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 1

Patient-derived SLFN14 mutant platelets show reduced signaling in response to major platelet agonists assessed by Western blotting.

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Patient-derived SLFN14 mutant platelets show reduced signaling in respon...
Major platelet agonists were used to activate platelets and assess downstream signaling of platelet receptors GPVI, PAR1, P2Y1, and P2Y12. (A) Platelets stimulated with low (3 μg/mL) and high doses (10 μg/mL) of collagen and CRP were used to assess P-Syk Tyr525/526. V220D and K219N patients showed reduced levels of phosphorylation compared with healthy control. (B) Platelets stimulated with low and high doses of collagen and CRP were used to assess P-LAT Tyr200. V220D and K219N patients showed reduced phosphorylation with increasing dose of agonist against control. (E) Downstream signaling of PAR1, P2Y1, and P2Y12 receptors assessed by P-ERK1/2 Tyr202/204 (MAPK) after stimulation with low and high doses of PAR1 peptide (30–100 μM) and ADP (30–100 μM). SLFN14 mutants showed reduced phosphorylation of ERK1/2 compared with control. (F) Reduced phosphorylation of P-AKT Tyr473 was observed in both patients with SLFN14 mutations. (C, D, G, and H) Quantification of Western blots normalized to GAPDH housekeeping gene. The ratio of pan/phosphorylation was measured and plotted. Biological replicates shown for healthy controls and patient samples; n = 2. Please note that the GAPDH images for control in A and B, V220D in A and B, and control in E and F are the same across the panels as they are derived from the same samples.