Platelet ageing: a review

Platelet ageing is an area of research which has gained much interest in recent years. Newly formed platelets, often referred to as reticulated platelets, young platelets or immature platelets, are defined as RNA-enriched and have long been thought to be hyper-reactive. This latter view is largely rooted in associations and observations in patient groups with shortened platelet half-lives who often present with increased proportions of newly formed platelets. Evidence from such groups suggests that an increased proportion of newly formed platelets is associated with an increased risk of thrombotic events and a reduced effectiveness of standard anti-platelet therapies. Whilst research has highlighted the existence of platelet subpopulations based on function, size and age within patient groups, the common intrinsic changes which occur as platelets age within the circulation are only just being explored. By understanding the changes that occur during the natural ageing processes of platelets, we may be able to identify the triggers for alterations in platelet life span and platelet reactivity. Here we review research on platelet ageing in the context of health and disease, paying particular attention to the experimental approaches taken and the robustness of conclusions that can be drawn.


Introduction
Studies into platelet ageing, or more generally how platelets of different ages respond to various stimuli and conditions, can be easily found in the literature.However, finding consensus in these studies is substantially more difficult.This is the consequence of differences in assumptions, techniques, analyses and even language brought to bear over the many years that these studies have been conducted.
It is well established that in healthy humans the population of circulating platelets turns over approximately every seven to ten days.Considering the platelet life span and the continuous nature in which platelets are produced, this will mean that each day approximately 10-15% of the population is refreshed with new platelets while another 10-15% of old platelets is removed.Therefore, when taking a sample of blood, it will contain a mix of platelets that can be readily separated into various population groups defined by size, surface markers and, depending upon the assay used, function.
Over the years some of these definitions have also been taken as indicative of circulatory age and used to delineate the characteristics of young versus old platelets, although such associations are generally only weakly proven.Here we will review this literature, and the reliability of the approaches taken, through an even-handed approach to evidence and interpretation and in doing so derive some overarching conclusions.We will be careful about the terms used to describe different types of platelets, especially terms such as immature, juvenile, mature, and senescent, which imply judgement about the nature of the platelets.We will preferentially use terms relevant to the age of circulating platelets, such as newly formed, young and old, which do not carry any other weight of judgement, unless otherwise proven.

Platelet turnover and ageing
[6] The term 'reticulated platelet' was first coined in the late 1960s during the study of platelets after acute blood loss in dogs, in which a small population of platelets was identified which had punctate condensations, resembling those seen in reticulocytes.However, this term is now more generally used to define a population with higher levels of ribonucleic acid.Recent work has suggested an intermediate stage, during which, under specific conditions, a small subset of preplatelets form large platelet barbells, a subpopulation described as being akin to reticulated or immature platelets, which later undergo separation to form two platelets. 7It remains unclear, however, which mechanisms during platelet biogenesis determine platelet size.
As for many nucleated cells, it has been proposed that platelets contain an internal clock that determines their life span.2][13][14] Whilst mechanisms central to these changes remain unclear, clinical associations with an increased risk of thrombosis have been demonstrated.To better understand the reasons for altered turnover in disease, further investigation into the processes occurring during the natural ageing process of a platelet are required.

The challenge: How to isolate differently aged platelets?
Whilst the mechanisms involved in platelet biogenesis are fairly well described, the inherent changes that occur to platelets as they age within the circulation are less well characterised.As touched upon above, this poor characterisation has been due to a lack of adequate techniques to separate platelet subpopulations by age.However, in recent years several different protocols have been developed which allow for the isolation of functional platelet subpopulations for subsequent characterisation (Figure 1).

Differential centrifugation: size and density isolation
To date, platelet ageing has been generally associated with a reduction in size or mean platelet volume, despite a number of dissenting studies and particular recent literature that calls this notion into question.If one starts from the view that platelets reduce in size as they age then apparent populations of young and old platelets can be separated by differential centrifugation.As centrifugation produces relatively little platelet activation, this method provides a robust approach to produce two subpopulations for characterisation studies. 15,16However, as there is contradictory evidence as to whether larger platelets are younger and smaller platelets older, these methods may not provide useful separation of differently aged platelets.Notably, for instance, studies some forty years apart indicate that while both platelet age and size are determinants of platelet function, they are independent determinants. 17,18Given the ease of centrifugation, this method has the advantages of reduced risk of activation and quicker processing times.However, as detailed above, it may not be the most accurate of methods due to conflicting literature on whether platelet size changes with age.

