Proteome changes in platelets after pathogen inactivation--an interlaboratory consensus.

Transfusion Medicine Reviews 28 (2014) 72–83

Contents lists available at ScienceDirect

Transfusion Medicine Reviews journal homepage: www.tmreviews.com

Proteome Changes in Platelets After Pathogen Inactivation—An Interlaboratory Consensus Michel Prudent a,⁎, 1, Angelo D’Alessandro b, 1, 2, Jean-Pierre Cazenave c, Dana V. Devine d, 1, Christian Gachet c, 1, Andreas Greinacher e, Niels Lion a, Peter Schubert d, 1, Leif Steil f, Thomas Thiele e, 1, Jean-Daniel Tissot a, Uwe Völker f, Lello Zolla b a

Service Régional Vaudois de Transfusion Sanguine, Unité de Recherche et Développement, Lausanne, Switzerland Department of Ecological and Biological Sciences, Tuscia University, Largo dell’Università, Viterbo, Italy c UMR_S949 INSERM, Université de Strasbourg, Etablissement Français du Sang-Alsace, Strasbourg, France d Centre for Innovation, Canadian Blood Services and Department of Pathology and Laboratory Medicine, Centre for Blood Research, University of British Columbia, Vancouver, BC, Canada e Institut für Immunologie und Transfusionsmedizin, Universitätsmedizin Greifswald, Greifswald, Germany f Interfakultäres Institut für Genetik und Funktionelle Genomforschung, Universitätsmedizin Greifswald, Greifswald, Germany b

a r t i c l e

i n f o

Available online 25 February 2014

a b s t r a c t Pathogen inactivation (PI) of platelet concentrates (PCs) reduces the proliferation/replication of a large range of bacteria, viruses, and parasites as well as residual leucocytes. Pathogen-inactivated PCs were evaluated in various clinical trials showing their efficacy and safety. Today, there is some debate over the hemostatic activity of treated PCs as the overall survival of PI platelets seems to be somewhat reduced, and in vitro measurements have identified some alterations in platelet function. Although the specific lesions resulting from PI of PCs are still not fully understood, proteomic studies have revealed potential damages at the protein level. This review merges the key findings of the proteomic analyses of PCs treated by the Mirasol Pathogen Reduction Technology, the Intercept Blood System, and the Theraflex UV-C system, respectively, and discusses the potential impact on the biological functions of platelets. The complementarities of the applied proteomic approaches allow the coverage of a wide range of proteins and provide a comprehensive overview of PI-mediated protein damage. It emerges that there is a relatively weak impact of PI on the overall proteome of platelets. However, some data show that the different PI treatments lead to an acceleration of platelet storage lesions, which is in agreement with the current model of platelet storage lesion in pathogen-inactivated PCs. Overall, the impact of the PI treatment on the proteome appears to be different among the PI systems. Mirasol impacts adhesion and platelet shape change, whereas Intercept seems to impact proteins of intracellular platelet activation pathways. Theraflex influences platelet shape change and aggregation, but the data reported to date are limited. This information provides the basis to understand the impact of different PI on the molecular mechanisms of platelet function. Moreover, these data may serve as basis for future developments of PI technologies for PCs. Further studies should address the impact of both the PI and the storage duration on platelets in PCs because PI may enable the extension of the shelf life of PCs by reducing the bacterial contamination risk. © 2014 Elsevier Inc. All rights reserved.

Contents Pathogen Inactivation of Platelet Concentrates. Riboflavin Plus UV Light . . . . . Amotosalen Plus UV-A Light . . . UV-C Treatment . . . . . . . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

73 74 74 74

Conflict of interest: NL received conference honorarium on 2 occasions from Cerus Corp, and JDT is a customer panel member at Terumo BCT. LZ and ADA have received funds from the Italian National Blood Centre within the framework of the Italian Platelet Technology Assessment Study, in collaboration with Cerus Corp and Terumo BCT. CG and JPC have received research funding from Cerus and JPC honoraria from Cerus Corp and Fresenius, Terumo BCT, Haemonetics, and MacoPharma and consultancy fees from Cerus Corp. DVD and PS received research support from Terumo BCT and MacoPharma for work on PRT. AG received research support from MacoPharma and performs consultant service for MacoPharma, and TT received research support and speaker honorarium from MacoPharma. MP, LS, and UV declare no conflict of interest. ⁎ Corresponding author. Michel Prudent, Service Régional Vaudois de Transfusion Sanguine, Unité de Recherche et Développement, 1066 Epalinges, Switzerland. E-mail address: [email protected] (M. Prudent). 1 Members of the writing committee. 2 Current address: Departments of Biochemistry and Molecular Genetics, University of Colorado - Anschutz Medical Campus - Aurora, CO, USA. 0887-7963/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.tmrv.2014.02.002

M. Prudent et al. / Transfusion Medicine Reviews 28 (2014) 72–83

Clinical Studies of PI-PCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Proteomic Tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Proteomic Analyses of PSLs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Proteomic Analyses of PI-PCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Riboflavin Plus UV Light . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Comparison to γ-Irradiated Platelets . . . . . . . . . . . . . . . . . . . . . . . . . . Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis and LC-MS/MS Analysis . . . Amotosalen Plus UV-A Light . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Two-Dimensional Gel Electrophoresis Analysis . . . . . . . . . . . . . . . . . . . . . Liquid Chromatography–MS/MS Analysis . . . . . . . . . . . . . . . . . . . . . . . . UV-C Technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biological Functions Affected by PI Discovered by Proteomic Studies . . . . . . . . . . . . . . . . . . . . . Riboflavin plus UV Affects Proteins Involved in Adhesion and Activation,… . . . . . . . . . . . . . … In Granule Secretion and Platelet Shape Change,… . . . . . . . . . . . . . . . . . . . . . . … And in Aggregation of Platelets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Amotosalen plus UV-A Affects Proteins Mainly Involved in the Mechanisms of Platelet Aggregation Further Studies That Will Supplement the Present Picture . . . . . . . . . . . . . . . . . . . . Conclusions and Expert Commentary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Platelet concentrates (PCs) are an essential supportive treatment for hemato-oncology, major surgery, and intensive care patients. Platelet concentrates are obtained either by apheresis or isolated from whole blood donations as pooled PCs from buffy coat (Europe and Canada) or from platelet-rich plasma (United States). The production processes of PCs have not undergone major alterations since their original development other than leukocyte reduction or the use of additive solutions replacing plasma as storage media in PCs. A fundamental change in PC production is the introduction of measures to reduce the risk of pathogen transmission by PCs. The development of pathogen inactivation (PI) of PCs 2 [1] has been driven by the emergence of new transfusion transmissible agents or those expanding their traditional range such as West Nile virus, Chikungunya virus, and dengue virus as well as the comparably high risk of bacterial contaminations in PCs [2]. New approaches for PC production are routinely validated by conventional in vitro methods. These methods are able to identify selective features of storage lesions in platelets [3–5]. After PI treatment, metabolic deterioration, impaired mitochondrial function, accelerated spontaneous platelet activation, and altered platelet function occur. These changes may be reflected in a loss of platelet aggregation responses to adenosine 5′-diphosphate (ADP) or to the thrombin receptor activation peptide [6–15]. These observations suggest that either the PI processes have intrinsic deleterious impacts on platelets or they just accelerate the development of platelet storage lesions (PSLs) that normally alter the functional integrity and structure of platelets after they have been withdrawn from the circulation [5]. Changes in the cellular proteins are particularly relevant for platelets as they have limited capacity to translate messenger RNA (mRNA) [16,17] to replace proteins affected by PI. However, neither the conventional methods of platelet function analysis nor routine quality control tests provide information on which changes in platelet proteins are the underlying cause of impaired platelet function induced by manipulations such as photochemical treatments. A comprehensive assessment of the impact of new ways of production and storage of PCs through the assessment of proteins on a large scale could provide this information and may guide additional functional studies.

