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Abstract

  1. Top of page
  2. Abstract
  3. Disclosure of Conflict of Interests
  4. References

See also Booth CJ, Brooks MB, Rockwell S, Murphy JW, Rinder HM, Zelterman D, Paidas MJ, Compton SR, Marks PW. WAG-F8m1Ycb rats harboring a factor VIII gene mutation provide a new animal model for hemophilia A. This issue, pp 2472–7.

During the Middle Ages, Europe’s rulers established menageries at their courts, wherein unusual or exotic animals were collected and displayed for private amusement or contemplation. In the eighth century, Nijmegen in the present-day Netherlands housed the Emperor Charlemagne’s menagerie. The menagerie of the Tower of London, England, established in 1204, was perhaps the last great royal menagerie. When the Tower Menagerie was closed in 1835, 2 years before Queen Victoria ascended the English thrown and 5 years before the birth of her first child, there is no evidence that any of the menagerie animals were subsequently used for the study of hemophilia, the condition inherited by Victoria’s descendents. Nevertheless, it is undoubtedly the case that the establishment of colonies of hemophilia A dogs one century later, along with the careful experimental observation of these animals, greatly accelerated the understanding of the mode of inheritance, the pathophysiology, the diagnosis (including the development of the aPTT) and the treatment of hemophilia [1,2]; naturally-occurring or engineered mouse, pig and other models of bleeding and thrombotic disorders have since been added to the fold. In this issue of the Journal of Thrombosis and Haemostasis, Booth et al. [3,4] describe the underlying genetic basis and the coagulation profile of a novel animal model of hemophilia A, which has arisen as a spontaneous mutation in an established colony of laboratory rats. Along with the re-established line of sheep with hemophilia A, reported in this journal by Porada et al. [5] earlier this year, investigators can now choose from a menagerie of hemophilia models to find the most ideal to answer a given research question.

In reporting the hemophilia A rat, the authors suggest the rat model will be valuable for assessing novel therapies. For decades, therapeutic efficacy in hemophilic dogs has proved to be an excellent predictor of human clinical efficacy. The availability of hemophilic mice for screening pharmacokinetics and pharmacodynamics of potential protein and gene therapeutics prior to scaling up to dogs has recently proven valuable. These mouse studies, however, need to limit repetitive collection volumes because of the small plasma volume of the mouse, and a rat severe hemophilia model with a larger plasma volume and the favorable breeding capacity of mice is likely to be valuable.

Nevertheless, the characterization of the factor VIII deficiency in this rat model as reported in the accompanying article is not complete. Interestingly, the authors report a marked discrepancy between very mildly deficient factor VIII levels measured with a one-stage factor VIII activity assay and severely deficient levels measured with a two-stage chromogenic assay, although the observed bleeding phenotype is severe. Although hemophilia A associated with discrepancies between one- and two-stage activity assays is well described, that phenotype is associated with factor VIII mutations that cause accelerated disassociation of the factor VIII at or closely adjacent to the interface of subunits A1 and A2 (e.g. FVIII Arg531His; FVIII Ala284Glu) [6,7]. Instead, the factor VIII exon IV missense mutation identified in the rat model and the resulting Leu176Pro substitution disrupts the A1 subunit at considerable distance from the A1 to A2 interface. Probably, the one-stage assay is instead sensitive to artefacts arising from the use of available human plasma reagents to perform the assay, in particular in the construction of the standard curve. Such an artefact led to the appearance of up to 8% activity in the one-stage factor assay in factor IX knockout mice (despite the mouse having no factor IX mRNA transcript) [8], and the artefact was eliminated after factor IX-deficient mouse plasma became available for inclusion in the assay dilutions. Additionally, reagents need to be identified to establish whether the rat model has circulating defective but antigenically reactive factor VIII protein (CRM+ hemophilia) and whether this rat strain tends to develop inhibitory antibodies after exposure to human factor VIII, as the hemophilia A mouse and dog models do. Until these parameters are established, the ability to perform meaningful in vivo recovery studies, or repeat dose kinetics, will be hampered. The potential for inhibitor development in both the rat and sheep models following a clinically significant number of exposure days must be established, not only to determine whether PK/efficacy studies will be confounded, but also to judge the niche for these animals in the study of factor VIII immunologic tolerance and gene correction.