Fluorescent activated cell sorting: Size gating
Building on the use of size as an index of platelet age, fluorescent activated cell sorting of platelets based upon CD61 fluorescence and forward scatter has been established.This technique allows for gating and sorting of the large (top 10% based upon forward scatter) 'young' platelets, and small bottom 10% based upon forward scatter) 'old' platelets for subsequent analysis. 16The same caveats as for 3.1 above apply to this approach.

Fluorescent activated cell sorting: Nucleic acid dye labelling
0][21][22][23] Originally demonstrated in the 1960s, newly formed platelets contain the highest levels of ribonucleic acids (RNA) originated from their parent megakaryocytes.As is normal in cells, this RNA is degraded or translated but as platelets are lacking a nucleus the RNA cannot be replaced.[26][27][28][29] Based on this process, nucleic acid dyes have been used for decades to define newly formed platelets, and are used in clinical analytical machines to determine what is often termed the 'immature platelet fraction'.1][32] We would note in this regard that 'immature platelet fraction' is a term that indicates that these platelets are is some way not fully formed or functional, however aside from containing higher levels of RNA it is not defined in which way they are 'immature'.We would also note that there is a lack of consensus between commercial analysis machines due to their proprietary dyes and different algorithms for identifying the 'immature platelets', sometimes weighted by platelet size, such that this is not a definitive measure. 33,34rthermore, platelet RNA content is a continuum, such that any cut off point is necessarily arbitrary, although consistent and reproducible across a machine type.This implies that while analyses from such machines are robust and qualitatively useful in clinical diagnoses, they do not provide quantitatively definitive findings.In this latter regard, the use of nucleic acid dyes to provide in depth description of subpopulations has now begun to be more widely adopted.
2][23] The gating strategies applied to define young and old platelets differ slightly between laboratories, but the general analytical approach is to define newly formed, young platelets as the top 10-20% of thiazole orange/SYTO13 bright and the bottom 20-30% of thiazole orange/SYTO13 dim as old platelets. 19,22,23As highlighted above, the level of RNA stain in such studies presents a continuum with the cut offs decided upon by individual researchers.Therefore, data from different research laboratories can be compared less well than that from matching diagnostic machines, but there is definitive separation between the highest and lowest RNA containing platelets.These techniques allow for the accurate isolation of platelets based on RNA content, however one caveat with the use of these nucleic acid dyes is that they do not allow for normalisation based on platelet size and, therefore, there may be an element of bias based on size.Furthermore, because fluorescence activated cell sorters work under high pressure, samples require the inclusion of inhibitors to ensure that the platelets remain inactivated.

Biotin labelling in vivo
Ex vivo separation of differently aged platelets based upon RNA content provides a fairly robust approach for the characterisation of differently aged populations, certainly more so than sorting by size alone, but there still are potential pitfalls and confounders; for instance, are all platelets formed containing a similar amount of megakaryocyte RNA, and do platelets of different sizes contain proportionately the same amount of RNA.
6][37] Even earlier than this, 75 Se-methionine was used to label baboon megakaryocytes and to follow the platelets that later appeared within the circulation. 18These methods have been further developed to allow the isolation of young and old platelets by magnetic separation following antibody labelling; biotinylated labelled antibodies and streptavidin coated magnetic beads have been employed to isolate young and old thrombocytes from zebrafish. 38Whilst these approaches are useful to provide insights into platelet ageing in animal models, they cannot readily be applied to human subjects.