73

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

74 74 75 76 76 76 77 77 77 78 78 78 78 80 80 80 80 81 81

Proteomic approaches have filled this gap and were successfully used to unravel protein changes caused by PI and storage of PCs. However, the variability of platelet products and proteomic methods used by different laboratories generated a variety of data sets dependent on the particular details of the approach. This complicates the interpretation of proteomic results and their relevance in the context of transfusion medicine. Therefore, this consensus paper authored by 5 laboratories that work in the platelet proteomic field in transfusion medicine aims to establish the current state of the art of proteomic findings in pathogen-inactivated PCs (PI-PCs).

Pathogen Inactivation of Platelet Concentrates At the present time, PCs can be pathogen inactivated by 3 photo/ photochemical methods, the riboflavin (RF) + UV light (Mirasol PRT; Terumo BCT Biotechnologies, Lakewood, CO), the amotosalen hydrochloride (AS) + UV-A (Intercept Blood System; Cerus Europe BV, Amersfoort, The Netherlands; Concord, CA), and the UV-C technology (Theraflex UV-C; Macopharma, Mouvaux, France). These PI treatments will be termed RF + UV, AS + UV-A, and UV-C for clarity. All 3 PI treatments inactivate a large range of bacteria, viruses, parasites, and also residual leukocytes, but none inactivates all pathogens, and there are significant differences in efficacy in regard to specific pathogens. In general, the currently available PI treatments have limited efficacy in the inactivation of nonenveloped viruses and very limited effects on bacterial spores, and they are ineffective against prions [1]. Current PI of PCs blocks the replication of pathogens by irreversible modification of nucleic acid strands. All PI treatments illuminate PCs with UV light (see Table 1) in the presence or absence of photoactive chemicals but subsequently rely on different chemical mechanisms. Therefore, their potential to interfere with molecules other than nucleic acids, for example, proteins, may also differ. Oxidized amotosalen reacts with DNA and RNA strands, which are then crosslinked by photocycloaddition. Moreover, free amotosalen molecules generate radicals upon irradiation, which interfere with nonnucleic Table 1 Wavelength ranges of UV light [18]

2

PI will be used throughout the manuscript. Pathogen reduction is also used in the literature but is somewhat misleading as it does not reduce the level of pathogens, it reduces their proliferation, and it may not totally eliminate them. However, treatment leads to a safer platelet product.

Name

Abbreviation

Wavelength range

Notes

Ultraviolet A Ultraviolet B Ultraviolet C

UV-A UV-B UV-C

380-320 nm 320-290 nm 290-190 nm

Long wave, black light Medium wave Short wave, germicidal

74


acid molecules. Riboflavin also forms radicals upon irradiation reacting with nucleic acids and other molecules. UV-C light irradiation directly induces intrastrand and interstrand reactions and has the potential to induce water oxidation and radical formation. Beside their ability to inhibit proliferation of pathogens, PI treatments effectively inactivate residual leukocytes in PCs and may thus prevent transfusion associated graft-versus-host disease [19–21]. Hence, PI can substitute for the most widely applied γ-irradiation procedure or the less often applied UV-B irradiation used for this indication. On the other hand, PI might affect residual platelet RNA or mitochondrial DNA, which is relevant in the light of recent evidence suggesting a potentially important biological role of platelet RNA for protein neosynthesis [22] and proper mitochondria function [23]. To which extent inhibition of these functions impacts the biological effects of PCs remains to be determined. Moreover, PI might also affect platelet microRNA (miRNA), which is known to regulate mRNA translation in platelets [24]. Riboflavin Plus UV Light Riboflavin + UV uses RF (vitamin B2) and UV light (6.24 J/mL, 280400 nm) for photochemical damage of nucleic acids by photon transfer and oxidative chemistry. Direct electron transfer, production of singlet oxygen, and generation of hydrogen peroxide with formation of hydroxyl radicals induce irreversible nucleic acid strand breaks [25,26]. Riboflavin is added at a final concentration of 50 μM to the PC, and UV-illumination is performed under back and forth motion with 120 cycles per minute [25]. As RF and its photoproducts naturally occur in the circulation, these substances are not removed before transfusion [27]. A clinical study revealed no specific antibody formation after administration of RF + UV PCs suggesting no relevant neoantigen formation [28]. Mirasol has the CE mark, and it is in routine use in at least 18 countries [29]. Amotosalen Plus UV-A Light Amotosalen + UV-A applies UV-A light (3.9 J/cm 2, 320-400 nm) in the presence of the photoactive psoralen AS (S59-HCl). Amotosalen is a tricyclic molecule consisting of a furan and a pyrone moiety [30]. Amotosalen penetrates cell membranes and reversibly intercalates into nucleic acids. Upon UV-A illumination, covalent bonds between the reactive groups of amotosalen and pyrimidine bases are formed leading to intrastrand and cross-strand reactions. This allows inactivation of both single- and double-stranded nucleic acids [31]. Amotosalen is added to the PC at a final concentration of 150 μM. The suspension is illuminated with UV-A light, and afterwards, residual amotosalen and free photoproducts are adsorbed on a compound adsorption device. Amotosalen and its photoproducts potentially bear a mutagenic risk; however, this risk is considered negligible at the low residual concentrations detectable in the PC upon AS + UV-A treatment [32]. Accordingly, toxicology assessment did not reveal any relevant effects [33]. In regard to immunogenic neoantigens, clinical trials do not show any evidence of antibody induction by AS + UV-A PCs [30,34]. This PI system is approved (CE mark) and used in at least 21 countries [29]. UV-C Treatment UV-C applies only UV-C light (254 nm) for PI. UV-C blocks replication of nucleic acids by triggering the formation of interstrand and intrastrand of cyclobutane pyrimidine and pyrimidine pyrimidone dimers [35,36]. Agitation in a light permeable bag forms UV-Cpermeable thin PC layers that increase the efficiency of the PI process [37]. A great challenge is the poor ability of UV-C light to penetrate turbid solutions such as plasma; therefore, the UV-C system requires

the use of nonturbid platelet additive solutions with a residual plasma content of ~ 35%, UV-light permeable bags, and agitation to facilitate effective UV-light irradiation within the PC. The UV-C technology is currently the only system without a photoadditive, and therefore, toxicological effects are not an issue. Repeated transfusions of autologous UV-C–treated PCs were well tolerated in a dog model and did not induce specific antibody responses [38]. Also in a phase I clinical trial on safety and tolerability of autologous UV-C-treated platelets, none of 10 volunteers developed antiplatelet antibodies after the transfusion of 3 UV-C–treated PCs over a period of 3 months [39]. Clinical Studies of PI-PCs To date, of 12 clinical studies conducted or underway with PI-PCs, 8 are published, 7 comparing AS + UV-A PCs to standard apheresis or pooled buffy coat PCs and 1 trial comparing RF + UV PCs to standard PCs [40–42]. Including preclinical toxicology studies, clinical studies, and postmarketing surveillance of AS + UV-A–treated products [33], more than a million units of PI-PCs and inactivated plasma were transfused to patients in Europe; no unexpected adverse toxic events or neoantibody formation has been reported [43]. Also in the RF + UV clinical trials, no adverse events have been reported, which might have been indicative for toxic effects or neoantigen formation [28]. Hence, the safety of recipients seems not to be compromised by PIPCs. However, the reduced risk of pathogen transmission by PI-PCs has to be balanced against potentially reduced efficacy. Amotosalen + UV-A PCs demonstrated acceptable viability of platelets in healthy volunteers [44], hemostatic efficacy in the SPRINT trial, a large phase III clinical trial [45], and corrected prolonged bleeding times in patients with thrombocytopenia [46]. However, 4 completed [45,47–49] and 1 prematurely stopped [50] phase III clinical trials consistently reported reduced recovery, as reflected by corrected count increment (CCI) values, from 3% to 32% as compared with conventional PCs as well as increased numbers of transfusions. In the MIRACLE trial [51], RF + UV PCs failed to reject noninferiority based on the predefined CCI inferiority margin (20% of the mean). However, total utilization of PCs and red blood cell concentrates and safety outcomes did not differ between the groups. Further studies are needed to show whether the lower CCI observed with RF + UV– treated PCs is associated with an increased risk of bleeding. Indeed, 1 such study is underway for treatment of buffy coat platelets prepared in plasma. For the UV-C method, no clinical data are available at this point in time. Proteomic Tools Proteomics relies on the qualitative and quantitative study of large sets of proteins based on protein/peptide separation (either via electrophoresis or liquid chromatography [LC]) and identification (via mass spectrometry [MS] and tandem mass spectrometry [MS/MS]) [52–54]. The major challenges in proteomics and particularly in platelet proteomics [55–60] are the wide dynamic range of protein concentrations and the highly unbalanced abundance of protein species [61]. No single proteomic strategy can address the entire proteome (either cytoplasmic or membrane proteins, high- or lowabundance species) at the same time. Besides sample preparation, sample collection and handling procedures also influence proteomics results [62–65], and commonly accepted standards for sample preparation for platelet proteomics are lacking. In this regard, the minimum information about a proteomics experiment initiative has been started to “define community standards for data production and representation in proteomics, which should reduce technical variability and ease data comparison, exchange, and verification” [66]. Contemporary proteomic methods are reviewed elsewhere [52,54,67] (see also Fig 1). Briefly, the approach currently used is a


