Omic Approaches to Quality Biomarkers for Stored Platelets: Are WeThere Yet?

Sandhya Kulkarni1, Meganathan Kannan1, and Chintamani D. Atreya

At present, there is no single biomarker that serves asthe “gold standard” predictive of the quality of storedplatelets used for transfusion. Some of the measurablefeatures of platelets such as morphology, biochemicalstatus, physiologic response to osmotic stress andagonist-induced changes, and measurement of pro-cess-associated activation indicators of platelets areconsidered useful in assessing the in vitro quality ofstored platelets. Such in vitro measurements combinedwith in vivo survival estimations using radiolabeledplatelets in healthy volunteers provide reasonableestimates of in vivo platelet function after transfusion.Thus, the current practice of estimating the quality andfunctional aspects of ex vivo stored platelets involvesutilization of a battery of tests that dates back to pre-

Transfusion Medicine Reviews, Vol 24, No 3 (July), 2010: pp 211-21

omic era. On the other hand, during the last decade,seminal discoveries have been made in plateletmolecular and cell biology by using “omic”-basedapproaches such as proteomics, genomics, and tran-scriptomics. Can we mobilize some of these discov-eries toward developing reliable quality biomarkers forstored platelets? To address this topic, we brieflyreview current practices and provide insights into someof the omic approaches that could be helpful inidentifying quality storage biomarkers of platelets inthe near future. We also briefly discuss here some ofthe challenges in using proteomic approaches andadvantages of using one of the transcriptomicsapproaches toward platelet biomarker development.Published by Elsevier Inc.

From the Section of Cell Biology, Division of Hematology,Office of Blood Research and Review, Center for BiologicsEvaluation and Research, US Food and Drug Administration,Bethesda, MD.

The findings and conclusions in this report have not beenformally disseminated by the Food and Drug Administration andshould not be construed to represent any agency determinationor policy; The authors do not have any conflicts of interests.

1Both authors made equal contribution to the manuscript.Address reprint requests to Chintamani D. Atreya, Suit

400N, WOC1, FDA, HFM-300 1401 Rockville Pike Rockville,MD 20852. E-mail: chintamani.atreya@fda.hhs.gov

0887-7963/$ - see front matterPublished by Elsevier Inc.doi: 10.1016/j.tmrv.2010.03.003

DURING STORAGE, PLATELETS undergomorphologic and physiologic changes such as

loss of surface glycoproteins and reduced aggrega-tion response to agonist. These collectively arereferred to as “the platelet storage lesion.” Becauseplatelets are one of the major cellular componentsof blood that play a crucial role in transfusionmedicine, there has been a tremendous interest infinding ways to extend the currently short (5-7days) shelf-life of stored platelets at room temper-ature while maintaining the quality of the product.In this direction, there have been 2 distinct types ofstudies reported in the literature: one has aimed atimproving the shelf-life of platelets and the otherhas focused on trying to understand the underlyingmechanisms of the platelet storage lesion. In theliterature, a variety of modalities including addi-tives and buffer systems have been examined.These include subjecting platelets to cold storage,the addition of second messengers to allow coldstorage, or the chilling of platelets followed byincubation at 22°C.1-12 These studies have mademarginal contributions to our understanding onhow to prolong the shelf-life of platelets. Dissectionof “the platelet storage lesion” at the cellular andmolecular level could provide specific clues towardreversing this phenomenon, that is, improve plateletshelf-life during storage. These approaches havebeen the primary driving force behind recent studiestrying to understand the underlying mechanismsassociated with the platelet storage lesion.13

CURRENT QUALITY ESTIMATES OFSTORED PLATELETS

There has been no single in vitro marker thatserves as the “gold standard” predictive of thequality of stored platelets after transfusion. Cur-rently, some of the measurable features of plateletssuch as (a) morphology, (b) biochemical status, (c)physiologic responses to osmotic stress and ago-nist-induced changes, and so on, and (d) measure-ment of platelet process-associated activationindicators have been considered useful in estimat-ing the in vitro quality of stored platelets. In vitromeasurements combined with in vivo survivalmeasurements using radiolabeled platelets inhealthy volunteers provide reasonable estimates ofin vivo platelet function after transfusion.

