Abstract Number: PB0141
Meeting: ISTH 2020 Congress
Background: Inherited mono-allelic RUNX1 variants are associated with Familial Platelet Disorder with predisposition to Acute Myeloid Leukemia (FPD/AML), characterized by abnormal platelet function, and/or thrombocytopenia and up to 40% predisposition to neoplasia. Animal/cellular models would help to elucidate the pathogenicity and biological consequences of RUNX1 variants.
Aims: Generate a RUNX1 knock-in murine model by CRISPR/Cas9 technology, and assess its platelet and hemostatic phenotype.
Methods: Two gRNA against exon 2 of Runx1, Cas9 nickase protein, and DNA template carrying the mutation c.127-128>TC were microinjected into zygotes of C57BL/6J strain. Platelet dysfunction was evaluated by tail-bleeding time and flow cytometry assays: 1) CD41+ platelet count, 2) α and δ granules secretion (anti-CD62P, anti-CD63), 3) αIIbβ3 integrin activation (JON/A, fibrinogen-AF488), 4) platelet aggregation (anti-CD9). Adhesion and spreading were performed by confocal microscopy with phalloidin-514. Thrombin (0,5-1U/mL), PMA (3µM), and ADP (30µM) were used as agonists.
Results: We generate a murine model carrying the mutation c.127-128>TC, which leads to mice RUNX1 L43S variant, mimicking human L56S alteration. Twenty-five mice for genotype were investigated (WT, heterozygous: WT/L43S; homozygous: L43S/L43S).
RUNX1WT/L43S and RUNX1L43S/L43S animals showed statistically significant increase of bleeding-tail times compared to control animals. Homozygous mice displayed thrombocytopenia (Table1). RUNX1WT/L43S and RUNX1L43S/L43S vs. RUNX1WT/WT displayed impaired α-granule release, while there were no differences in δ-granule secretion. Moreover, agonist-induced αIIbβ3 integrin activation, fibrinogen-binding, and aggregation were significant impaired in platelets from RUNX1WT/L43S and RUNX1L43S/L43S (Table1, Figure1A,B,C). No differences in adhesion were found, while spreading was significantly reduced after thrombin stimulation in platelets from RUNX1WT/L43S and RUNX1L43S/L43S vs. control mice (Figure1D).
Conclusions: A knock-in RUNX1 murine model was successfully generated by CRISPR/Cas9, which allowed assessing the deleterious effect of the relatively common RUNX1 L56S variant. Noteworthy, both heterozygosis and homozygosis status associates with defective αIIbβ3 integrin activation, α-granule secretion and platelet spreading in the mice model.
Funding: PI17/01966,GRS2061A/19,FMM AP172142019,Premio López Borrasca 2019
|Bleeding time (s)||94,24 ± 14,16||154,4 ± 26,03; p = 0,048||170,08 ± 26,8; p = 0,016|
|Platelet count (x1000/µL)||587,16 ± 29,22||563,26 ± 28,17; p = 0,559||480,94 ± 28,19; p = 0,012|
|% α-granule secretion (thrombin 1U/mL)||31,88 ± 1,69||25,65 ± 1,76; p = 0,014||25,17 ± 0,78; p = 0,001|
|% α-granule secretion (PMA 3µM)||15,24 ± 0,8||12,53 ± 0,93; p = 0,032||11,77 ± 0,71; p = 0,002|
|% δ-granule secretion (T1U/mL)||5,19 ± 0,32||5,55 ± 0,26; p = 0,376||5,02 ± 0,5; p = 0,778|
|% δ-granule secretion (PMA 3µM)||2,08 ± 0,14||2,24 ± 0,27; p = 0,611||2 ± 0,18; p = 0,739|
|% αIIbβ3 integrin activation (T1U/mL)||36,11 ± 2,03||25,44 ± 2,17; p = 0,001||26,71 ± 1,06; p < 0,001|
|% αIIbβ3 integrin activation (PMA 3µM)||16,75 ± 0,9||12,03 ± 1,19; p = 0,003||10,6 ± 0,61; p < 0,001|
|% Active αIIbβ3 – fibrinogen binding (ADP 30µM)||9,51 ± 0,85||7,4 ± 0,53; p = 0,041||4,78 ± 0,38; p < 0,001|
[Table 1.Results of functional platelet assay. All comparisons were made between two experimental groups; p value is referenced to WT mice. Mean ± SEM]
To cite this abstract in AMA style:Marín-Quílez A, Benito R, García-Tuñón I, Fernández-Infante C, Hernández-Cano L, Vega-Marín A, Méndez-Sánchez L, del Rey M, Ordoñez JL, Palma-Barqueros V, Lozano ML, Sánchez-Martín M, Guerrero C, González Porras JR, Rivera J, Hernández-Rivas JM, Bastida JM. CRISPR/Cas9-Mediated Generation of a RUNX1 Knock-In Mice Model and Platelet Characterization [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/crispr-cas9-mediated-generation-of-a-runx1-knock-in-mice-model-and-platelet-characterization/. Accessed May 6, 2021.
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