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Timely and large dose of clotting factor IX provides better joint wound healing after hemarthrosis in hemophilia B mice

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Abstract

Bleeding into the joints represents the major morbidity of severe hemophilia and predisposes it to hemophilic arthropathy (HA). In a reproducible hemarthrosis mouse model, we found distinct changes in thrombin activity in joint tissue homogenate following exposure of the joint to blood in wide type (WT) and hemophilic B mice. Specifically, at early time points (4 h and 24 h) after hemarthrosis, thrombin activity in WT mice quickly peaked at 4 h, and returned to baseline after 1 week. In hemophilia B mice, there was no/minimal thrombin activity in joint tissues at 4 h and 24 h, whereas at 72 h and thereafter, thrombin activity kept rising, and persisted at a higher level. Nevertheless, prothrombin had not decreased in both WT and hemophilia. The pattern was also confirmed by Western blotting and immunostaining. To optimize the protection against development of HA, we tested different treatment regimens by administration of clotting factor IX into hemophilia B mouse after hemarthrosis induction, including a total of 600 IU/kg FIX within the first 24 h or the whole 2-week period. We concluded that timely (in the first 24 h) and sufficient hemostasis correction is critical for a better protection against the development of hemophilic arthropathy.

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Acknowledgements

The authors acknowledge the Animal Histopathology and Laboratory Medicine Core at UNC-CH for histology processing. This work was partly supported by a research grant from Asklepios BioPharmaceutical (to J.S.). H.B.L. is supported by Beijing Municipal Natural Science Foundation (No 7162151) and the Novo Nordisk Hemophilia China Research Fund. It is also supported by “the Fundamental Research Funds for the Central Universities”. Parts of work were supervised by Dr. Paul E Monahan during his work in UNC-Chapel Hill. We acknowledge the language editing from Editage (http://www.editage.com).

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  1. Ping He and Feixu Zhang have contributed equally to the study.

Authors and Affiliations

  1. Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Shanghai, China

    Ping He, Feixu Zhang, Chen Zhong, Min Li, Jing Zheng & Junjiang Sun

  2. Department of Hematology, Clinical Medical College, Yangzhou University, Yangzhou, China

    Baolai Hua

  3. Gene Therapy Center, University of North Carolina, Chapel Hill, NC, USA

    Junjiang Sun

  4. Division of Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC, USA

    Junjiang Sun

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  1. Ping He
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12185_2019_2639_MOESM1_ESM.pptx

Supplemental Figure: Dynamic changes of Tissue Factor expression in synovium after hemarthrosis induction in hemophilic mice Joint hemorrhage was induced in FIX KO mice. Day 1 to 14 d post joint bleeding, joints were collected, fixed, decalcified and sectioned for immunostaining for tissue factor. Representative images were shown. Naïve FIX KO mice served as “D0” controls. Upper panels: × 4 magnification, lower panels: × 40 magnification

Supplementary Materials: Materials and Methods: TF IHC staining

: Antigen retrieval was performed by heating the sections at 95 ºC in 10 mM citrate buffer (pH 6.0) for 20 min. Endogenous peroxidase activity was blocked by incubation in Dako Dual endogenous enzyme Block solution (Dako, Carpinteria, CA, USA). Binding of primary antibody, rabbit anti-mouse tissue factor IgG (American Diagnositca, Stanford, CT, USA), was detected with a biotinylated goat anti-rabbit antibody (Vector Labs, Burlingame, CA, USA), which employed avidin and biotin and was visualized with diaminobenzidine (DAB) substrate from Dako

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He, P., Zhang, F., Zhong, C. et al. Timely and large dose of clotting factor IX provides better joint wound healing after hemarthrosis in hemophilia B mice. Int J Hematol 110, 59–68 (2019). https://doi.org/10.1007/s12185-019-02639-5

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