Temporal labelling of murine platelets in vivo
A common approach to studying newly formed platelets in mice is by causing acute destruction of circulating platelets through injection of anti-GPIba antibodies and studying the platelets that subsequently repopulate the circulation.Such models do not reflect physiological platelet removal and replacement, and therefore we cannot assume that the nature of the burst of platelets that appears in the circulation following such a dramatic insult is similar to the nature of those that are produced normally.Whilst these models may inform us on platelet production seen in humans after pathological insult such as chemotherapy, or traumatic injury with extensive blood loss, we cannot assume that they are representative of platelet formation under steady state.Indeed, research has demonstrated that the platelets that repopulate following anti-GPIba platelet depletion are 1.6 times larger, suggesting a dysregulation in the platelet biogenesis response. 39ilding on biotin labelling techniques, fluorescently-conjugated antibodies have recently been used to define populations of differently aged platelets within mice. 40,41This 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

Age related changes in platelet composition
As platelets are anucleate, the historical perspective was that the platelet transcriptome and proteome were relatively stable.However, recent studies have shown that platelet stimulation causes alterations in both the platelet transcriptome and proteome, suggesting that they may also change throughout a platelet's life span.
When discussing the nature of changes in platelet composition, it is pertinent to clarify the nomenclature to be used.Platelets differ from nucleated cells in that they cannot transcribe new RNA and their translational capacity is extremely low.Therefore, any changes that are identified occur against a background of general loss and decline of RNA and protein content.Consequently, it may be confusing or even misleading to discuss alterations in transcripts and proteins as up-or downregulated when the changes are all relative to overall very marked declines in content.We have recently reported, using platelets separated on the basis of their RNA content, that the oldest 30% of platelets contain half the protein per platelet of the youngest 10%. 22Therefore, we will simply use 'higher' or 'lower' levels to define the transcriptomic and proteomic changes demonstrated in each study, with 'enrichment' and 'decline' to represent alterations in biological pathways determined by bioinformatics approaches.

