Page 266 - Haematologica April 2020

L. Zheng et al.
event.19,20 A13-/- mice rarely develop spontaneous TTP unless they are challenged by a bacterial shigatoxin21,22 or a large dose of recombinant VWF.23,24 Baboons with acquired A13 deficiency develop features of TTP, but not a fatal condition.25 These findings indicate that additional envi- ronmental or genetic factors may be necessary for trigger- ing severe TTP on top of A13 deficiency.
The potential triggers may be infection, inflammation, and pregnancy, etc.26 Infections or inflammation, including systemic lupus erythematosus, may cause activation of neutrophils, resulting in cell death – a process termed NETosis.27 This may lead to release of neutrophil granular contents including human neutrophil peptides (HNP),28 myeloperoxidase,29 and histone-DNA complexes.27 The plasma levels of HNP1-3,30,31 histone-DNA complexes,29 and other inflammatory mediators are significantly increased in patients with acute immune-mediated TTP. HNP1-3 may be prothrombotic31,32 or anti-thrombotic,33 depending on the context and their redox status. Increased plasma levels of histone-DNA complexes correlate with low platelet counts and disease severity.29 However, no direct evidence is available to date to support a causative role for any of these inflammatory mediators in the patho- genesis of TTP.
To test a hypothesis that inflammatory mediators, such as histones, may play a crucial role in triggering TTP when there is a severe deficiency of A13, we generated several novel zebrafish lines with a null mutation in a13, vwf, and both using CRISPR/Cas9. These novel models were then used to assess the role of a lysine-histone, which is known to activate endothelial exocytosis, in triggering acute TTP. Zebrafish have been extensively used for modeling human diseases,34 including thrombosis and hemostasis.35- 37 Zebrafish provide advantages over other animal models in terms of speed, cost, and high-throughput capability, enabling rapid assessment of the role of various environ- mental and genetic factors in triggering TTP and identifi- cation of novel therapeutics for TTP and perhaps other arterial thrombotic or inflammatory disorders.
Methods
Zebrafish
Zebrafish (Danio rerio) were used according to the protocol approved by Institutional the Animal Care and Use Committee. The guide RNA (gRNA) was designed using the CRISPR design tool (http://crispr.mit.edu/). A 69-nt oligonucleotide, consisting of a T7 promoter, a target sequence, and a gRNA scaffold, was synthe- sized (ThermoFisher, Waltham, MA, USA). The gRNA was then generated using a Guide-it sgRNA transcription kit (Takara- Clontech, Mountain View, CA, USA). The Cas9 mRNA was syn- thesized from pT3TS-nCas9n using the mMESSAGE mMACHINE T3 kit (Life Technologies, Carlsbad, CA, USA). The final products (2 nL with 12.5 pg/nL gRNA and 300 pg/nL Cas9 mRNA) were co-injected into one-cell stage embryos of a double transgenic zebrafish (gata1-dsRed and fli1-eGFP).38,39
Western blotting
A capillary-based western blotting system (ProteinSimple, San Jose, CA, USA) was used to determine the presence of a13 protein in plasma and embryo lysate of zebrafish. The antibody was gen- erated commercially (ABmart, Shanghai, China) by immunization of mice with nine synthetic peptides of zebrafish a13 protein (Online Supplementary Table S2).
Agarose gel electrophoresis
Plasma VWF multimers were determined by western blotting with anti-vwf IgG raised against zebrafish vwf peptides (ABclonal, Woburn, MA, USA) (Online Supplementary Figure S2) after elec- trophoresis on a 1.5% sodium dodecylsulfate-agarose gel.31
Microfluidic assay
Microchannels (Fluxion Bioscience, San Francisco, CA, USA) were coated with a fibrillar collagen (100 μg/mL). The surface was blocked with 0.5% bovine serum albumin. Pooled whole blood collected from ten adult zebrafish and anticoagulated with PPACK (100 μM) was diluted with 50 μL of phosphate-buffered saline (PBS) and perfused under 15 dyne/cm2 over the collagen surface. The digital images were collected every 3 seconds for 120 seconds.
Administration of a lysine-rich histone
PBS or lysine-rich histone (H5505) (Sigma-Aldrich) (200 mg/kg body weight) was injected intraperitoneally into adult zebrafish.
Blood cell counts
Zebrafish whole blood was diluted (1:51) with PBS (containing 4 mM EDTA). The total cell counts (per μL of blood) were deter- mined using a Hemavet 950FS Hematology Analyzer (Drew Scientific, Miami Lakes, FL, USA). Additionally, flow cytometry (BD Biosciences, San Jose, CA, USA) was performed to differenti- ate erythrocytes, immature thrombocytes, and mature thrombo- cytes. The percentages of erythrocytes and thrombocytes were determined in 50,000 cells per sample. The number of thrombo- cytes per liter of whole blood was calculated using the formula: Thrombocyte count=Total cell counts per liter x Thrombocyte percentage x Dilution factor.
Histological assessments
Zebrafish were fixed in 4% paraformaldehyde in PBS and embedded in paraffin for hematoxylin and eosin staining. Immunohistochemistry was then performed on the fixed tissue sections with a costumer-made rabbit anti-zebrafish vwf IgG (ABclonal, Woburn, MA, USA), followed by a horseradish perox- idase-conjugated anti-rabbit IgG and color reaction.
Statistical analysis
All data are presented as the mean ± standard errors (SEM) or the median in a box-whisker plot unless specified in the figure leg- ends. Kaplan-Meier survival analysis was performed with a log- rank test. The Mann-Whitney test was used to determine the dif- ference between two groups, while Krüskal-Wallis analysis was used to test the significance for more than three groups. All statis- tical analyses were carried out using Prism 7 software.
Results
Generation and characterization of a13-/- zebrafish
To generate a stable a13-/- zebrafish line, an in vitro tran- script consisting of gRNA and Cas9 mRNA that targets a signal peptide of zebrafish a13 protein (Figure 1A) was injected into one-cell embryos of a double transgenic zebrafish (gata-1/dsRed and fli-1/eGFP) which expresses a red fluorescent protein under the gata-1 promoter in ery- throcytes and immature thrombocytes and a green fluo- rescent protein under the fli-1 promoter in the entire vas- culature and thrombocytes. Of 22 F0 founders randomly selected, 19 demonstrated the formation of heteroduplex- es consistent with a heterozygous allele of a13 (Online Supplementary Figure 1A). The F0 founders were then out-
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