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Updated: Aug 30, 2025
In Vivo Two-photon Imaging of Megakaryocytes and Proplatelets in the Mouse Skull Bone Marrow
Published on: July 28, 2021
Temporal in vivo platelet labeling in mice reveals age-dependent receptor expression and conservation of specific
Paul C Armstrong1, Harriet E Allan1, Nicholas S Kirkby2
1Centre for Immunobiology, Blizard Institute, Faculty of Medicine and Dentistry, Queen Mary University of London, London, United Kingdom.
Insights
Young platelets exhibit heightened reactivity and contribute more to thrombus formation than older platelets. This study introduces a novel method to track platelet age and function, revealing age-dependent changes in molecular characteristics and thrombotic potential.
Area of Science:
- Hematology
- Cardiovascular Biology
- Molecular Biology
Background:
- The clinical significance of young platelets (reticulated platelets) in cardiovascular outcomes is recognized, but their distinct functional and molecular characteristics remain incompletely understood.
- Existing technical limitations hinder precise age-based analysis of platelet populations.
Purpose of the Study:
- To develop and validate an in vivo temporal labeling method for subdividing platelets by age.
- To assess age-dependent differences in platelet functional and molecular characteristics.
Main Methods:
- Developed an in vivo temporal labeling approach using injectable fluorescent antiplatelet antibodies.
- Utilized sequential sampling to track platelet size and function over their lifespan.
- Analyzed changes in calcium flux, degranulation, thrombus formation, and surface receptor expression (GPVI, CD31, CD9).
- Quantified age-associated changes in platelet messenger RNA (mRNA) content.
Main Results:
- Young platelets (<24 hours old) demonstrated greater calcium flux, degranulation, and thrombus formation capacity in vitro and in vivo compared to older platelets.
- Altered functionality was independent of platelet size, with size distribution consistent across the platelet population.
- Thrombotic function decreased with age, accompanied by reduced GPVI and CD31 expression and increased CD9 expression.
- Platelet mRNA content decreased with age, with differential rates for specific mRNAs, suggesting conservation of granule protein encoding mRNAs.
Conclusions:
- The developed pulse-chase method effectively defines circulating platelet age and function.
- Young platelets possess distinct functional and molecular properties, challenging previous assumptions about platelet size and reactivity.
- This validated tool offers novel insights into age-associated platelet heterogeneity in health and disease, with implications for cardiovascular research.
Abstract:
The proportion of young platelets, also known as newly formed or reticulated, within the overall platelet population has been clinically correlated with adverse cardiovascular outcomes. However, our understanding of this is incomplete because of limitations in the technical approaches available to study platelets of different ages. In this study, we have developed and validated an in vivo temporal labeling approach using injectable fluorescent antiplatelet antibodies to subdivide platelets by age and assess differences in functional and molecular characteristics. With this approach, we found that young platelets (<24 hours old) in comparison with older platelets respond to stimuli with greater calcium flux and degranulation and contribute more to the formation of thrombi in vitro and in vivo. Sequential sampling confirmed this altered functionality to be independent of platelet size, with distribution of sizes of tracked platelets commensurate with the global platelet population throughout their 5-day lifespan in the circulation. The age-associated decrease in thrombotic function was accompanied by significant decreases in the surface expression of GPVI and CD31 (PECAM-1) and an increase in CD9. Platelet messenger RNA (mRNA) content also decreased with age but at different rates for individual mRNAs indicating apparent conservation of those encoding granule proteins. Our pulse-chase-type approach to define circulating platelet age has allowed timely reexamination of commonly held beliefs regarding size and reactivity of young platelets while providing novel insights into the temporal regulation of receptor and protein expression. Overall, future application of this validated tool will inform age-based platelet heterogeneity in physiology and disease.