Transcriptome changes
The platelet transcriptome was originally described in 2002, with later studies demonstrating marked similarities among healthy individuals.3][44] To date, two comprehensive transcriptome analyses have been performed on young and old platelets isolated from healthy individuals. 20,23These studies, using platelets isolated by nucleic acid dye staining and cell sorting as described above, confirmed that young platelets have a significantly higher RNA content than old platelets (0.88-1.2fg per platelet vs. 0.35-0.4fgper platelet) and demonstrated that platelet ageing is accompanied by changes in the transcriptome.
Whilst these studies report significant reductions in RNA content, it is striking that they identified an increase in a large number of transcripts in old platelets.Bongiovanni et al. demonstrated that there were higher levels of 1074 transcripts and lower levels of 670 transcripts in young compared to old platelets.Further, Hille et al. identified higher levels of 1212 transcripts and lower levels of 1264 transcripts in young compared to old platelets. 20,23It is unclear whether these alterations are representative of changes in the absolute transcript level or are relative changes.As these results are determined against a background of general RNA loss, with most of the RNA degraded within 24 hours of platelets entering the circulation, it is surprising to see reports that older platelets have higher levels of so many transcripts.During such analyses, internal validation measurements using housekeeping transcripts such as GAPDH are commonly used, however in circumstances of general RNA decline, caution should be used as these are likely also degraded during platelet ageing.Even accepting the well-established notion that platelets are capable of endocytosing molecules from the circulation, which may account for higher levels of non-megakaryocytic transcripts, it raises the question as to how these other transcripts are quantitively higher in old platelets.It is therefore essential to be clear regarding qualitative versus quantitative differences in such studies.
Notably, the top transcripts altered in both studies are largely distinct from one another (Table 1), however common to both transcriptome studies is the finding of higher levels of transcripts for surface integrins important for haemostatic function including integrin alpha-IIb (Itga2b) and integrin beta-3 (Itgb3), as well as the glycoproteins GPV and GPVI in young platelets.In addition, these studies highlight relatively higher levels of transcripts for the thromboxane A2 receptor and the thrombin receptor, PAR-4, and Hille et al, report similarly regarding transcripts for P-selectin (SELP). 20,23Gene ontology terms associated with the higher levels of these transcripts include haemostasis, platelet aggregation and activation, platelet degranulation and blood coagulation.In support of an enhancement in these functions, both studies indicate young platelets contain greater numbers of transcripts than old platelets for proteins involved in calcium homeostasis (STIM1, Orai1 and Orai2) which is central to most platelet activation pathways. 20,23 Supporting the idea that pathways associated with platelet activation are transcriptionally related to platelet age, analysis of a small number of transcripts in temporally labelled murine platelets demonstrated a greater than mean reduction in 25 transcripts in older platelets, with a particular loss of cytoskeletal (TUBB1, COF1), signalling (Atp2a3, Alox12) and receptor (Itga2b, Pecam-1) transcripts.
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. 41spite 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. 20,23Furthermore, several transcripts associated with inflammatory processes -including interleukin 7, complement protein C5, C-C motif chemokine 5 and 17 -were found to be relatively higher in old platelets which might indicate an alteration in platelet function from a haemostatic responder to an immune regulator. 20,23It remains unclear where the majority of these transcripts are coming from, however it is well established that platelets can act as sponges, taking up substances from the circulation. 45,46In line with this idea, Clancy et al, show that platelets co-incubated with endothelial cells and vascular cells readily take up nonmegakaryocytic/platelet transcripts. 16In confirmation of the presence of endocytic pathways being active during platelet ageing, Hille et al, identified increases in transcripts from other blood cell origins including haemoglobin protein components (HBA1, HBA2, HBB). 16,23,47,48other transcriptomic study defining young and old platelets based on size identified a total of 5037 transcripts common to both populations, with a further 378 unique transcripts found in larger young platelets and 2314 distinct to smaller older platelets. 16Similar to samples sorted based on nucleic acid content, gene ontology of these transcripts revealed that larger platelets have an enrichment in haemostasis and coagulation.This is consistent with functional studies indicating that larger platelets are generally more reactive, further discussed below.Interestingly, processes involved in chromosome and nucleosome assembly as well as DNA packaging were also shown to be higher in larger platelets.On the other hand, the transcripts higher in smaller platelets were associated with varied gene ontology terms including apoptosis, leukocyte and lymphocyte activation, erythrocyte development and mitochondrial membrane organisation. 45 addition to long RNAs, just under 400 small RNAs have been detected in platelets following sorting based on nucleic acid dye fluorescence, with the majority of these consisting of microRNAs. 20,23Whilst one study demonstrated no difference in expression of small RNAs between young and old platelets, Bongiovanni et al, highlight a difference in the expression of 18 small RNAs, all of which are less abundant in young platelets. 20Nine of these small RNAs were miRNAs which have over 1900 validated interaction partners, 128 of which were found to be higher in young platelets. 20 general terms, we can conclude that during biogenesis, platelets are packaged with a finite quantity of megakaryocytic RNA and as they age these levels rapidly decline due to degradation or a very limited amount of translation.Transcriptomic studies highlight that although there is bulk RNA loss across platelet life span, there are subtler and potentially controlled changes in transcript levels.
Overall, whether the data is showing slower rates of decay for specific transcripts or higher absolute levels, the general relevance of these transcripts must be questioned as platelets only have a very limited translational capacity; differences in transcripts will not necessarily equate to differences in protein content.Indeed, ribosomal signatures including ribosomal 18S and 28S have been shown to decrease with platelet age, which may suggest that the levels of translation may also decline throughout a platelet's life span. 23

