PB0367 - Temporal labelling of ageing platelets in mice indicates differential regulation and retention of granule protein encoding mRNAs

Sunday, July 10, 2022

6:30 PM – 7:30 PM

Location: Exhibition

Presentation Format: In-Person

Presenting Author(s)

PA

Paul C. Armstrong, PhD

Assistant Professor
Blizard Institute, Queen Mary University of London
London, England, United Kingdom

Co-Author(s)

NK

Nicholas Kirkby

Senior Research Fellow
Imperial College London
London, England, United Kingdom
CG

Clemens Gutmann

Research Student
King's College London
London, England, United Kingdom
Harriet E. Allan, PhD

Postdoctoral Researcher
Blizard Institute, Queen Mary University of London
London, England, United Kingdom
AJ

Abhishek Joshi

Cardiology Registrar
King's College London
London, England, United Kingdom
MC

Marilena Crescente, PhD

Senior Lecturer
Manchester Metropolitan University
Manchester, England, United Kingdom
JM

Jane Mitchell

Professor of Pharmacology in Critical Care Medicine
Imperial College London
London, England, United Kingdom
MM

Manuel Mayr

BHF Professor of Cardiovascular Proteomics
King's College London
London, England, United Kingdom
TW

Timothy D. Warner

Professor
Blizard Institute, Queen Mary University of London
London, England, United Kingdom

Background: Platelets inherit mRNA from their parent megakaryocyte. Young newly formed platelets are characterized as those with the highest levels of inherited mRNA, as this is rapidly lost once platelets are released, and age, within the circulation. Previous studies have identified the general decay half-life of platelet mRNA to be approximately 6 hours, but it is unclear if this is decay is similar for all mRNAs.

Aims: To use in vivo temporal labelling to investigate platelet mRNA dynamics.

Methods: Fluorescent anti-CD42c antibodies (Emfret) were injected (i.v) at various timepoints to C57Bl6 mice (12-14wk) to identify age-specific platelet populations: younger ( < 24h), older (>1 days old) and global. Following isolation by cell sorting, RNA was extracted (2.5million per sample) and qRT-PCR performed for a panel of highly expressed platelet mRNAs. Unbiased clustering analysis was performed to compare age and abundance of individual RNAs.

Results: Principal component analysis demonstrated each analyzed population clustered individually but with differential directionality for individual mRNAs; indicating potential for individual patterns of decreased abundance. Focused comparison and grouping of mRNAs revealed the amount of loss varied by specific mRNA (median fold change ranging from -5.3 to -66.4) but was not contingent on starting abundance. In particular, those mRNAs classified as associated with signaling proteins (Gnas, Atp2a3, Alox12, Rgs18, Sh3bgrl3) demonstrated the greatest reduction in abundance, whilst those associated with granule proteins (Srgn, Nrgn, B2m, Pf4, Pbpp) demonstrated the least (mean ±s.e.m fold change: -35.6 ±6.8 versus -9.2 ±1.9 respectively, p< 0.05, n=4).

Conclusion(s): The variable rates of loss observed in this dataset strongly suggest that platelet mRNA is under more refined control than previously appreciated. Further research is required to determine the mechanisms and purpose of such regulation, particularly if retention of granule protein encoding mRNAs correlates with the phenomenon of downstream de novo protein production previously reported by some groups.