75

Fig 1. Techniques and methods in proteomic analysis (only related to reviewed research articles). Abbreviations: PTM, posttranslational modifications; SEC, size-exclusion chromatography; SCX, strong cation exchange; RP, reverse phase; ESI, electrospray ionization; MALDI, matrix-assisted laser desorption ionization; ToF, time of flight; iTRAQ, isobaric tags for relative and absolute quantification; MRM, multiple reaction monitoring.

bottom-up or shotgun approach where proteins, before or after separation, are digested into peptides, which are sequenced by MS/MS. The separation is performed either by LC (gel free) or using a gel-based approach (sodium dodecyl sulfate–polyacrylamide gel electrophoresis [SDS-PAGE] or 2-dimensional gel electrophoresis [2DE]). The detection of proteins is usually performed online by coupling LC to mass spectrometer (LC-MS/MS). The different separation techniques are based on various physicochemical properties and, therefore, may obtain different sets of proteins. This variability is aggravated by various technical differences (i) in the instruments used, for example, capillary or nanoflow LC, MS type—ion trap, orbitrap, time of flight—(ii) in the methods used, for example, specific pI range or staining methods in gel-based approaches, and by using various design or analysis criteria, for example, detection cutoff, number of technical replicates, and P value. Beyond these considerations, the different techniques applied in proteomics are complementary. Proteomic Analyses of PSLs Platelet storage lesions [3–5] encompass all changes leading to progressive damage in the platelet structure and function that arise

from the time blood is drawn from a donor to the time PCs are transfused into a recipient [5]. Platelet storage lesions affect platelet activation, metabolism, and platelet morphology [68]. Some of these changes may be reversible upon transfusion, whereas others result in the accelerated clearance of the platelets from the circulation [5]. Comprehensive proteomic approaches were applied to identify PSL at the protein level during PC storage [69–71]. Proteins identified are linked to platelet activation, cytoskeletal reorganization, vesicle trafficking, and apoptosis pathways (Table 2). Because there is only poor correlation between in vitro platelet function assays and the in vivo recovery and survival of transfused platelets, the question arises whether proteomic results correlate better with the transfusion outcome. In a pilot study, differences in the protein profile between healthy volunteers with either acceptable or reduced platelet recovery and survival were assessed using SDS-PAGE and MS [78]. This study showed a significant correlation between protein levels of Rap1 and RhoGDI during storage and platelet recovery and survival and provides the first evidence that protein profiling may predict the platelet transfusion outcome. This finding suggests that proteomic analysis of PIplatelets might provide information to guide further optimization of PI of therapeutic PCs.

Table 2 Summary of proteomic analyses of stored PC Studies

Affected proteins during storage of untreated PC

References

Intercept (buffy coat)

Coronin 1B, gelsolin, ILK, PCBP1, pleckstrin, prohibitin, UROD, VASP, CXCL7, dynamin 2, fibrinogen, cytoplasmic protein NCK2, SPARC (proteins from untreated and treated) Glycoprotein Ibβ chain, glycoprotein IX, Rab-1A (list not exhaustive) Cofilin 1, actin, CLIC4 Actin, 14-3-3 protein, gelsolin, DJ-1, lactate dehydrogenase, adenine phosphoribosyl transferase Rap1, talin, glycoprotein IIIa 14-3-3 protein, cofilin Early focal adhesion signaling, integrin αIIb βIIIa signaling Accumulation of proteins in supernatant during storage Superoxide dismutase, septin 2, zyxin Accumulation of proteins in supernatant during storage: tremlike transcript 1, ILK, CXCL8, TNF-α Septin 2, gelsolin, β-actin

Hechler et al [9]

Intercept (buffy coat) Intercept (buffy coat) Mirasol (apheresis) Mirasol (apheresis) Mirasol (buffy coat and apheresis) Storage Storage (buffy coat) Storage Storage (buffy coat) Storage

Thiele et al [72] Prudent et al [73] Marrocco et al [74] Schubert et al [75] Schubert et al [76] Thiele et al [71] Egidi et al [65] Thon et al [70] Glenister et al [77] Thiele et al [69]

76


Table 3 Summary of proteomic analyses of PI-PCs PI

Process of inactivation

Proteomic approach

No. of proteins affected

Proteins of interest

Function potentially affected

References

Theraflex UV-platelets process (buffy coat– derived PC) γ Irradiation

UV-C UV-B γ Irradiation

DIGE (gel) MALDI-MS/MS LC-MS/MS

48 67 87

RasGTPase-activating-like protein (↗) ERp72 (disulfide isomerase) (↗)

Cytoskeletal organization PLT aggregation

Mohr et al [10]

Mirasol (buffy coat and apheresis)

RF + UV

SDS-PAGE (gel) LC-MS/MS

26 at day 6 (no difference before)

VASP P Ser239 (↗)

Maintenance of the actin structure and regulation of its dynamics

Schubert et al [76]

Mirasol (apheresis)

RF + UV

SDS-PAGE (gel) LC-MS/MS

5 after treatment 12 changed during storage, 7 of these strongly expressed in treated

Rap1, RhoA, moesin, RhoGDI, HSP27

Relocalization of proteins to cytoskeleton Intracellular vesicle transport

Schubert et al [75]

Mirasol (apheresis)

RF + UV

iTRAQ LC-MS/MS

52 treated 105 untreated

Talin, vinculin, filamin, zyxin 14-3-3 isoforms, Rab GTPases (↗)

Cytoskeleton Signal transduction regulating vesicle trafficking

Schubert et al [79]

Mirasol (apheresis) γ Irradiation

RF + UV γ Irradiation

2DE MALDI-MS/MS LC-MS/MS MRM on metabolites

5 at day 1 9 at day 5 (γ: 4 at day 1 10 at day 5)

Actin (↘) Pleckstrin (↘) ATP5B (↘) BH3 interacting domain death agonist isoform2 (↘)

Oxidative stress PLT metabolism and activation Cell signaling Cytoskeleton

Marrocco et al [74]


AS + UV-A

DIGE (gel) MALDI-MS/MS

3 at Day 2.5 (13 related to storage)

Integrin-linked protein kinase (↗) Cytoplasmic protein NCK2 (↘) Pleckstrin (↗)