Proteome changes
The vast majority of protein within a platelet is synthesised within the parent megakaryocyte and packaged during its formation.However, recent research has highlighted that platelets contain essential components for protein synthesis, including ribosomes and polyribosome complexes, as well as translational initiation regulatory factors. 25,49,50Following physiological stimulation, there is an initiation of translation facilitating changes in the platelet proteome. 51Interestingly, activation with thrombin, collagen or adenosine diphosphate (ADP) promotes the synthesis of Bcl-3 and interleukin-1β (IL-1β), with levels maintained for several hours after activation. 52Indeed levels of IL-1β are barely detectable in quiescent platelets, but rise to approximately 600-800pg/ml following stimulation. 53rther, de novo protein synthesis has been demonstrated during storage of platelet concentrates used in transfusion, which may assist in cellular repair mechanisms. 54However, against the large general loss of protein seen in anucleate platelets and limited translational machinery these changes may at most be small variations against a marked overall change.
As sorting of human platelets by age is still in its infancy, there is only one study that we are aware of, from our own laboratory, analysing the protein content of platelets of different ages following separation based on nucleic acid content. 22We found that platelet ageing is associated with a progressive decline in total protein content such that old platelets contain around 50% less protein than young platelets. 22There are particular variations in protein loss set against this overall reduction.
Of particular note, we identified a significantly greater decrease in cytoskeletal-associated proteins twinfilin-2, emerin, and gelsolin, which are fundamental for cytoskeletal rearrangement, a process underpinning all platelet activation pathways. 22Interestingly, platelet specific knockout of twinfilin-2 has been shown to cause increased platelet turnover, suggesting a potential role for the cytoskeleton and cytoskeletal-associated proteins in the maintenance of platelet life span. 55Furthermore, old platelets have an enhanced loss of mitochondrial-associated proteins such as citrate synthase, ATP synthase and ADP/ATP translocase 2, suggesting that platelet ageing is associated with marked alterations in metabolism.7][58] Consistent with alterations in the platelet transcriptome, old platelets had a relatively lower abundance of proteins fundamental for platelet activation and aggregation including ITGA2B and ITGB3, STIM1 and Pselectin. 59ilst there is still debate regarding whether platelets change in size as they age, proteomic analysis based on fractions of different sized platelets identified a total 894 proteins.This analysis showed that large platelets had a 1.5-fold higher abundance of 38 proteins compared to small platelets, including a number of cytoskeletal-and mitochondrial-associated proteins. 15On the other hand, small platelets had 1.5-fold higher abundance of 42 proteins, most of which were comprised of circulating plasma proteins. 15Although highlighting a similar trend of differences in protein levels as found in platelets separated on the basis of thiazole orange staining, it is important to emphasize that the proteins identified in these two proteomic studies are largely distinct from one another.This is consistent with the previously noted finding that while both platelet age and size are determinants of platelet function, they are independent determinants. 17,18,60nally, the subsets of proteins that were in relatively higher than mean abundance in old platelets were circulating proteins such as haemoglobin (HBA), fibrinogen (fibrinogen alpha chain; FGA), as well as complement proteins (C1S, C5, C8G). 22As noted for RNA transcripts, it may well be that these proteins are endocytosed throughout the platelet's life span.An alternative possibility is that they are selectively maintained, suggestive of platelets taking on a more inflammatory phenotype as they age.

Surface receptor content
To date, newly formed platelets have been largely characterised by their higher RNA content, however recent work has shown that surface receptor expression changes with platelet age.Studies using murine platelets have shown that young platelets have higher levels of MHC-I and PECAM-1. 41,61istent 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.Investigation into the levels of surface GPVI has provided contradictory results.Using temporal labelling in a murine model, our group demonstrated that as platelets age there is a significant reduction in GPVI. 41Supporting this work, Veninga et al. have recently shown that young platelets identified by HLA-I expression have higher GPVI levels than old platelets. 17Conversely, 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. 62In this model, the levels of aIIbβ3 and CLEC-2 were higher on young platelets. 62Although as already noted, we have no reason to suppose that platelets that appear rapidly in the circulation following an extreme acute collapse in platelet numbers are similar to those that appear during normal processes.Furthermore, the surface levels of CD9 have been shown to increase as platelets age within a murine model. 41

