Platelet-specific SLFN14 deletion causes macrothrombocytopenia and platelet dysfunction through dysregulated megakaryocyte and platelet gene expression
Rachel J. Stapley, Xenia Sawkulycz, Gabriel H.M. Araujo, Maximilian Englert, Lourdes Garcia-Quintanilla, Sophie R.M. Smith, Amna Ahmed, Elizabeth J. Haining, Nayandeep Kaur, Andrea Bacon, Andrey V. Pisarev, Natalie S. Poulter, Dean Kavanagh, Steven G. Thomas, Samantha J. Montague, Julie Rayes, Zoltan Nagy, Neil V. Morgan
Rachel J. Stapley, Xenia Sawkulycz, Gabriel H.M. Araujo, Maximilian Englert, Lourdes Garcia-Quintanilla, Sophie R.M. Smith, Amna Ahmed, Elizabeth J. Haining, Nayandeep Kaur, Andrea Bacon, Andrey V. Pisarev, Natalie S. Poulter, Dean Kavanagh, Steven G. Thomas, Samantha J. Montague, Julie Rayes, Zoltan Nagy, Neil V. Morgan
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Research Article Genetics Hematology

Platelet-specific SLFN14 deletion causes macrothrombocytopenia and platelet dysfunction through dysregulated megakaryocyte and platelet gene expression

Abstract

Schlafen 14–related (SLFN14-related) thrombocytopenia is a rare bleeding disorder caused by SLFN14 mutations altering hemostasis in patients with platelet dysfunction. SLFN proteins are highly conserved in mammals where SLFN14 is specifically expressed in megakaryocyte (MK) and erythroblast lineages. The role of SLFN14 in megakaryopoiesis and platelet function has not been elucidated. Therefore, we generated a murine model with a platelet- and MK-specific SLFN14 deletion using platelet factor 4 (PF4) Cre-mediated deletion of exons 2 and 3 in Slfn14 (Slfn14 PF4-Cre) to decipher the molecular mechanisms driving the bleeding phenotype. Slfn14 PF4-Cre+ platelets displayed reduced platelet signaling to thrombin, reduced thrombin formation, increased bleeding tendency, and delayed thrombus formation as assessed by intravital imaging. Moreover, fewer in situ bone marrow MKs were present compared with controls. RNA-Seq and Gene Ontology analysis of MKs and platelets from Slfn14 PF4-Cre homozygous mice revealed altered pathways of ubiquitination, adenosine triphosphate activity, and cytoskeleton and molecular function. In summary, we investigated how SLFN14 deletion in MKs and platelets leads to platelet dysfunction and alters their transcriptome, explaining the platelet dysfunction and bleeding in humans and mice with SLFN14 mutations.

Authors

Rachel J. Stapley, Xenia Sawkulycz, Gabriel H.M. Araujo, Maximilian Englert, Lourdes Garcia-Quintanilla, Sophie R.M. Smith, Amna Ahmed, Elizabeth J. Haining, Nayandeep Kaur, Andrea Bacon, Andrey V. Pisarev, Natalie S. Poulter, Dean Kavanagh, Steven G. Thomas, Samantha J. Montague, Julie Rayes, Zoltan Nagy, Neil V. Morgan

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Figure 3

In vitro assessment of platelet size and glycoprotein levels and TEM of platelets in Slfn14 PF4-Cre mice.

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In vitro assessment of platelet size and glycoprotein levels and TEM of ...
(A–C) Slfn14 PF4-Cre mice show macrothrombocytopenia. Flow cytometry confirms increased platelet size and granularity (FSC-A and SSC-A) in Slfn14fl/+ PF4-Cre and Slfn14fl/fl PF4-Cre. Data are mean ± SEM; 2-way ANOVA with correction for multiple comparisons; *P < 0.05, **P < 0.01. (D) Resting surface glycoprotein expression levels measured by flow cytometry. GPIbα+ platelets were costained using indicated surface markers in a whole-blood flow cytometry assay. Data presented are MFI ± SEM from n = 5–11 mice per genotype; significance assessed using 2-way ANOVA with correction for multiple comparisons. (E) Representative TEM images of each genotype of Slfn14 PF4-Cre platelets. Scale bars: 2,000 nm. (F–H) Quantification of area (μm2), α granule (blue arrows in E), and dense (δ) granule (orange arrows in E) content in Slfn14 PF4-Cre platelets using TEM.