Cytoskeletal organization PLT function

Hechler et al [9]


AS + UV-A

LC-MS/MS (gel free)

23 at day 1 58 at day 5 3 show consistent changes

PEAR-1 (↘) Protein-tyrosine sulfotransferase 2 (↘) CLIC4 (↗)

Catalytic activity PLT function Oxidative stress

Thiele et al [72]


AS + UV-A

2DE LC-MS/MS

3 at day 2 6 at day 8

DJ-1 (↘) Glutaredoxin 5 (↘) G(i)α2 (↗) (C ter)

Cytoskeletal organization Oxidative stress PLT function

Prudent et al [73]

Proteomic Analyses of PI-PCs Table 3 summarizes all published proteomic analyses to date on PIPCs. The following section elaborates studies of PC treated with various PI systems. Riboflavin Plus UV Light Comparison to γ-Irradiated Platelets Within the framework of the Italian Platelet Technology Assessment Study (IPTAS), Marrocco et al [74] investigated the variations of the protein profiles of apheresis PCs following treatment with RF + UV. Control, γ-irradiated (35 Gy), and RF + UV–treated apheresis PCs were analyzed on days 1 and 5 of storage by means of gel-based analytical approaches (2DE) and subsequent MS-based identification of proteins showing statistically significant (fold change at least ≥ 1.5; P b .05, analysis of variance) differences. Supernatants were also assayed for metabolites and oxidative stress-related changes using multiple-reaction monitoring MS (see Fig 1). Both γ-irradiation and RF + UV treatment resulted in decreased levels of glutathione (GSH) and promoted increases in oxidized GSH, a trend that was further exacerbated by storage. Supernatant glucose consumption, lactate accumulation, and pH drop correlated with progressive release into the supernatant of the enzyme lactate dehydrogenase, both in untreated controls and treated units. Changes continued with storage of RF + UV–treated units supporting prior

reports that these units are more metabolically active than untreated controls [76,80–82]. Both the analysis of Marrocco et al [74] and the study by Thiele et al [69] showed that storage of untreated apheresis platelets resulted in the alteration of cytoskeletal proteins (actin, gelsolin) and signaling proteins (14-3-3) as well as in an increase in oxidative stress proteins (DJ-1) similar to that reported by Prudent et al [73] for AS + UV-A–treated platelets. Both γ-irradiation and RF + UV treatments accelerated oxidative stress accumulation in comparison with untreated controls. DJ-1 was up-regulated, and ER-60 protease (which is involved in redox-based modifications of protein disulfide bonds) was down-regulated soon upon γ-irradiation treatment right at day 0 (in comparison with untreated controls). Riboflavin + UV–treated platelets displayed several features of longer stored control counterparts, as suggested by treatment-dependent day 0 decrease of in vitro aging markers such as actin and pleckstrin [71]. Although platelet in vitro aging has been often linked to apoptosis [83], in the IPTAS investigation, a decrease in the levels of the proapoptotic marker BH3-interacting domain death agonist isoform 2, an apoptosis inducer that counteracts the Bcl-2 protective effect, was observed. This led to the consideration that PI treatments based upon RF and UV exposure might trigger other mechanisms than canonical apoptotic pathways. However, Reid et al [84] found specific apoptosislike features in RF + UV–treated units, such as phosphatidylserine exposure (annexin V), cytochrome C release, and increased Bak and Bax levels and caspase 3 activity.


Overall, this proteomics/targeted metabolomics investigation conducted as part of the IPTAS did not reveal major variations in the platelet proteome of RF + UV–treated platelets in comparison with γirradiated counterparts (the currently issued blood therapeutics), except for a more sustained metabolic activity indicated by a higher glucose consumption and lactate accumulation and increased oxidative stress as gleaned by monitoring the GSH/oxidized GSH ratio. Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis and LC-MS/ MS Analysis Proteomic studies suggest that RF + UV–induced protein changes affect specific cell compartments and signal transduction pathways [75,76,79,85]. These changes may contribute to the decreased platelet function as reported by in vitro assays. Schubert et al [76] compared protein changes in RF + UV–treated apheresis and buffy coat–derived PCs stored in plasma. Using SDSPAGE, they identified several changes in proteins associated with the structure and regulation of the cytoskeleton. These findings were confirmed and complemented by Marrocco et al [74] who found changes in proteins linked to oxidative stress, platelet activation, and cell signaling, as described above. Some of these effects were more pronounced in the γ-irradiated products suggesting a less invasive effect of the RF + UV treatment on the PC. The different proteomic approaches reveal only partial agreement primarily due to the nature of the techniques [70,86]. A gel-free proteomic analysis based on iTRAQ labeling [79] identified only a small number of pronounced protein changes, which is in agreement with Marrocco et al [74]. In addition, SDS-PAGE was used to analyze RF + UV–treated platelets that were lysed and subjected to differential centrifugation to obtain crude fractions enriched in proteins of the membrane cytoskeleton, actin cytoskeleton, and cytoplasm [75]. This analysis revealed that the most notable change consisted of relocalization of proteins from the cytoplasm to the membrane cytoskeleton following RF + UV treatment. This observation supports a model in which the GTPase RhoA and moesin relocalize to the platelet membrane in response to RF + UV treatment, while at the same time, RhoA might regulate the kinase ROCK (see section on “biological functions”). Further investigation in RF + UV–treated apheresis platelets identified treatment-dependent phosphorylation at Ser-239 of the vasodilator-stimulated phosphoprotein (VASP), a phenomenon that may impair the platelet shape change, but which occurred to a much lesser degree in untreated buffy coat–derived PCs [76]. This study also revealed an exponential correlation between VASP phosphorylation and platelet activation as measured by P-selectin expression. The finding that posttranslational modifications are affected by the RF + UV treatment triggered a study analyzing changes in the phosphorylation profile of a subset of protein kinases [85]. Among the candidates identified, the kinase p38MAPK increased 4-fold in phosphorylation after RF + UV treatment and incubation of PCs with a p38MAPK-specific inhibitor before PI significantly improved in vitro platelet function measures. Phosphorylation levels of the p38MAPK substrates AKT, VASP, and the heat shock protein 27 (HSP27) also decreased with inhibitor treatment. Reduction of phospho-HSP27 levels correlated with a decrease in platelet activation. These biochemical alterations are consistent with a signaling model for PCs treated with the RF + UV system in which the treatment triggers activation of p38MAPK either directly or via an as yet unidentified indirect mechanism. Phosphorylation of p38MAPK leads to AKT activation or phosphorylation probably via the MAPK-activated protein kinase 2. Either p38MAPK directly via the MAPK-activated protein kinase 3 or via Akt then activates HSP27 by phosphorylation. An alternative or additional signaling pathway branch involves VASP, which might be activated or phosphorylated by p38MAPK following RF + UV treatment, although the precise details remain to be determined. This signaling model provides a testable set of hypotheses to pinpoint