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Platelet Morphology

Platelet physical features that can easily bevisually estimated such as disk vs sphere shapeand quantification of different forms of platelets bylight microscopy as well as electron microscopy areuseful. However, for stored platelets, generally thein vitro tests do not correlate well with in vivoperformance after transfusion.14-17

Biochemical Status of Platelets

Because visual observations of platelets instorage do not accurately estimate their performancein vivo, a series of biochemical measurements havebeen used to complement morphologic observationsto correlate stored platelet functions in vivo. Plateletsuspension medium pH (6.2-7.6); cellular levels ofadenosine triphosphate, glucose, and lactate; and anestimate of overall drop in the level of lactatedehydrogenase in the platelet medium all provide aglimpse of an opportunity to predict howwell storedplatelets may perform in vivo after transfusion.18,19

Physiologic responses

As ex vivo storage causes stress to platelets, it hasbeen recognized that some estimates of the markersof osmotic stress and agonist-induced changes inplatelets could be correlated with their function invivo after transfusion.20 Platelets aggregate toincreasing concentrations of individual physiologicagonists such as collagen, epinephrine, adenosinediphosphate, or their combinations. Therefore,measuring platelet aggregation after treatment withagonists provides an opportunity to predict their invivo responsiveness to some extent. Another set ofuseful measurements of stored platelets is theirserotonin uptake capability, agonist-induced sero-tonin secretion capability, and the expression ofactivation markers such as platelet alpha-granulemembrane protein (GMP-140).18

Process-Associated Platelet Activation Indicators

Platelets are easily activated by external stimuli.Several procedural steps are involved in plateletconcentrate preparation and no matter how theplatelets are processed (ie, by buffy coat orapheresis), during such preparation platelets mayundergo activation. Activation of platelets includessurface expression of GMP-140 (also named P-selectin or CD62) and the fibrinogen-binding formof glycoprotein IIb/IIIa that can be detectable by a

monoclonal antibody, named PAC-1.21 Othermeasurable indicators are 2 platelet-specific pro-teins, β-thromboglobulin and platelet factor 4,which are released into the medium when plateletsare activated,22 as is platelet factor 3, whose levelsare increased during activation.23

OMIC-BASED INSIGHTS INTO THE CELL ANDMOLECULAR BIOLOGY OF PLATELETS

Omics refers to the study of biological systems ona large scale (http://www.nature.com/nrc/journal/v5/n2/glossary/nrc1547_glossary.html). Accordingto 1 etymological analysis, the suffix “ome” isderived from the Sanskrit word OM (“completenessand fullness”).24 Although standard DNA-basedgenomics is not applicable to anucleate platelets,other omic approaches such as proteomics (large-scale high-throughput study of proteins with respectto their structure and function, collectively known astheir proteome) and transcriptomics (large-scalehigh-throughput profiling of mRNA expression thatincludes profiling of microRNAs, the negativeregulators of mRNA expression) have recentlytaken center stage in understanding the functionalbiology of platelets.

Platelet Proteomics

When the excitement of using the “omic”approach to the vast field of cell biology was a fastgrowing field, studies with stored platelets weremoving rather slowly and cautiously due to thehistorical perception that a platelet is a bagful ofproteins and nothing else! However, subsequently,the same perception of platelets was also veryhelpful in identifying the platelet as the modelenucleate cell to study its proteome. With proteomictools in hand, stored platelets have recently beenunder intense investigation.25-27 These elegantstudies, in general, demonstrated that the expressionlevels of most platelet proteins (approximately 97%)remain unchanged during storage and only a handfulof themost high-abundance proteins (approximately3%) appear to have their expression levels altered,which potentially would make them useful asquality markers of stored platelets.25,27 However,soon it was realized that the nature of these handfulof platelet potential “storage marker” proteinsidentified varies from report to report dependingon the methods and tools used by each investigator.

For example, by using differential in-gel electro-phoresis and mass spectrometry to analyze changes

Fig 1. Western blots showing the effect of the buffer on theprotein extraction. Variation in the detection of low-molecular-weight (45 kd) Gelsolin when using different buffer systems isillustrated here: (A) buffer 1 (SDS-NEM), (B) buffer 2 (HEPESsupplemented with 1.8 mmol/L CaCl2), and (C) buffer 3 (HEPESwithout CaCl2). GAPDH served as an internal control (D).

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in the platelet proteome during the storage ofplatelets, Thiele et al25 reported that the expressionlevels of platelet β-actin as observed in 2-dimen-sional electrophoresis images and the 45-kdfragment of gelsolin observed in immunoblotanalysis appeared to increase from day 1 throughday 15 during platelets storage, suggesting thatthese protein profiles could serve as potentialmarkers for changes in the platelet proteome. Inanother report, by using a wide variety of proteomicapproaches while β-actin expression level wasreported to be unchanged during platelets storage(which makes this protein somewhat irrelevant tothe context of platelets storage lesion marker),proteins such as Superoxide desmutase, Rho-GDPdissociation inhibitor, Septin-2, and Zyxin weresuggested to be useful as platelets storage mar-kers.27 These 2 reports clearly indicate that there issome room for further investigation with regard tothe determination of which protein or a panel ofplatelet proteins could serve as potential qualitymarkers associated with platelets storage.

