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 2

Slfn14 PF4-Cre mice generated using a Cre/LoxP conditional KO system have mild macrothrombocytopenia.

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Slfn14 PF4-Cre mice generated using a Cre/LoxP conditional KO system ha...
(A) Clones (P1B8 and P1F1) were generated containing the Slfn14 transgene. Germline testing and transmission were performed to identify different coat colors of the embryonic stem (ES) cells and the host blastocysts. (B) Targeting strategy used to generate the SLFN14-floxed mouse model. Green brackets highlight regions to be deleted upon Cre expression by loxP sites. Slfn14cre shows Slfn14 gene structure upon conditional KO of exons 2 and 3 after PF4-Cre recombinase activity. FRT, flippase recognition target; DTA, diphtheria toxin subunit A. (C) Detection of Slfn14fl alleles by PCR and agarose gel electrophoresis. Genotypes 1–3 are Slfn14fl/fl, Slfn14fl/+, and Slfn14+/+, respectively. (D) Detection of PF4-Cre expression by PCR and agarose gel electrophoresis. Genotypes 1 and 2 are negative and positive for PF4-Cre, respectively. (E) Verification of SLFN14-KO in platelets shown by lack of SLFN14 expression in Slfn14fl/fl PF4-Cre mice and reduced expression in Slfn14fl/+ PF4-Cre mice using quantitative RT-PCR. Data are mean ± SEM; 3 mice per genotype; n = 3. *P < 0.05. (F) Slfn14fl/fl PF4-Cre mice show significantly reduced platelet count (P = 0.0012). (G) Slfn14fl/fl PF4-Cre mice have a tendency to have enlarged platelets (increased MPV) compared with controls but did not reach significance. Data are mean ± SEM; n = 21–30 mice per genotype. Significance was assessed by 1-way ANOVA with multiple comparisons. (H) RBC count. (I) Mean corpuscular volume (MCV). (J) Slfn14fl/fl PF4-Cre mice show significant increase in percent lymphocytes. (K) Percent monocytes, (L) percent neutrophils, (M) percent eosinophils, and (N) percent basophils in mice were consistent across all genotypes. Data are presented as mean ± SEM; 1-way ANOVA with correction for multiple comparisons. n = 11–30 mice per genotype.