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
1  Cancer Research Center - CSIC, Instituto de Investigación Biomédica de Salamanca, University of Salamanca, Salamanca, Spain
,
1  Cancer Research Center - CSIC, Instituto de Investigación Biomédica de Salamanca, University of Salamanca, Salamanca, Spain
,
Cristina Fernández-Infante
1  Cancer Research Center - CSIC, Instituto de Investigación Biomédica de Salamanca, University of Salamanca, Salamanca, Spain
,
Luis Hernández-Cano
1  Cancer Research Center - CSIC, Instituto de Investigación Biomédica de Salamanca, University of Salamanca, Salamanca, Spain
,
Verónica Palma-Barqueros
2  Servicio de Hematología y Oncología Médica, Hospital Universitario Morales Meseguer, Centro Regional de Hemodonación, University of Murcia, Murcia, Spain
,
Elena Vuelta
1  Cancer Research Center - CSIC, Instituto de Investigación Biomédica de Salamanca, University of Salamanca, Salamanca, Spain
3  Transgenic Facility, Nucleus, University of Salamanca, Salamanca, Spain
,
Manuel Sánchez-Martín
1  Cancer Research Center - CSIC, Instituto de Investigación Biomédica de Salamanca, University of Salamanca, Salamanca, Spain
3  Transgenic Facility, Nucleus, University of Salamanca, Salamanca, Spain
4  Department of Medicine, University of Salamanca, Salamanca, Spain
,
José Ramón González-Porras
4  Department of Medicine, University of Salamanca, Salamanca, Spain
5  Department of Hematology, University Hospital of Salamanca - IBSAL, Salamanca, Spain
,
Carmen Guerrero
1  Cancer Research Center - CSIC, Instituto de Investigación Biomédica de Salamanca, University of Salamanca, Salamanca, Spain
4  Department of Medicine, University of Salamanca, Salamanca, Spain
,
Rocío Benito
1  Cancer Research Center - CSIC, Instituto de Investigación Biomédica de Salamanca, University of Salamanca, Salamanca, Spain
,
José Rivera*
2  Servicio de Hematología y Oncología Médica, Hospital Universitario Morales Meseguer, Centro Regional de Hemodonación, University of Murcia, Murcia, Spain
6  On behalf of the “Grupo Español de Alteraciones Plaquetarias Congénitas (GEAPC)”, Hemorrhagic Diathesis Working Group, SETH
,
Jesús María Hernández-Rivas*
1  Cancer Research Center - CSIC, Instituto de Investigación Biomédica de Salamanca, University of Salamanca, Salamanca, Spain
4  Department of Medicine, University of Salamanca, Salamanca, Spain
5  Department of Hematology, University Hospital of Salamanca - IBSAL, Salamanca, Spain
,
4  Department of Medicine, University of Salamanca, Salamanca, Spain
5  Department of Hematology, University Hospital of Salamanca - IBSAL, Salamanca, Spain
6  On behalf of the “Grupo Español de Alteraciones Plaquetarias Congénitas (GEAPC)”, Hemorrhagic Diathesis Working Group, SETH
› Author Affiliations
Funding This work was partially supported by grants from Instituto de Salud Carlos III (ISCIII) and Feder (PI17/01311, PI17/01966, and CB15/00055), Fundación Séneca (19873/GERM/15), Gerencia Regional de Salud (GRS 2061A/19 and 1647/A/17), Fundación Mutua Madrileña (FMM, AP172142019), and Sociedad Española de Trombosis y Hemostasia (SETH-FETH; Premio López Borrasca 2019 and Ayuda a Grupos de Trabajo en Patología Hemorrágica 2019). The authors' research on IPDs is conducted in accordance with the aims of the Functional and Molecular Characterization of Patients with Inherited Platelet Disorders Project, which is supported by the Hemorrhagic Diathesis Working Group of the Spanish Society of Thrombosis and Haemostasis. A.M.-Q., C.F.-I., and L.H.-C. were supported by predoctoral grants from the Junta de Castilla y León, Spain. E.V. was supported by the predoctoral grant from the University of Salamanca, Spain. IG-T and RB were supported by "Contratos postdoctorales Programa II) from the University of Salamanca, Spain.

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 (RUNX1WT/WT, RUNX1WT/L43S, and RUNX1L43S/L43S). Platelet phenotype was assessed by flow cytometry and confocal microscopy. On average, RUNX1L43S/L43S and RUNX1WT/L43S mice had a significantly longer tail-bleeding time than RUNX1WT/WT mice, indicating the variant's involvement in hemostasis. However, only homozygous mice displayed mild thrombocytopenia. RUNX1L43S/L43S and RUNX1WT/L43S displayed impaired agonist-induced spreading and α-granule release, with no differences in δ-granule secretion. Levels of integrin αIIbβ3 activation, fibrinogen binding, and aggregation were significantly lower in platelets from RUNX1L43S/L43S and RUNX1WT/L43S using phorbol 12-myristate 13-acetate (PMA), adenosine diphosphate (ADP), and high thrombin doses. Lower levels of PKC phosphorylation in RUNX1L43S/L43S and RUNX1WT/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 αIIbβ3 activation, aggregation, α-granule secretion, and platelet spreading, mimicking the phenotype associated with RUNX1 variants in the clinical setting.

* These authors share senior authorship.


Supplementary Material



Publication History

Received: 29 July 2020

Accepted: 28 December 2020

Publication Date:
18 February 2021 (online)

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