77

molecular mechanisms of changes in the intraplatelet signaling cascade triggered by the RF + UV treatment. Amotosalen Plus UV-A Light Two-Dimensional Gel Electrophoresis Analysis Prudent et al used a gel-based proteomic strategy to study the effect of AS + UV-A treatment on PCs from pooled buffy coats based on a pool and split model, that is, 2 bags were pooled and split in 2 identical units, where one is treated and the other one kept as untreated control (n = 5) [9,73]. Samples were withdrawn at day 1 (before treatment) and at days 2 and 8 from the treated and untreated PCs. Total protein extracts were separated by 2DE (pI 4-7), and spots of interest were analyzed by LC-MS/MS for protein identification. The number of spots detected fits well with the number of proteins reported using these techniques of separation and silver staining (see reference map on Swiss-2DPAGE database [87]). A detailed analysis of the 2DE maps revealed the low impact of the AS + UV-A treatment on the proteome. Only 3 proteins were affected by the AS + UV-A treatment: DJ-1, glutaredoxin 5, and guanine nucleotide-binding protein G(i) subunit α2 (G(i)α2). As for the impact of storage, 3 other proteins increased in concentration with time and independent of AS + UV-A treatment: chloride intracellular channel protein 4 (CLIC4), actin, and cofilin 1. The AS + UV-A treatment induced a significant increase of oxidized DJ-1 [88] (3-fold increase at day 2 vs control and 4-fold increase at day 8 vs control), and the amount of the native protein tends to decrease. As reported by Marrocco et al [74] DJ-1 is related to oxidative stress and is a partner in chaperone machinery. Duan et al [88] found that oxidized DJ-1 slightly diminished, whereas newly synthesized DJ-1 rescues the basal protein pattern after the removal of the oxidative stress. In neurons, it is implicated in chaperone and proteasome-targeting activities [89–92], and DJ-1 interacts with antioxidant enzymes [93]. In platelets, the function of this protein is unknown. Nevertheless, DJ-1 that is linked to oxidative stress is not rescued at day 2 after AS + UV-A treatment nor after 8 days of storage. Glutaredoxin 5 is another protein related to oxidative stress that decreases upon treatment with AS + UV-A (with the concomitant appearance of an acidic form). This monothiolglutaredoxin is poorly studied and is passively recycled in the presence of GSH [94,95]. Thus, the appearance of an oxidized form upon treatment may be related to decreased GSH system activity [96]. Signal transduction and aggregation activity of the platelet may also be affected by the treatment. Of note, the G-protein G(i)α2 was altered and the formation of a C-terminal fragment was observed (according to MS data) after irradiation. This protein is linked to ADPmediated activation through its interaction with the ADP receptor P2Y12 [97–102]. This modification may have only a minor impact on the promotion of platelet aggregation (see section on “biological functions”), since Hechler et al [9] reported that ADP activation is well preserved in AS + UV-A–treated platelets [9]. Moreover, this Gprotein activates PI3-K, which leads to αIIbβ3 activation. Finally, 3 proteins were affected in stored PCs independently of AS + UV-A treatment, that is, CLIC4, actin, and cofilin 1. Cofilin 1 showed a significant decrease over the storage period only in untreated units. Actin fragments accumulated to statistically significant levels, consistent with this known effect of platelet storage and the resultant alterations in the platelet cytoskeleton [10,70]. Chloride intracellular channel protein 4 also increased over time during storage. The function of this protein, which inserts into nuclear membranes in a redox sensitive manner [103–105], is unknown in platelets. The presence of CLIC4 in 2DE is indicative of an altered function of the transporter. Moreover, several studies have reported the CLIC/actin interactions are a key regulator of channel activity [73,106] and play a role in the stress-induced death pathway by converging on mitochondria upon DNA damage [105]. This suggests

78


that a modification of these interactions can alter the function of platelets stored for 8 days. This work, based on a 2DE approach, mainly shows that a limited number of proteins is affected by the AS + UV-A treatment. Two of them are related to oxidative stress lesion, and 1 is linked to the aggregation properties of platelets. These modifications persisted during storage, which suggests that no rescue is possible. In addition, 3 proteins were modified over 8 days of storage independently of the storage time. Of particular interest, CLIC4 links cytoskeleton organization, apoptotic mechanisms, and oxidative stress. Hechler et al [9] also applied a gel-based approach in their work on the functional and biochemical properties of platelets from AS + UVA-treated buffy coat PCs compared with those from conventional PCs. Four AS + UV-A–treated PCs and 4 conventional PCs were stored for 6.5 days, and platelet function and proteomic profiles were examined at various time points during storage. To evaluate their intrinsic properties, samples of stored platelets were taken, washed, and suspended in Tyrode's buffer before testing. Platelet counts and morphology were conserved, although a slight increase in the platelet volume was observed after PI. Glycoproteins (GP) IIbIIIa, IaIIa, and VI expression remained stable, whereas GPIbα declined similarly in both types of PCs. A steep decrease of 50% in GP-V levels occurred by day 1.5 in AS + UV-A–treated PCs and by day 2.5 in control PCs. For both treated and control PCs, P-selectin expression and activated GPIIbIIIa remained low during storage. Treated and control PCs were fully responsive to aggregation agonists up to day 4.5 and exhibited similar functionality in assays under flow conditions. Mitochondrial membrane potential and annexin V binding of AS + UV-A–treated PCs and control PCs were comparable. As for the proteomic study using difference gel electrophoresis (DIGE) and MS, only 13 proteins (among 1886 protein spots, |fold change| N 1.5, pI 3-11; P b .05) were altered indicating overall stability in the proteome of PCs during storage. Moreover, comparing treated PCs to the controls, only 3 proteins changed in protein levels by AS + UV-A–treated PCs, namely, an integrin-linked protein kinase, a cytoplasmic NCK2 protein, and the protein kinase C (PKC) substrate pleckstrin. Additional studies are required to further establish the relationship between the molecular modifications of these proteins and their functional consequences. Overall, the AS + UV-A procedure does not substantially alter the proteomic profile as observed by DIGE. Of course, this does not rule out posttranslational modifications that could not be detected due to specific protein properties and limitation of the technique. Thus, washed AS + UV-A–treated and AS + UV-A–untreated PCs have similar functional, morphological, and proteomic characteristics provided platelets are suspended in an appropriate medium during testing.

indicates that AS + UV-A treatment and γ-irradiation alter different platelet proteins. A second important observation of this study is the different dynamics of protein changes between AS + UV-A–treated and γ-irradiated PCs. The relative stronger increase in altered protein levels in AS + UV-A–treated platelets suggests that enhanced storage lesions occur upon this treatment. Platelet aggregation receptor 1 precursor (PEAR-1), CLIC4, and protein-tyrosine sulfotransferase 2 were identified as changing after treatment and storage of AS + UV-A–treated PCs. Identification of PEAR-1 as a protein, which changes during AS + UV-A treatment, is a good example for the necessity to apply complementary proteomic techniques. This membrane protein was not observed in 2DE. Platelet aggregation receptor 1 precursor interacts with proteins in the integrin αIIbβ3 pathway during platelet aggregation [108], and protein-tyrosine sulfotransferase 2 contributes to the chemokinebinding function of chemokine receptors such as C-X-C chemokine recpetor type 4 (CXCR-4), which promote platelet activation [109]. These alterations may be involved in the functional defects observed in AS + UV-A–treated PCs.

Liquid Chromatography–MS/MS Analysis Highly sensitive LC-MS/MS was used to profile protein alterations in platelets induced by the AS + UV-A treatment, compared with γirradiation over 5 days of storage [72]. Three biologically identical buffy coat–derived PCs, obtained from 15 donors by a pool and split approach, were treated by AS + UV-A or irradiation with 30 Gy, while the third unit was used as control. In total, 721 platelet proteins were identified by at least 2 peptides and could be quantified by a label-free approach [107]. Accepted differences were at least 1.5-fold (P b .01). Amotosalen + UV-A triggered 23 protein alterations compared with the control, whereas γ-irradiation induced 49 changes. The higher number of altered proteins as compared with the study described above reflects the greater coverage of gel-free–based methods (which is mainly due to the specificity of 2DE that focuses on particular protein sets, see the section “Proteomic tools”). After 5 days of storage, more proteins had changed in the AS + UV-A–treated PCs than in γ-irradiated platelets (58 vs 50 proteins) compared with the control at day 1. The overlap of changed proteins was small between the groups. This

Figure 2 displays the role of platelets in hemostasis and thrombus formation. The platelet proteins involved in these processes, which have been identified by proteomic analyses as changing during different PI of PCs, are highlighted in Figure 3; proteins involved in aging and oxidative stress are not discussed here. As shown by the blue boxes, RF + UV impacts mainly actin polymerization, cytoskeleton organization, and platelet shape change, whereas AS + UV-A (red boxes) seems to impact platelet activation and aggregation pathways. As for UV-C (green boxes), the 2 proteins of interest are associated with different parts of hemostasis and thrombus formation, that is, platelet shape change through an IQGAP2 [113] and aggregation through the activation of αIIbβ3 via ERp72 [114,115] upon thrombin activation.

