OC 68.2 - SLFN14 deletion causes macrothrombocytopenia and thrombus instability by dysregulating ubiquitination and degradation pathways in platelets and megakaryocytes.

Tuesday, June 25, 2024

15:00 – 15:15 ICT

Room: 211 A-D

Presenting Author(s)

Neil V. Morgan, PhD

Professor of Cardiovascular Genetics
University of Birmingham
Birmingham, England, United Kingdom

Co-Author(s)

XS

Xenia Sawkulycz

Postdoctoral Scientist
University of Birmingham
Birmingham, England, United Kingdom
RS

Rachel Stapley

Postdoctoral Scientist
University of Birmingham
Birmingham, England, United Kingdom
LG

Lourdes Garcia Quintanilla

Research Technician
University of Birmingham
Birmingham, England, United Kingdom
AA

Amna Ahmed

PhD student
University of Birmingham
Birmingham, England, United Kingdom
EH

Elizabeth Haining

Postdoctoral Scientist
University of Birmingham
Birmingham, England, United Kingdom
NK

Nayandeep Kaur

Research Technician
University of Birmingham
Birmingham, England, United Kingdom
AB

Andrea Bacon

Research Technician
University of Birmingham
Birmingham, England, United Kingdom
DK

Dean Kavanagh

Assistant Professor
University of Birmingham
Birmingham, England, United Kingdom
NP

Natalie Poulter

Assistant Professor
University of Birmingham
Birmingham, England, United Kingdom
Julie Rayes, PhD

Associate Professor
University of Birmingham
Birmingham, England, United Kingdom
ST

Steve Thomas

Associate Professor
University of Birmingham
Birmingham, England, United Kingdom

Background: SLFN (Schlafen) proteins are highly conserved in mammals where SLFN14 is specifically expressed in the Megakaryocytic and Erythroblast lineages. Mutations in the Schlafen 14 (SLFN14) cause an inherited thrombocytopenia with platelet dysfunction in human patients. However, the role of SLFN14 in megakaryopoiesis and platelet function has not been fully elucidated.

Aims: To define the role of SLFN14 in megakaryocytes (MK) and platelet function, we generated a novel murine model with a platelet and megakaryocyte specific SLFN14 deletion and employed extensive haematological analysis and RNA sequencing in platelets and megakaryocytes from these mice.

Methods: SLFN14 conditional knockout mice were generated by crossing PF4-Cre+ transgenic mice with SLFN14-flox mice using the Cre/LoxP system. SLFN14+/+ PF4Cre were used as controls compared to SLFN14fl/+ PF4Cre and SLFN14fl/fl PF4Cre mice. Hematological and functional assays were conducted including transmission electron microscopy, platelet function assays, laser induced injury model, isolation of native MKs for flow cytometry and RNA sequencing.

Results: PF4 Cre-mediated deletion of exons 2 and 3 in SLFN14 results in mild macrothrombocytopenia with normal levels of platelet glycoprotein receptors and granule content. However αIIbβ3 activation and thrombin-mediated platelet function and thrombus stability was reduced. SLFN14fl/fl PF4Cre mice had fewer in situ MKs in the bone marrow compared to controls. RNA sequencing analysis of MKs and platelets from SLFN14-PF4Cre mice and subsequent gene ontology analysis revealed altered pathways of ubiquitination, ATP activity and cytoskeletal processes in homozygous vs control mice. Targeted RNA expression analysis in MKs and platelets confirmed up- and down-regulated genes known to be key contributors in the production and functionality of MKs and platelets.

Conclusion(s): SLFN14fl/fl PF4 Cre mice presented with macrothrombocytopenia and reduced platelet function in response to thrombin-mediated activation. Gene ontology analysis indicates a dysregulation of pathways linked to gene transcription and/or translation which is a potential mechanism for SLFN14-related thrombocytopenia in mice and humans.