Thon et al27 clearly pointed out that no singleapproach is sufficient to identify the changesassociated with platelets storage in arriving atselecting a few proteins as useful storage markers.Although it is given that the technological toolsused will certainly make a difference in theidentification and interpretation of results, itappears that apparently one core issue fundamentalto the discordance could be simply the buffersystem that each laboratory is using in extractingproteins for the profiling of platelet proteins duringstorage. To illustrate this point further, in ourlaboratory, we chose the platelet gelsolin-related45-kd protein as our protein of interest for thestudy, which was previously suggested as one ofthe potential platelets storage markers by others28

and the housekeeping protein GAPDH as theinternal control. Three different protein extractionbuffers were used in this study (buffer 1: sodiumdodecyl sulfate and N-ethylmaleimide buffer,which is routinely used for protein extraction;buffer 2: 4-(2-hydroxyethyl)-1-piperazineethane-sulfonic acid [HEPES] supplemented with 1.8mmol/L CaCl2; and buffer 3: HEPES withoutCaCl2) to test our hypothesis that the bufferconstituents used in protein extraction affects theprofiling outcome of platelet proteins over a periodduring storage. Standard polyacrylamide gel elec-trophoresis followed by immunoblot analysis

(using gelsolin-specific antibodies) was used inthe study. We observed that results are variableand the time of appearance of 45-kd gelsolin-related protein band in the platelet extracts clearlydepended on the buffer system used (Fig 1).Although the protein of interest (45-kd gelsolin-related protein) was not detected either in buffer 1or 2 until day 15 in storage and only started toshow up in buffer 2 from day 16 onward,interestingly, the same protein was evident fromday 7 onward when buffer 3 was used.

Platelet Transcriptomics: mRNA ExpressionProfiling New Insights

Gene activity in platelet was reported by Bugertet al.29 These authors found that about 15.5% ofthe protein-coding genes are represented in the formof mRNA in platelets. Platelets contain rough

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endoplasmic reticulum and polyribosomes indica-tive of protein synthesis capabilities, and in fact,there is evidence of protein synthesis in humanplatelets upon signal-dependent activation, which isreported to have a strong correlation between theabundance of mRNA and protein synthesis inplatelets.30-32 As part of functional genomics thatallows the analysis of the expression of thousandsof genes, transcriptomics, that is, mRNA expres-sion profiling, uses 2 standard methods: (a)microarray and (b) serial analysis of gene expres-sion (SAGE). In various types of hematopoieticcells including platelets, SAGE analysis has beenreported that it was able to molecularly define thesubset and function of blood cells. The advantage ofSAGE analysis is the fact that it can cover thenumber of expressed genes that are unequaled byany other mammalian DNA microarray systems yetavailable. Specific to platelets, the SAGE analysis,although consistent with microarray data, furtherdetected more details of the transcriptome. Thesedetails of the platelet transcriptome include identi-fication of the longer untranslated regions ofmRNA, more stable folding nature of enrichedmRNAs, and biologically relevant mRNA, withregulatory elements for RNA stabilization ortranslational control such as cytoplasmic polyade-nylation elements, as well as enrichment of signaltransduction activities.33 Although the functionalrelevance of the translation mechanism in plateletsis still poorly understood, Dittrich et al33 reportedthat ongoing translation is much more important toproteins involved in signal transduction than tostructural proteins. Many platelet proteins involvedin signal transduction are either transmembraneproteins or soluble cytosolic proteins, which aremore susceptible to proteolytic degradation thanstructurally bound cytoskeleton proteins. SAGEanalysis resulted in identifying high expression ofthe regulators of G-proteins (RGS18 and RGS10)in the SAGE library. It has been recently reportedthat RGS18 is most abundantly expressed inplatelets34 and the phosphorylation of theseregulator proteins in TRAP activated plateletsreveal their active role in platelets.