UV-C Technology DIGE was applied to monitor UV-C–related proteome changes in platelets [10]. UV-B-irradiation and γ-irradiation at doses applied in clinical practice served as reference treatments [110]. In total, 763 protein spots before and after treatment with UV-C (0.4 J/cm2), UV-B (0.5 J/cm2), or γ-irradiation (25 Gy) were monitored. UV-C treatment induced changes in 107 protein spots (48 distinct proteins), UV-B induced changes in 140 protein spots (67 distinct proteins), and γ-irradiation triggered alterations in 161 protein spots (87 distinct proteins). The overlap between the different irradiation procedures involved 92 common spots (42 proteins). Only 2 protein spots were changed by UV-C irradiation alone (13 shared with UV-B, and 92 in common with both UV-B and γ-irradiation): IQGAP2 (RasGTPase-activating-like protein) and ERp72 (protein disulfide-isomerase A4). Although IQGAP2 was also identified in other protein spots altered by UV-B and γirradiation, ERp72 was changed exclusively by UV-C treatment. UV-C treatment changed the platelet proteome far less than the other irradiation procedures. Of note, the UV-C dose now applied in clinical trials is 0.2 J/cm 2 and lower than the dose evaluated by this proteomic study. This may result in even less protein changes than observed here. However, further data need to be collected to access the impact of different UV-C dosages on the proteome and subsequent platelet storage. Biological Functions Affected by PI Discovered by Proteomic Studies

Riboflavin plus UV Affects Proteins Involved in Adhesion and Activation,… von Willebrand factor (vWF) released from injured endothelial cells will bind to platelets and facilitate platelet adhesion and concomitant phosphorylation of the kinase p38MAPK. This kinase is activated after


79

Fig 2. Platelet in hemostasis and thrombosis.

binding of vWF to GP Ib-IX-V and indirectly induces the release of thromboxane A2 for platelet activation [116]. p38MAPK is one of the proteins affected by the RF + UV treatment [85] and is involved in several signaling pathways leading to platelet adhesion/activation, granule release, and platelet shape change. p38MAPK is also involved in

thrombin-mediated platelet activation [117]. Cytosolic phospholipase A2, responsible for thromboxane A2 release [118], is targeted by p38MAPK via PKCδ upon thrombin activation [119]. p38MAPK also interacts with the fibrinogen receptor αIIbβ3 in a complex regulated system. Initially, αIIbβ3 inhibits the p38 kinase,

Fig 3. Platelet activation/aggregation pathways and proteins potentially affected by PI. Only potentially affected pathways are pictured. Part of the mechanisms was based on Ref [101,111], and cited references in italic. Illustrations used elements from Servier Medical Art [112].

80


but later, in the platelet activation cascade, it enhances its activity (see bottom right of the main cell in Fig 3). Therefore, upon binding to fibrinogen, this outside-in signaling is a key step in platelet activation. Thus, 2 different mechanisms are involved to induce the activation of MAPK in platelet activation, an agonist-mediated MAPK activation and an integrin-mediated MAPK activation [120].

aggregation and secretion of platelets. Lian et al [129] emphasized the role of pleckstrin on exocytosis [129]. Indeed, they found that pleckstrin regulates the fusion of granules in a PKC-dependent manner. In addition, they observed that a PI3-K–dependent signaling pathway compensates for the loss of this protein in pleckstrin null platelets from mice.

… In Granule Secretion and Platelet Shape Change,…

Amotosalen plus UV-A Affects Proteins Mainly Involved in the Mechanisms of Platelet Aggregation

Talin, vinculin, and zyxin all play a role in cytoskeleton organization and link actin polymers to integrins. Actin polymerization, which is required for platelet shape change, is controlled at least in part by tryptophan-aspartic acid (WD) repeat-containing protein 1 isoform 1 (WDR1) mediating the disassembly of actin polymers in conjunction with actin depolymerizing factor (ADF)/cofilin proteins. In addition, VASP that is phosphorylated at Ser 239 impairs actin filament formation [121]. This phosphorylation is particularly pronounced in apheresis platelets after RF + UV treatment, as reported by Schubert et al [76]. They also reported the increase of VASP phosphorylation at Ser 157 in RF + UV–treated buffy coat PCs, which blocks VASP binding to αII integrin and impairs integrin αIIbβ3 activation [122]. The phosphorylation on Ser 157 is also achieved through PKC-dependent and Rho-kinases–dependent mechanisms in platelets activated by thrombin [123]. Among other signaling pathways, VASP phosphorylation is also triggered by p38MAPK [85]. Polanowska-Grabowska and Gear [124] reported that HSP27 is phosphorylated after thrombin stimulation following the activation of a protein kinase cascade controlling actin polymerization. Modifications of this set of proteins consequently may impair the actin polymerization and thus the platelet shape change and the release of granules as well as the clot retraction. Another set of proteins related to platelet shape change appears to be affected by RF + UV treatment [75]. GTPaseRhoA and moesin relocalize to the platelet membrane in response to RF + UV treatment. RhoA plays an important role in platelet shape change, spreading, and clot retraction. RhoGDI releases RhoA after its activation by a guanine nucleotide exchange factor (GEF) triggered by stimulation with thrombin [125]. It then activates myosin for platelet shape change through ROCK, which also regulates actin filaments via moesin, a protein that is affected by the RF + UV treatment [75]. Myosin can also be activated by p38MAPK and then activates an integrin-mediated Rac1 pathway that is important for platelet retraction [120]. Rap1, another GTPase, seems to move to the membrane cytoskeleton following RF + UV treatment. This protein is involved in intracellular vesicle transport, and the relocalization dynamics correlated well with α granule release as determined by P-selectin surface expression. Thus, modification of proteins related to platelet shape change can directly affect hemostasis (see Fig 2). … And in Aggregation of Platelets Rap1, which initiates the activation of αIIbβ3 for platelet aggregation, is also affected by RF + UV. The activation of Rap1 by the Ca 2+-dependent CalDAG-GEFI and PKC induces the recruitment of different proteins to the membrane such as RIAM and talin, which are required for integrin activation and subsequent binding to fibrinogen and other activated platelets necessary for aggregation [115,126]. Moreover, the activation of Rap1 is dependent on PI3-K [127]. It has to be pointed out that the PI3-K inhibitor LY294002 used in this study seemed to improve platelet integrity during storage. Finally, pleckstrin (or p47), a substrate of PKC, is affected by RF + UV treatment [9,74]. Baig et al [128] have shown, using pleckstrin null platelets from knockout mice, that upon activation, pleckstrin is translocated to the plasma membrane, a process that is facilitated by serum deprivation response protein (SDPR). Subsequently, it is phosphorylated by PKC, and the phosphorylated form induces both

