Characterization of the Platelet Phenotype Caused by a Germline RUNX1 Variant in a CRISPR/Cas9-Generated Murine Model

Ana Marín-Quílez; Ignacio García-Tuñón; Cristina Fernández-Infante; Luis Hernández-Cano; Verónica Palma-Barqueros; Elena Vuelta; Manuel Sánchez-Martín; José Ramón González-Porras; Carmen Guerrero; Rocío Benito; José Rivera; Jesús María Hernández-Rivas; José María Bastida

doi:10.1055/s-0041-1723987

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2021; 121(09): 1193-1205
DOI: 10.1055/s-0041-1723987

Cellular Haemostasis and Platelets

Characterization of the Platelet Phenotype Caused by a Germline RUNX1 Variant in a CRISPR/Cas9-Generated Murine Model

Ana Marín-Quílez

¹Cancer Research Center - CSIC, Instituto de Investigación Biomédica de Salamanca, University of Salamanca, Salamanca, Spain

,

Ignacio García-Tuñón

¹Cancer Research Center - CSIC, Instituto de Investigación Biomédica de Salamanca, University of Salamanca, Salamanca, Spain

,

Cristina Fernández-Infante

¹Cancer Research Center - CSIC, Instituto de Investigación Biomédica de Salamanca, University of Salamanca, Salamanca, Spain

,

Luis Hernández-Cano

¹Cancer Research Center - CSIC, Instituto de Investigación Biomédica de Salamanca, University of Salamanca, Salamanca, Spain

,

Verónica Palma-Barqueros

²Servicio de Hematología y Oncología Médica, Hospital Universitario Morales Meseguer, Centro Regional de Hemodonación, University of Murcia, Murcia, Spain

,

Elena Vuelta

¹Cancer Research Center - CSIC, Instituto de Investigación Biomédica de Salamanca, University of Salamanca, Salamanca, Spain

³Transgenic Facility, Nucleus, University of Salamanca, Salamanca, Spain

,

Manuel Sánchez-Martín

¹Cancer Research Center - CSIC, Instituto de Investigación Biomédica de Salamanca, University of Salamanca, Salamanca, Spain

³Transgenic Facility, Nucleus, University of Salamanca, Salamanca, Spain

⁴Department of Medicine, University of Salamanca, Salamanca, Spain

,

José Ramón González-Porras

⁴Department of Medicine, University of Salamanca, Salamanca, Spain

⁵Department of Hematology, University Hospital of Salamanca - IBSAL, Salamanca, Spain

,

Carmen Guerrero

¹Cancer Research Center - CSIC, Instituto de Investigación Biomédica de Salamanca, University of Salamanca, Salamanca, Spain

⁴Department of Medicine, University of Salamanca, Salamanca, Spain

,

Rocío Benito

¹Cancer Research Center - CSIC, Instituto de Investigación Biomédica de Salamanca, University of Salamanca, Salamanca, Spain

,

José Rivera^*

²Servicio de Hematología y Oncología Médica, Hospital Universitario Morales Meseguer, Centro Regional de Hemodonación, University of Murcia, Murcia, Spain

⁶On behalf of the “Grupo Español de Alteraciones Plaquetarias Congénitas (GEAPC)”, Hemorrhagic Diathesis Working Group, SETH

,

Jesús María Hernández-Rivas^*

¹Cancer Research Center - CSIC, Instituto de Investigación Biomédica de Salamanca, University of Salamanca, Salamanca, Spain

⁴Department of Medicine, University of Salamanca, Salamanca, Spain

⁵Department of Hematology, University Hospital of Salamanca - IBSAL, Salamanca, Spain

,

José María Bastida ^*

⁴Department of Medicine, University of Salamanca, Salamanca, Spain

⁵Department of Hematology, University Hospital of Salamanca - IBSAL, Salamanca, Spain

⁶On behalf of the “Grupo Español de Alteraciones Plaquetarias Congénitas (GEAPC)”, Hemorrhagic Diathesis Working Group, SETH

› Author Affiliations

Abstract

RUNX1-related disorder (RUNX1-RD) is caused by germline variants affecting the RUNX1 gene. This rare, heterogeneous disorder has no specific clinical or laboratory phenotype, making genetic diagnosis necessary. Although international recommendations have been established to classify the pathogenicity of variants, identifying the causative alteration remains a challenge in RUNX1-RD. Murine models may be useful not only for definitively settling the controversy about the pathogenicity of certain RUNX1 variants, but also for elucidating the mechanisms of molecular pathogenesis. Therefore, we developed a knock-in murine model, using the CRISPR/Cas9 system, carrying the RUNX1 p.Leu43Ser variant (mimicking human p.Leu56Ser) to study its pathogenic potential and mechanisms of platelet dysfunction. A total number of 75 mice were generated; 25 per genotype (RUNX1^WT/WT, RUNX1^WT/L43S, and RUNX1^L43S/L43S). Platelet phenotype was assessed by flow cytometry and confocal microscopy. On average, RUNX1^L43S/L43S and RUNX1^WT/L43S mice had a significantly longer tail-bleeding time than RUNX1^WT/WT mice, indicating the variant's involvement in hemostasis. However, only homozygous mice displayed mild thrombocytopenia. RUNX1^L43S/L43S and RUNX1^WT/L43S displayed impaired agonist-induced spreading and α-granule release, with no differences in δ-granule secretion. Levels of integrin α_IIbβ₃ activation, fibrinogen binding, and aggregation were significantly lower in platelets from RUNX1^L43S/L43S and RUNX1^WT/L43S using phorbol 12-myristate 13-acetate (PMA), adenosine diphosphate (ADP), and high thrombin doses. Lower levels of PKC phosphorylation in RUNX1^L43S/L43S and RUNX1^WT/L43S suggested that the PKC-signaling pathway was impaired. Overall, we demonstrated the deleterious effect of the RUNX1 p.Leu56Ser variant in mice via the impairment of integrin α_IIbβ₃ activation, aggregation, α-granule secretion, and platelet spreading, mimicking the phenotype associated with RUNX1 variants in the clinical setting.

Keywords

RUNX1 - inherited platelet disorders - platelet function - mouse model - CRISPR/Cas9

Full Text

References

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Supplementary Material

Supplementary Material