Complete homozygous gene-edited mouse models can be prepared directly from embryonic stem cells in as little as two months.
 

MingCeler Biotech​ can customize various hemophilia-related mouse models according to client needs, such as F8 KO mice, F9 KO mice, F11 KO mice, R333Q-hFIX mice, and R29X-hFIX mice. Inquiries are welcome.