Page 141 - Haematologica Vol. 107 - September 2022

ARTICLE - A mouse model of humanized type 2B VWD
S. Kanaji et al.
orated hGPIba shedding and the hGPIba expression was recovered on VWF2Bhet hGPIba mouse platelets (Figure 6B). It has been known that NMC-4 blocks VWF binding to GPIba, and we have previously reported that administra- tion of NMC-4 (0.8 mg/kg) prolongs bleeding time even in mice having normal sequence of humanized VWF exon 28 (VWFhA1 hGPIba).13 Thus, as predicted, despite correction of thrombocytopenia and hGPIba expression, tail bleeding time was still prolonged in VWF2Bhet hGPIba administered with NMC-4 (see Figure 1E). Interestingly, BM MK analysis performed after 4 doses of NMC-4 injection showed that MK ploidy shift observed with VWF2Bhet hGPIba mice was reversed by anti-VWF A1 blocking antibody (Figure 6C, compare with Figure 4B).
Discussion
Mouse models of type 2B VWD have been described using hydrodynamic injection of mutated mouse VWF ex- pression plasmids into VWF-/- mice.10,11 In these models, mutations were introduced into mouse VWF cDNA, and expressed ectopically in the liver, not in EC or MK. Subse- quently, a knockin mouse harboring a type 2B VWD mu- tation (p.V1316M) in mouse Vwf gene has been described.12,23 This model enabled physiologic expression in EC and MK and provided valuable information. However,
introduction of a single point mutation in the context of mouse VWF did not reproduce platelet phenotype in autosomal dominant manner and had to be bred into homozygous, suggesting that mutagenesis in mouse VWF A1 in association with mouse GPIba may not be identical to that of human. Here, we generated a type 2B VWD model with humanized VWF A1-GPIba interaction. Using the knockin strategy to replace Vwf exon 28 - encoding domains A1 and A2 – with that of the human homologue, we have generated a strain with a mutation (p.V1316M) in the sequence of human VWF A1. Subsequent cross-breed- ing with hGPIba transgenic mice successfully established a mouse model of type 2B VWD that present macrothrom- bocytopenia in autosomal dominant manner. Reciprocal HSC transplantation determined VWF of endothelial syn- thesis as the cause of platelet abnormalities. An open question is whether ADAMTS13 sensitivity is altered by Vwf exon28 replacement encoding A1 and A2 (ADAMTS13 cleavage) domains. Enhanced susceptibility of type 2B VWF by ADAMTS13 has been previously reported and the roles of ADAMTS13-mediated proteolysis in the loss of HMW VWF multimers, regulation of platelet aggregates, and impaired hemostasis have been well established.4,10 Further study is warranted to explore the alteration of ADAMTS13 sensitivity by humanizing VWF A1/A2 domains in VWFhA1 hGPIba mice and potential contribution to the phenotype of type 2B VWD model mice.
AB
C
Haematologica | 107 September 2022
2140
Figure 6. Inhibition of VWF A1-GPIbα reverses the effect of gain-of-function mutant von Willebrand factor on platelets and megakaryocytes. (A) A recombinant monoclonal antibody against von Willebrand factor (VWF) A1 which blocks binding to GPIba (NMC-4) was intravenously administered into VWF2Bhet hGPIba mice (2 mg/kg, n=6) daily, and platelet counts were monitored. Data were analyzed by RM one-way ANOVA without assuming sphericity and with Holm-Sidak’s post-test for com- paring pretreatment values with those at each day post injec- tion. (B) Representative profiles of blood flow cytometry analysis showing recovery of hGPIba expression on VWF2Bhet hGPIba mouse platelets after 4 doses of NMC-4 administration. (C) Bone marrow (BM) cells of VWF2Bhet hGPIba mice treated with daily NMC-4 administration were harvested on day 4 and analyzed by flow cytometry as in the Figure 4B. Data are shown as 25th-75th percentile boxes with min-to-max-whisker and analyzed by two-way ANOVA with Šidák’s multiple comparisons test. **P<0.01, ***P<0.001,****P<0.0001; only significant differ- ences are shown.