Page 135 - Haematologica Vol. 107 - September 2022

ARTICLE - A mouse model of humanized type 2B VWD
S. Kanaji et al.
VWF A1-GPIba interaction in vivo. In addition, mouse VWF A1-GPIba interaction was found not to be identical to that of human.10-12 In this study, we report the first mouse model of type 2B VWD having gain-of-function mutation in humanized VWF A1-GPIba interaction. We have em- ployed the strategy to target Vwf exon 28 to humanize VWF A1/A2 domains,13 and generated type 2B VWD model by mutagenizing the human VWF A1 with the p.V1316M substitution. This strain was bred with humanized GPIba transgenic strain to humanize VWF A1-GPIba interaction in vivo. As expected, this model closely recapitulated pla- telet phenotype of human type 2B VWD in autosomal dominant manner and was proven to be a valuable tool to study the biological effect of VWF A1-GPIba interaction in vivo.
Methods
Animal experiments
All animal procedures were performed in accordance with the National Institute of Health Guideline for the Care and Use of Laboratory Animals. All protocols for animal studies were approved by the Institutional Animal Care and Use Committees (IACUC) of The Scripps Research Institute and Medical College of Wisconsin.
Generation of human von Willebrand factor exon 28 knockin mice with p.V1316M mutation and cross- breeding with human GPIbα transgenic mice
A new knockin strain having type 2B VWD mutation was generated at the Transgenic Core Facility, Blood Research Institute/Medical College of Wisconsin. The targeting vec- tor previously used to generate a knockin mouse with Vwf exon28 replaced by human homolog (VWFhA1) was used with additional mutagenesis of p.V1316M in the A1 do- main.13 Correctly targeted embryonic stem cells were in- jected into blastocyst stage embryos to generate chimeric mice. In order to remove the loxP-flanked Neo cassette, these mice were bred with B6.FVB-Tg (EIIa-Cre) C5379Lmgd/J mice (The Jackson Laboratory) and then crossed with the human GPIba transgenic mouse strain in which platelets express only human GPIba in the GPIb- IX-V complex. The knockin strain having human VWF exon 28 with p.V1316M substitution was bred into homozygous (VWF2Bhomo hGPIba). VWF2Bhomo hGPIba strain was bred with previously generated VWFhA1 hGPIba strain to generate VWF2Bhet hGPIba mice having heterozygous p.V1316M mu- tation.
Platelet count analysis
For blood cell counting, blood obtained by retro-orbital bleeding were analyzed using the IDEXX ProCyteTM (IDEXX Laboratories Inc., Westbrook, ME).
NMC-4 injection study
Monovalent Fab fragment14 expressed in D. melanogaster S2 cells was purified from culture supernatant and ad- ministered (2 mg/kg) by retro-orbital vein injection for 4 consecutive days.
Bone marrow hematopoietic stem cell transplantation
Bone marrow (BM) cells were collected from femurs and tibia of donor mice, and BM mononuclear cells (BM MNC) were isolated using ficoll as described previously.15,16 Six- to 8-week-old recipient mice were conditioned with a lethal dose of 1,100 cGy total body irradiation using a Gam- macell 40 Exactor cesium irradiator. Twenty-four hours after irradiation, a dose of 1×107 BM MNC was infused by retro-orbital vein injection. Recipients were analyzed be- ginning at 6 weeks after transplantation.
Plasma von Willebrand factor analysis
VWF antigen (VWF:Ag) was measured by enzyme-linked immunosorbent assay (ELISA) using a rabbit anti-human VWF polyclonal antibody (IgG) cross-reacting with mouse VWF (produced at the Scripps Research Institute).16 Nor- mal pooled plasma from C57BL/6J wild-type (WT) mice was used as a reference and defined as 1 U/mL. VWF multimers were analyzed by electrophoresis through 1% HGT(P) agarose containing 0.1% sodium dodecyl sulfate followed by western blotting with the same antibodies used for ELISA.13
Flow cytometry analysis
Mouse blood samples were collected from the retro-or- bital plexus using sodium citrate as anti-coagulant. Blood samples were diluted in phosphate-buffered saline (pH 7.4) containing 2 mM ethylenediaminetetraacetic acid. Samples were stained with anti-CD41 (integrin aIIb) (MWReg30, Biolegend, San Diego, CA), anti-GPIba (LJ-P3 for hGPIba, 5A7 for mGPIba17, MERU-VasImmune, San Diego, CA), anti-VWF [polyclonal antibodies13]). Samples were analyzed using a NovoCyte flow cytometer (ACEA Biosciences). The results were analyzed with FlowJo v.10.7.1.
Results
Generation and characterization of type 2B von Willebrand disease model mice with humanized von Willebrand factor A1-GPIbα interaction
We have previously generated a mouse strain expressing human VWF A1/A2 in the context of mouse VWF and human GPIba (VWFhA1 hGPIba).13 In the current study, the same strategy was employed to generate a strain having type 2B VWD mutation (p.V1316M) in human VWF exon 28 (encoding A1/A2 domains). The substitution p.V1316M
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