Structural changes
As touched upon above, there are contradictory opinions regarding the associations between platelet size and ageing. 23,37Clinical observations of increased platelet turnover, hyper-reactivity and risk of thrombosis, as well as platelet depletion murine models, have associated younger platelets with larger mean platelet volumes. 62Under such pathological conditions, megakaryocyte maturation may be affected, with studies showing higher megakaryocyte number and ploidy thereby allowing for an increase in platelet mass. 63More recently, using electron microscopy, Hille et al, observed a decrease in size of older platelets in healthy human blood samples, with older platelets having around half the cross sectional area of young platelets. 23Conversely, our group and others have found that under steady state conditions, platelet ageing is not associated with a reduction in platelet cross sectional area. 7,17,18,22,37,41,60,64,65Furthermore, a recent study supports data from the 1980s indicating that platelet ageing and size are independent determinants of platelet reactivity.This recent research separating platelets into two fractions, large and small, demonstrated that whilst RNA-rich platelets had a tendency to be in the large-size fraction, they were not found exclusively in this fraction. 17nsistent with this data, using micro-sampling of temporal labelled murine platelets, our group has shown that based on forward and side scatter, newly formed platelets are dispersed amongst the global population with varying sizes and follow this pattern at each time point measured.When comparing the geomean of the young vs. old platelets there is a minor shift indicating a slight reduction in size as platelets age, but not sufficient to allow separation of platelets of different ages on this basis. 41e contradictory literature regarding changes in size across platelet life span may result from the use of different platelet preparation techniques, equipment used to separate the subpopulations, as well as of approaches to measure platelet size.However, it is hard to rationalize that there is a ready association between platelet size and age if this is not easily and consistently demonstrable by different research groups.
Despite conflicting observations regarding platelet size during ageing, it is clear that as platelets age there are extensive changes in their ultrastructure, as would be expected from the loss of protein.
Notably, there is a deterioration in the actin and microtubule cytoskeleton, which has been proposed to contribute to changes in the buoyant density of the platelet and to cause structural instability. 22,64rthermore, electron microscopy has demonstrated that a small proportion of newly formed platelets contain remnants of the rough endoplasmic reticulum and Golgi apparatus, whilst these components are entirely absent from old platelets. 23Consistent with the loss of the rough endoplasmic reticulum, the size and complexity of the dense tubular system is reduced in old platelets, and there is a reduction in the number of a-granules, which may provide an explanation for the decrease in P-selectin protein and transcript levels. 20,23Furthermore, microscopy has highlighted a higher number of open canalicular system openings in young platelets consistent with their greater ability to undergo shape changes. 23terestingly, the number of mitochondria in platelets markedly declines with age, with approximately half the number in old platelets compared to newly formed platelets. 22,23,65Consistent with their greater reactivity, larger platelets have also been reported as being more metabolically active than smaller platelets. 66Further supporting the notion of mitochondrial loss during platelet ageing, barbell shape platelets, which may be precursors to young platelets, show high levels of MitoTracker fluorescence. 7Whilst the reasons for mitochondrial loss as platelets age remain unknown, mechanistically it may be due to release of mitochondria in microvesicles or mitophagy pathways. 22 platelets are highly metabolically active this loss of mitochondria raises the questions as to whether metabolic exhaustion is an important factor in triggering platelet clearance.
The extensive changes in protein composition that occur as platelets age, including a degraded cytoskeletal structure and the loss of a number of intracellular structures, may well lead to a reduction in their overall buoyant density.Indeed, studies from the 1980s suggested that buoyant density is a more accurate indicator than size of platelet age. 18,64,67

Functional changes associated with platelet ageing
Associations between platelet age and function have long been established, generally based on studies conducted under circumstances of altered platelet turnover.From such studies, increases in the proportion of newly formed platelets have been linked to increased risk of thrombotic events, resistance to anti-platelet therapeutics and higher P-selectin levels.With the development of platelet cell sorting protocols, it has been possible to demonstrate more precisely that young platelets formed under physiological conditions have a more reactive phenotype than older platelets with alterations in both their primary and secondary haemostatic functions.

Primary Haemostasis
The initial response to exposed extracellular matrix or soluble mediators requires a rapid response from platelets.Young platelets rapidly adhere and spread on fibrinogen and collagen forming large lamellipodia-like structures.Conversely, old platelets arrest at the filopodia stage of adhesion forming spherical structures with small protrusions. 22In line with these observations, the recently described actin nodule is present in a far higher proportion of young platelets than old platelets consistent with higher levels of actin polymerisation. 68These impairments may result from loss of cytoskeletal proteins, which would limit the dynamic remodelling needed to form large protrusions as there would be a limited reserve of cytoskeleton to support the increased surface area.
Consistent with a more robust adhesion response, newly formed human and murine platelets demonstrate a heightened maximal calcium flux in response to thrombin receptor activating peptide 6 (TRAP-6) amide or PAR-4 amide, suggesting they have larger calcium stores than old platelets. 22,41pporting the notion that young platelets are more reactive, analysis of aggregates formed in vitro from mixed populations of platelets demonstrated that newly formed platelets contribute disproportionately to aggregates, being present in approximately 95% of thrombi and tending to form the core of aggregates, with older platelets binding to the periphery. 22,69Furthermore, in a murine pulmonary embolism model, we found that newly formed platelets contributed more than old platelets to in vivo thrombus formation. 41