As mentioned above, phosphorylated pleckstrin induces platelet aggregation through αIIbβ3. Pleckstrin was also observed to be affected by the AS + UV-A, but in a different manner (see Table 3). It decreases in RF + UV, whereas it increases in AS + UV-A–treated PCs. The differences may be explained either by a different effect of the PI or a difference in details of the 2DE analyses. The latter can influence the results depending how the analysis deals with several spots that can contain the phosphorylated form. Indeed, the increase in pleckstrin intensity reported by Gachet et al takes in consideration 2 spots, very close to each other [9]. On the other hand, in their RF + UV study, Marrocco et al observed a decrease [74]. Although it is difficult to unravel the exact impact on the aggregation of platelets (inhibition/ activation in function of PI treatments), pleckstrin remains a protein of interest, and a phosphoproteomic analysis could provide new insights. The decrease in PEAR1 abundance was found by a gel-free approach in AS + UV-A–treated PCs [72]. This membrane protein, not detected by Prudent et al [73] in their gel-based approach, plays a role in sustaining platelet aggregation via the amplification of αIIbβ3 activation. Kauskot et al [108] described a role in which PEAR1 and its ligand increase at the platelet surface after platelet activation. Once linked to its ligand on an adjacent platelet, PEAR1 is phosphorylated, which induces the activation of Akt by phosphorylation on Ser 473. This phosphorylation leads to the activation of integrin αIIbβ3 and thus platelet aggregation. Thiele et al also observed the decrease of protein-tyrosine sulfotransferase 2. This type of enzyme controls the interaction of proteins with partners by the sulfation of tyrosine residues, which then interact with chemokine receptors [109]. G-coupled proteins are also affected by AS + UV-A, and a fragment of G(i)α2 was detected in 2DE by Prudent et al [73] upon this PI treatment. This α subunit of G-protein is coupled to the ADP receptor P2Y12 [97–102]. Upon ADP binding to its receptor, the C-terminal part binds to adenylyl cyclase [130] and inhibits the formation of cyclic adenosine monophosphate (cAMP), thereby facilitating platelet aggregation (of note, cAMP is also required for VASP phosphorylation). Gi is also involved in the regulation of PKC activity [131]. Finally, 2 other proteins were either up-regulated or downregulated in DIGE after AS + UV-A treatment, that is, integrin-like protein kinase (ILK) and NCK2, respectively [9]. Integrin-like protein kinase is a kinase that links to integrins [132]. It phosphorylates Akt on Ser 473 and thus regulates the integrin αIIbβ3 by phosphorylation of the β3 subunit [133,134]. Integrin-like protein kinase is also involved in coupling different receptors and links integrins to actin filaments [132]. Moreover, it is coupled to growth factor receptors through PINCH and NCK2 [135], proteins that are also affected by AS + UV-A. Impairment of these proteins may thus hamper normal platelet aggregation. Further Studies That Will Supplement the Present Picture First of all, the above discussion rests primarily on proteomic data, which should serve to target mechanisms in aggregation and not to draw direct conclusion on hemostatic properties. Our current understanding of the impact of PI on proteins covers a wide range of platelet functions from platelet adhesion and activation to platelet aggregation via platelet shape change. In AS + UV-A and RF + UV


studies, complementary proteomic approaches demonstrate that RF + UV tends to affect more the cytoskeleton organization than the AS + UV-A treatment, which, in turn, affects primarily the aggregation pathways. Because of the limited number of proteomic analyses, it is difficult to draw conclusions about the impact of UV-C on platelet protein changes. However, the remaining question is: to what extent do these modifications influence thrombus formation? It should be emphasized that the degree of the impact at the protein level (independently of the protein synthesis, where the impact is still unknown to our knowledge) is quite small and, in general, PI does not totally suppress the properties and functions of platelets. The worst case seems to be the reduction in platelet activity, as reported by hemostatic activity (see the section “clinical studies”). Future studies should focus on further unraveling the biochemical processes that are affected by PI to identify ways of compensating the potential decrease in hemostatic activity caused by PI. As a final commentary, the transcriptome has not been hitherto approached in the context of platelet-directed PI. Indeed, it is known that platelets contain a pool of mRNAs [22]. These mRNAs are regularly translated when required upon activation, such as translation of actin, αIIbβ3, and vWF. The presence of miRNA was reported in platelets, and they are key regulators of mRNA [24]. The photochemistry of PI targets DNA and RNA strands, and thus, platelet miRNA/ mRNA might also be the target of the diverse inactivation systems. Thus, the translation regulation will be affected, up-regulating or down-regulating proteins depending on the mechanism. Conclusions and Expert Commentary Although PI has been introduced in different countries worldwide, some countries made different choices or are waiting for further hemovigilance data or complementary clinical studies that will enrich the actual knowledge on the impact of PI on PCs and on the efficiency of platelet transfusions. We reviewed here the proteomic analyses conducted by our different research groups to understand the impact of PI on PCs, including the RF + UV (ie, Mirasol), the AS + UV-A (ie, Intercept), and the UV-C (ie, Theraflex) for treatment of platelets, and the potential impact on platelet functions. The complementarities of the approaches applied enable coverage of a wide range of proteins and provide an overview of the potential for PI-mediated platelet damage. The reason for this damage is probably linked to oxidative properties of the photoactive compounds. Oxidative defenses overcome these oxidants and limit the damage. However, the reason why a particular set of proteins are preferentially affected is not clear. In some cases, proteins may be directly oxidized, which can either induce a direct loss of function or displace metabolic fluxes or signaling capacities [136]. In summary, there is a relatively weak impact of PI on the overall proteome of platelets, but some data show that the different PI treatments lead to an acceleration of storage lesions. Although a variety of proteins are affected (degraded, oxidized, phosphorylated), the number of altered proteins is low (in comparison with the whole proteome), and most proteins remain intact. Although these proteins affect relevant pathways, further studies are needed to make strong conclusions. These identifications provide the basis to understand the impact of different PI approaches on the molecular mechanisms of platelet function. Moreover, these data should serve as basis for future developments of PI technologies of PCs, and identified protein lesions are potential markers in PI-mediated platelet damage [85]. The next question concerns the storage time and the impact of PI on PSL. Although PI theoretically enables the extension of the shelf life of PCs, it will be pivotal to assess whether this might be hampered by the exacerbation of certain PSL in response to PI treatments (such as the release of specific proteins in the supernatant—differences beyond 5

81

days of storage [136]). One of the further steps should thus consider both the PI and the impact on storage duration. These analyses will undoubtedly be conducted and will provide more details. Of course, deeper proteomic analyses, such as phosphoproteomics and transcriptomics, biochemistry (even at the peptide level), and functional analyses will furnish new insights, will complete the current picture of PI-affected pathways, and will highlight the ways of compensating the potential decrease in hemostatic activity. This research is still ongoing, and we believe that these data will enable us to better understand PI, to make comparisons between PI treatments and will allow blood centers, administrations, and decision makers to choose a suitable technology to make platelet transfusion safer and more efficient. The joint platelet proteomic approach has already provided an unprecedented deep insight into the molecular structures of platelets produced for platelet concentrates.