Platelet Transcriptomics: mRNA Analysis ofStored Platelets

Recently, a new class of mRNA regulatorymolecules were identified and included as a subsetof functional genomics. These are cellular micro-

RNAs (miRNAs), a class of 19 to 25 nucleotideshort endogenous noncoding regulatory RNAs.Control of cellular gene expression in differentia-tion and apoptosis via mRNA degradation orinhibition of translation is one of the primaryfunctions of such miRNAs, and these have beenoften implicated in human diseases.35-42 Recentreports on miRNA profiling in cancer cells illustratethe fact that differential miRNA profiling is avaluable tool as biomarkers to identify the cellulartype and stage of a given type of cancer.43,44

Several investigations have focused on studyingplatelets in storage, and some of these studieswere aimed at specific aspects of apoptosis instored platelets. As a result, a wealth of informa-tion on few facets of major events and the proteinsthat represent the hallmark of apoptosis have beengenerated. Apoptosis represents the programmedprocess of cell death of nucleated cells, rich inmitochondria, and some artificially enucleatedcells also manifest this phenomenon.45,46 Becausemature platelets contain numerous mRNAs andundergo signal-dependent translational regula-tion,47,48 it is arguable that platelets havemiRNA as translational regulators which thusplay a crucial role in platelet biology duringstorage. There are several reports that support theexistence of and function for miRNAs in platelets.In general, pre-miRNAs are processed by RNaseIII Dicer and Argonaute 2 (Ago2) to form amature miRNA in cytoplasm. Landry et al49

recently provided evidence for the existence ofmiRNA pathway in platelets by identifying theDicer and Ago2 complexes in platelets and RNAsilencing function of some of the miRNAs. Theyalso validated the gene silencing properties ofsome of the matured miRNAs in platelet extractsand found that the silencing activity of miRNAswere proportional to their levels in platelets.49 Allthese studies strongly suggest the existence ofmiRNAs and their function in platelets. However,so far, there have been no reports of miRNAprofiling of platelets during storage to evaluatetheir value as product biomarkers of storage,similar to the cancer cell miRNA profiling thathelps in identifying the cancer stage.

Recently, for the first time, we have demonstrat-ed that subtle but global changes do occur in themiRNA population of platelets during storage. Weperformed membrane array-based apoptosis asso-ciated 52 miRNA analysis in platelets during

Fig 2. Pie diagram representing the profiling of 52 plateletmiRNAs relevant to apoptosis. Note that only 4 miRNAs (Let-7b,miR-16, miR-145 and miR-7) among the 52 miRNAs tested instored platelets have shown some trend during storage fromday0to day 9. The others did not. In the pie chart, “mild” represents lowexpression ofmiRNAs (Let-7a, Let-7d, Let-7c, Let-7e, Let-7f, Let-7g, Let-7i, miR-15b, miR-216, miR-368), “high” representsmiRNAs that remain high throughout the storage period (miR-150, miR-151, miR-152, miR-184, miR-188, miR-196a, miR-197, miR-202), and “variable” represents variable expression ofmiRNAs throughout the storage period (miR-10a, miR15a,miR-16, miR-21, miR-24, miR-25, miR-28, miR-96, miR-101,miR-133b, miR-142, miR-144, miR-148, miR-151, miR-153,miR-193a, miR-193b, miR-210, miR-214, miR-216, miR-218,miR-224, miR-337, miR-338, miR-345, miR-361, miR-368, miR-369-3p, miR-369-5p, miR-371). RUN 48, a nonapoptotic miRNA,was used as an internal control in this experiment.

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storage. Our analysis revealed that during storage,although most of the miRNAs were variable, hardlydetectable, or remain very high throughout thestorage period, only 4 miRNAs demonstrated asignificant pattern from day 0 to day 9. Of these, 2miRNAs (Let-7b and miR-16) demonstrated anupward trend and another 2 (miR-7 and miR-145) a

downward trend50 (Fig 2). Because miRNA path-ways have been identified in platelets,49 we believethat active splicing of pre-miRNA to maturedmiRNA might be the modus operandi for theobserved upward trend of Let-7b and miR-16, andthe possible degradation over time explains theobserved downward trend with miR-7 and miR-145. However, each of these 2 distinct trends needsindependent experimental verification. BecausemiRNAs regulate their target mRNAs, we alsoidentified the potential target mRNAs, includingCaspase-3, Bcl-2, and other apoptosis regulatorgenes upon which these 4 miRNAs could exert theirregulatory effect. Overall, we hope that miRNA–target mRNA interactions as reported50 will openup a new paradigm in the experimental biology ofplatelets toward understanding the miRNA-basedmolecular regulatory mechanisms of plateletmRNAs relevant to the platelet storage lesion andwhich will also contribute toward identifyingbiomarkers of stored platelets, predictive of theirin vivo quality.

In conclusion, it is clear from the recentadvances made in the field of platelet molecularand cellular biology that by taking advantage ofsome of the available omic approaches, we passedover the hump of “can we ever identify a storagebiomarker for platelet using omic approaches” to astage where we are confident that it is only amatter of when!

ACKNOWLEDGMENT

This work in part is supported by the FDA,CBER Critical Path Research Initiative funds toCDA. MK is a recipient of Oak Ridge Institute forScience and Engineering fellowship.

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