Temporal in vivo platelet labeling in mice reveals age-dependent receptor expression and conservation of specific mRNAs

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. Our understanding of this is incomplete, however, 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 labelling' approach using injectable fluorescent anti-platelet antibodies to sub-divide platelets by age and assess differences in functional and molecular characteristics. With this approach we found that young platelets (<24h old) in comparison to 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 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 re-examination of commonly held beliefs regarding size and reactivity of young platelets whilst providing novel insights into the temporal regulation of receptor and protein expression. Overall, future application of this validated tool will inform on age-based platelet heterogeneity in physiology and disease.

Platelet ageing: A review

“…Studies using murine platelets have shown that young platelets have higher levels of MHC-I and PECAM-1. 41,61 Consistent with these murine studies, young thiazole orange bright human platelets express higher levels of surface HLA-I and have higher ribosomal contents than older platelets. Indeed, the authors of this study suggest that HLA-I may be a better marker than thiazole orange to determine the percentage of young platelets in patient samples.…”

“…Using temporal labelling in a murine model, our group demonstrated that as platelets age there is a significant reduction in GPVI. 41 Supporting this work, Veninga et al have recently shown that young platelets identified by HLA-I expression have higher GPVI levels than old platelets. 17 Conversely, following platelet depletion using an anti-GPIba antibody in mouse, the newly formed platelets had similar levels of surface GPVI, although with an impairment in the associated downstream signalling pathways.…”

“…39 Building on biotin labelling techniques, fluorescently-conjugated antibodies have recently been used to define populations of differently aged platelets within mice. 40,41 This technique involves the sequential injection of CD42c antibodies conjugated to different fluorophores at defined time points. Subsequent analysis can be performed by micro-sampling over the course of the whole 5-day platelet life span in a mouse, allowing for small volume assay analysis of time-associated differences such as surface receptor expression using flow cytometry or imaging analysis.…”

“…Alternatively, using the entire volume of blood coupled with fluorescent activated cell sorting allows for more in depth characterisation such as analysis of transcripts. 41…”

“…Interestingly, transcripts associated with granular proteins appear to experience the smallest relative loss during platelet ageing compared to other functional categories, suggesting that there may be selective retention of these transcripts. 41 Despite the general decline in RNA across platelet life span, it is interesting that these studies have identified that relative to the general loss of RNA older platelets have higher levels of a large number of transcripts. A subset of these transcripts is associated with the gene ontology terms RNA processing, binding, ribosome biogenesis and nuclear processes, which may indicate a selective retention to help facilitate the limited translational processes that occur within platelets.…”