Secondary haemostasis
In accordance with alterations in primary haemostasis, in vitro exposure to ADP, or selective peptides for PAR-1 (SFLLRN) or PAR-4 (AYPGKF) causes young platelets to express significantly higher levels of P-selectin on their surfaces than older platelets, indicating higher levels of α-granule secretion. 20,22,23,35,41,70Additionally, platelets isolated from arterial thrombi demonstrated higher staining for thiazole orange as well as higher surface P-selectin expression compared to platelets isolated from the circulating blood. 71Consistent with increased α-granule secretion, young platelets have a marked increase in dense granule secretion, as measured by adenosine triphosphate (ATP) release.Furthermore, old platelets have a reduction in the synthesis of pro-aggregatory mediators such as thromboxane A2 and prostaglandin E2, suggesting an impairment of the amplification response needed to promote clot formation. 22alysis of phosphatidylserine exposure, which can act as a marker of a procoagulant phenotype as well as marker of apoptosis, has produced contradictory results. 37One study reported that old platelets have higher phosphatidylserine exposure under basal conditions than young platelets, potentially being a marker of a senescent platelet that is undergoing preparation for clearance from the circulation. 22Similarly, biotinylation of rabbit platelets showed that under resting conditions a higher percentage of older platelets were annexin V-positive, indicative of higher phosphatidylserine exposure compared to younger platelets. 37Conversely, another study demonstrated that annexin V binding is higher in young platelets and is increased 1.5-fold following activation, whilst the lower annexin V binding in old platelets is unchanged by activation. 22,70This data may suggest that young platelets have a greater propensity to become procoagulant, which would support previous findings that younger platelets have a greater haemostatic function.

Significance of altered platelet turnover in disease
It is well documented that the proportion of circulating young platelets, generally termed the 'immature platelet fraction', is elevated in a number of pathological states such as cardiovascular disease, diabetes mellitus, chronic kidney disease, immune thrombocytopenia, sepsis and most recently SARs-CoV-2 infection. 11,12,72,73These observed increases in young platelets, indicative of reduced platelet life span, have been associated with increased risk of thrombotic events and in some cases a reduced effectiveness of anti-platelet therapies. 74,75 patients with coronary artery disease, a higher 'immature platelet count' has been associated with higher rates of subsequent major adverse cardiovascular events. 76,77Indeed, meta-analysis studies have demonstrated that a higher level of reticulated platelets is a good prognostic marker for future cardiovascular events in such individuals. 78The percentage of reticulated platelets was also found to be significantly higher in diabetic mellitus patients compared to both non-diabetic patients and healthy controls, and the percentage of reticulated platelets negatively correlated with the antiplatelet effectiveness of aspirin and clopidogrel. 75mune thrombocytopenia, an autoimmune condition characterised by low levels of circulating platelets, has also been shown to be associated with a significantly higher 'immature platelet fraction', indicative of altered platelet turnover. 79This increase in newly formed platelets may be a compensatory mechanism in which maturation of megakaryocytes is enhanced to compensate for the lower platelet counts.Interestingly, despite the low platelet counts and excessive bleeding, patients with immune thrombocytopenia also have a higher incidence of cardiovascular events, which may be explained by the higher levels of young platelets. 80Rs-CoV-2 infection is now recognised to have multiorgan effects, with reports of coagulopathies and thrombotic events.Indeed, numerous studies have demonstrated an increased immature platelet fraction compared to healthy individuals as well as patients previously documented to have higher levels of newly formed platelets, such as in stable coronary artery disease. 11,81The higher immature platelet fractions have been shown to be associated with disease severity, and proposed as a useful biomarker for disease severity, length of hospitalisation and mortality. 72nsistent with the use as a biomarker, recent work has shown that an increased immature platelet fraction is a good determinant between patients with sepsis and those without.However, there is contradictory literature on whether there are significant differences between patients with different sepsis severities. 82,83e mechanisms governing changes in the age profile of platelets in pathological conditions remain unknown, however in many cases they may be associated with an enhanced clearance of platelets leading to increased haematopoiesis.Given such dysregulations in the platelet production process, it is unclear as to whether the platelets produced under these stress conditions are 'normal' and they may perhaps be more akin to those found in animal models of acute platelet depletion.Platelets formed under such pathological conditions could be larger in volume and packaged with greater quantities of granular content as a compensatory mechanism for reduced platelet life span and so offer little help towards our understanding of the physiological processes of platelet ageing.This reinforces the need to continue our efforts to understand the normal life cycle of platelets relevant to the development of better treatment regimens to maximise platelet inhibition and reduce the risk of thrombosis in patient cohorts.