A significant research investment is required to develop species-appropriate reagents, assays and experience before the interpretation of results generated in rats and sheep is as comfortable as interpreting data generated in hemophilic dogs (60 years experience) or hemophilic mice (15 years). Nevertheless, the accompanying table (Table 1) [2,3,5,9–14] delineates differences between these model species in normal development and in clinical phenotype that suggest the new rodent and large animal models will reveal their own unique study opportunities. As an example, the study of hemophilic arthropathy in severely deficient hemophilia A mice and dogs is currently hampered by the relatively infrequent occurrence of clinically recognizable spontaneous joint bleeding. Induced joint bleeding in hemophilic mice does accurately reproduce the histopathologic sequelae of hemophilic joint bleeding, although requiring a significant trauma [15]. Additionally, the ability to study tissues of the articular space and synovial fluid over time and without sacrificing the animal is very challenging using mice. Hemophilia A rats and sheep frequently present with apparently spontaneous hemarthropathy and may find a special niche in advancing understanding and treatment of this major morbidity of hemophilia. These novel models are certainly a welcome addition to the zoo. An additional severe hemophilia A transgenic mouse model has been generated expressing human factor VIII mRNA carrying an Arg593Cys substitution. Although not discussed, the model is of interest, particularly for studies that investigate or require factor VIII tolerance [16].

Table 1.   Experimental animals with congenital factor VIII deficiency: characteristics guiding choice of hemophilia A model
 MouseRatDogSheep
  1. *Level in hemizygous male and homozygous female hemophilic animals. Level in homozygote males and females; human plasma standard curve. Human reference plasma used to construct the standard curve. §Pooled normal sheep plasma used to construct the standard curve. Authors report 2.3% = the level of sensitivity of their one-stage FVIII assay. Newly engineered colony; initial line derived from semen from single male founder.

Weight: birth≤1 g5–6 g300–500 g∼3.5 kg
Weight: adulthood25–30 g200–250 g10–15 kg (Queens) 25–30 kg (Chapel Hill, UAB)∼70–75 kg
Total blood volume (adult)1.8–2 mL∼14.5 mL>1.0–1.5 L (∼0.06 × body weight)∼4.5 L (∼0.06 × body weight)
Lifespan∼2 years3+ years>10 years w/plasma replacement therapy <4 years prior to era of hemostatic support≥8–10 years (normals ) Not established for hemophilic colony
Age at skeletal maturity∼20 weeks140–170 days, epiphyseal closure at some sites delayed >1 year6–12 months3 years
Age at sexual maturity
 Reproductive age: Male5–8 weeks50–60 days∼5 months∼6–8 months
 Reproductive age: Female5–8 weeks35–90 days∼5–9 months ∼8 months
Gestational period19–20 days20–22 days63 days145–155 days
Litter size (average)4–8 pups8–14 pups6–7 pups1–2 (per year)
Litters/year4–5+7–10 (lifetime)11–2 (per year)
Weaning age3–4 weeks3 weeks3–6 weeks2–3 months
FVIII gene locationSex chromosome (chr X)Autosome (chr 18)Sex chromosome (chr Xqter)Sex chromosome (chr X)
Degree of a.a. sequence identity With human factor VIII protein
 Overall74%51%>80%>71%
 A1 Region 85%67%84%81%
 A2 Region85%71%89%88%
 B Region55%26%62%47%
 A3 Region 90%69%87%87%
 C1 Region93%76%92%90%
 C2 Region84%66%83%86%
FVIII gene mutationdel exon 16 or exon 17Point mutation nt578(t[RIGHTWARDS ARROW]c) in exon 4Intron 22 inversion12 nt substitution and frameshift; creates 5 stop codons in exon 14
ResultFVIII major deletionmissense Leu193Pro (equiv human FVIII a.a.176)Large deletionLarge deletion
Reported FVIII Antigen*<5 ng/mLNot assayedNot detectableNot assayed
Reported FVIII activity (Assay)
 1-stage FVIII assay<0.01–0.02 U/mL FVIII*0.28 U/mL FVIII<0.01 U/mL*≤2.3% FVIII§
 2-stage FVIII assay<0.01 U/mL FVIII1*<0.02 U/mL FVIIINot detectable*Not assayed
Clinical bleeding
 Spontaneous0–20%YesYesYes
 Trauma-inducedYesYesYesYes
 HemarthrosisRareCommon (tarsal joint)Yes (hemarthropathy develops over years)Frequent
 Peripartum
  DamsUncommonMay occurRareNot observed
  NeonatesUncommonCommonCommon prior to plasma replacement therapyUmbilical, abdominal
Inhibitors after hFVIII exposureYesNot assayedYesUsually
Outbred/InbredInbredInbredOutbredRelatively inbred
Cost (per diem)LowLowHighHigh
Species-appropriate assays/reagentsAvailableIn developmentAvailableIn development

Disclosure of Conflict of Interests

  1. Top of page
  2. Abstract
  3. Disclosure of Conflict of Interests
  4. References

The authors states that he has no conflict of interest.

References

  1. Top of page
  2. Abstract
  3. Disclosure of Conflict of Interests
  4. References
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