Conclusions
Platelet ageing is a particularly interesting area of research as it is well documented that in disease there is dysregulation to the natural ageing process, associated with an increased risk of thrombosis and an apparent reduced effectiveness of anti-platelet drugs.This area of research warrants more extensive study, as improved in-depth characterisation of young and old platelets will allow us to better understand alterations in such pathological states and adapt treatment regimens to reflect these changes.
The advancement of cell sorting technologies has allowed the development of protocols to separate young and old platelets formed under physiological conditions.Research thus far has demonstrated that physiological platelet ageing is defined by a decline in overall RNA and protein content, accompanied by changes in the transcriptome and proteome.These changes are associated with a reduction in gene ontology terms associated with key platelet functional processes, including haemostasis, aggregation and coagulation.Supporting this data, functional analysis of young, reticulated platelets has shown they have a greater haemostatic function compared to old platelets, rapidly responding to stimuli and contributing more to thrombus formation than older platelets (Figure 2).Therefore, a shift in the age profile of platelets towards a population with a higher proportion of newly formed platelets results in a greater risk of thrombotic events and a reduced effectiveness of anti-platelet therapeutics.
Despite the overall decline in the RNA levels, it is interesting to note an increase in transcripts associated with other circulating cells, such as erythrocytes.It is well documented that platelets are able to endocytose both RNA and proteins, which explains some of these increases, however it remains unclear if there is a functional reason for their uptake. 45,84The accumulation of these transcripts may facilitate platelets taking on a more immune phenotype, or could be a removal mechanism from the circulation as aged platelets are subsequently cleared in the liver or spleen.
Literature often describes young and old platelets as if they are separate types of platelets with distinct characteristics rather than being at opposite ends of a common ageing population.It would benefit the field to clarify definition of platelet types and to be clear about markers which do and do not allow separation of platelets by age.Similarly, it is important to reflect upon animal models which allow insights to normal physiological turnover and those which reflect pathological upsets.This does not mandate for a hierarchy in which physiological studies are 'better', but rather a clarity regarding the conclusions which can be drawn from the study undertaken.The platelets that arise from acute platelet ablation may well provide us with insights into the platelets seen following acute injury and blood loss or chemotherapy.Characterisation of platelets of varying sizes can help us understand how differences in the size distribution of platelets in individuals could make them more or less at risk of thrombotic events.Fortunately, the emergence of new technologies and isolation methodologies will allow us to confidently discriminate platelets by characteristics such as age, size, reactivity and surface markers, and thus characterise the various subpopulations that make up circulating platelets in health and disease. Figures

Figure 1 :
Figure 1: Techniques to isolate differently aged platelet subpopulations 1A.Differential centrifugation of platelet rich plasma into large, young platelets and small old platelets 1B.Cell sorting based on forward scatter and CD61 fluorescence 2. Fluorescence activated cell sorting based on nucleic acid dye (SYTO-13 or Thiazole Orange; TO) fluorescence.Young platelets defined as the top 10-20% of SYTO-13/TO fluorescence and old platelets defined as the bottom 20-30% of SYTO-13/TO fluorescence 3.In vivo labelling with biotin or fluorescently conjugated antibodies.

Figure 2 :
Figure 2: Summary schematic of the changes that occur during the natural platelet ageing process Young, or reticulated, platelets are released from proplatelet elongations originating from precursor megakaryocytes.This release can occur directly, or through an intermediate stage in which nascent platelets form large platelet barbells which undergo subsequent fission to individual platelets.Between the beginning and end of their 7-10 day life span in healthy individuals, platelets lose endogenous ribonucleic acids (RNA) and proteins, and show substantial changes in their ultrastructure.These changes are associated with a deterioration in the platelets' haemostatic function.Furthermore, as platelets age, there are increases in the levels of exogenous intracellular RNA and proteins indicative of endocytosis of various circulating molecules.

Table 1 :
Top 10 transcripts identified as higher in young or old platelets following nucleic acid dye cell