References [1] Cazenave JP, Isola H, Kientz D. Pathogen inactivation of platelets. In: Sweeney J, Lozano M, editors. Platelet transfusion therapy. Bethesda, MD: AABB Press; 2013. p. 1–58. [2] Alter HJ, Klein HG. The hazards of blood transfusion in historical perspective. Blood 2008;112:2617–26. [3] Devine DV, Serrano K. The platelet storage lesion. Clin Lab Med 2010;30:475–87. [4] Ohto H, Nollet KE. Overview on platelet preservation: better controls over storage lesion. Transfus Apher Sci 2011;44:321–5. [5] Shrivastava M. The platelet storage lesion. Transfus Apher Sci 2009;41:105–13. [6] Apelseth TO, Bruserud O, Wentzel-Larsen T, Bakken AM, Bjorsvik S, Hervig T. In vitro evaluation of metabolic changes and residual platelet responsiveness in photochemical treated and gamma-irradiated single-donor platelet concentrates during long-term storage. Transfusion 2007;47:653–65. [7] Bashir S, Cookson P, Wiltshire M, Hawkins L, Sonoda L, Thomas S, et al. Pathogen inactivation of platelets using ultraviolet C light: effect on in vitro function and recovery and survival of platelets. Transfusion 2012;53:990–1000. [8] Goodrich RP, Li JZ, Pieters H, Crookes R, Roodt J, Heyns AD. Correlation of in vitro platelet quality measurements with in vivo platelet viability in human subjects. Vox Sang 2006;90:279–85. [9] Hechler B, Ohlmann P, Chafey P, Ravanat C, Eckly A, Maurer E, et al. Preserved functional and biochemical characteristics of platelet components prepared with amotosalen and ultraviolet A for pathogen inactivation. Transfusion 2013;53:1187–200. [10] Mohr H, Steil L, Gravemann U, Thiele T, Hammer E, Greinacher A, et al. A novel approach to pathogen reduction in platelet concentrates using short-wave ultraviolet light. Transfusion 2009;49:2612–24. [11] Perez-Pujol S, Tonda R, Lozano M, Fuste B, Lopez-Vilchez I, Galan AM, et al. Effects of a new pathogen-reduction technology (Mirasol PRT) on functional aspects of platelet concentrates. Transfusion 2005;45:911–9. [12] Picker SM, Tauszig ME, Gathof BS. Cell quality of apheresis-derived platelets treated with riboflavin-ultraviolet light after resuspension in platelet additive solution. Transfusion 2012;52:510–6. [13] van Rhenen DJ, Gulliksson H, Cazenave JP, Pamphilon D, Davis K, Flament J, et al. Therapeutic efficacy of pooled buffy-coat platelet components prepared and stored with a platelet additive solution. Transfus Med 2004;14:289–95. [14] Verhaar R, Dekkers DWC, De Cuyper IM, Ginsberg MH, de Korte D, Verhoeven AJ. UV-C irradiation disrupts platelet surface disulfide bonds and activates the platelet integrin alpha IIb beta 3. Blood 2008;112:4935–9. [15] Sigle J-P, Infanti L, Studt J-D, Martinez M, Stern M, Gratwohl A, et al. Comparison of transfusion efficacy of amotosalen-based pathogen-reduced platelet components and gamma-irradiated platelet components. Transfusion 2013;53:1788–97. [16] Schubert P, Devine DV. De novo protein synthesis in mature platelets: a consideration for transfusion medicine. Vox Sang 2010;99:112–22. [17] van der Meijden PE, Heemskerk JW. Platelet protein shake as playmaker. Blood 2012;120:2931–2. [18] Coohill TP. Uses and effects of ultraviolet radiation on cells and tissues. In: Waynant RW, editor. Lasers in medicine. Boca Raton, FL: CRC Press; 2002. p. 85–108. [19] Fast LD, DiLeone G, Marschner S. Inactivation of human white blood cells in platelet products after pathogen reduction technology treatment in comparison to gamma irradiation. Transfusion 2011;51:1397–404. [20] Seltsam A, Muller TH. UVC irradiation for pathogen reduction of platelet concentrates and plasma. Transfus Med Hemother 2011;38:43–54. [21] Grass JA, Hei DJ, Metchette K, Cimino GD, Wiesehahn GP, Corash L, et al. Inactivation of leukocytes in platelet concentrates by photochemical treatment with psoralen plus UVA. Blood 1998;91:2180–8. [22] Harrison P, Goodall AH. “Message in the platelet”—more than just vestigial mRNA! Platelets 2008;19:395–404. [23] Bruchmuller I, Losel R, Bugert P, Corash L, Lin L, Klutner H, et al. Effect of the psoralen-based photochemical pathogen inactivation on mitochondrial DNA in platelets. Platelets 2005;16:441–5. [24] Landry P, Plante I, Ouellet DL, Perron MP, Rousseau G, Provost P. Existence of a microRNA pathway in anucleate platelets. Nat Struct Mol Biol 2009;16:961–84.

82


[25] Goodrich RP, Edrich RA, Li J, Seghatchian J. The Mirasol PRT system for pathogen reduction of platelets and plasma: an overview of current status and future trends. Transfus Apher Sci 2006;35:5–17. [26] Klein HG. Pathogen inactivation technology: cleansing the blood supply. J Intern Med 2005;257:224–37. [27] Marschner S, Goodrich R. Pathogen reduction technology treatment of platelets, plasma and whole blood using riboflavin and UV light. Transfus Med Hemother 2011;38:8–18. [28] Ambruso DR, Thurman G, Marschner S, Goodrich RP. Lack of antibody formation to platelet neoantigens after transfusion of riboflavin and ultraviolet lighttreated platelet concentrates. Transfusion 2009;49:2631–6. [29] AABB. http://www.aabb.org/resources/bct/eid/Documents/prt-systems-inroutine-use-country-listing.pdf; 2013. [Accessed: October 2013]. [30] Irsch J, Lin L. Pathogen inactivation of platelet and plasma blood components for transfusion using the INTERCEPT blood system. Transfus Med Hemother 2011;38:19–31. [31] Lin L, Cook DN, Wiesehahn GP, Alfonso R, Behrman B, Cimino GD, et al. Photochemical inactivation of viruses and bacteria in platelet concentrates by use of a novel psoralen and long-wavelength ultraviolet light. Transfusion 1997;37:423–35. [32] Tice RR, Gatehouse D, Kirkland D, Speit G. The pathogen reduction treatment of platelets with S-59 HCl (Amotosalen) plus ultraviolet A light: genotoxicity profile and hazard assessment. Mutat Res 2007;630:50–68. [33] Ciaravino V, McCullough T, Dayan AD. Pharmacokinetic and toxicology assessment of INTERCEPT (S-59 and UVA treated) platelets. Hum Exp Toxicol 2001;20:533–50. [34] Lin L, Conlan MG, Tessman J, Cimino G, Porter S. Amotosalen interactions with platelet and plasma components: absence of neoantigen formation after photochemical treatment. Transfusion 2005;45:1610–20. [35] Sinha RP, Hader DP. UV-induced DNA damage and repair: a review. Photochem Photobiol Sci 2002;1:225–36. [36] Cadet J, Sage E, Douki T. Ultraviolet radiation-mediated damage to cellular DNA. Mutat Res 2005;571:3–17. [37] Seghatchian J, Tolksdorf F. Characteristics of the THERAFLEX UV-Platelets pathogen inactivation system—an update. Transfus Apher Sci 2012;46:221–9. [38] Pohler P, Lehmann J, Veneruso V, Tomm J, von Bergen M, Lambrecht B, et al. Evaluation of the tolerability and immunogenicity of ultraviolet C–irradiated autologous platelets in a dog model. Transfusion 2012;52:2414–26. [39] Thiele T, Pohler P, Kohlmann T, Sümnig A, Aurich K, Selleng K, et al. Open phase I clinical trial on safety and tolerance of autologous UVC-treated single donor platelet concentrates in healthy volunteers—dose escalation study. Transfus Med Hemother 2013;40(Suppl. 1):50. [40] Cid J, Escolar G, Lozano M. Therapeutic efficacy of platelet components treated with amotosalen and ultraviolet A pathogen inactivation method: results of a meta-analysis of randomized controlled trials. Vox Sang 2012;103:322–30. [41] Vamvakas EC. Meta-analysis of the randomized controlled trials of the hemostatic efficacy and capacity of pathogen-reduced platelets. Transfusion 2011;51:1058–71. [42] Vamvakas EC. Meta-analysis of the studies of bleeding complications of platelets pathogen-reduced with the Intercept system. Vox Sang 2012;102:302–16. [43] McCullough J. Pathogen inactivation of platelets. In: AuBuchon J, Prowse C, editors. Pathogen inactivation: the penultimate paradigm shift. Bethesda, MD: AABB Press; 2010. p. 99–123. [44] Snyder E, Raife T, Lin L, Cimino G, Metzel P, Rheinschmidt M, et al. Recovery and life span of (111)indium-radiolabelled platelets treated with pathogen inactivation with amotosalen HCl (S-59) and ultraviolet A light. Transfusion 2004;44:1732–40. [45] McCullough J, Vesole DH, Benjamin RJ, Slichter SJ, Pineda A, Snyder E, et al. Therapeutic efficacy and safety of platelets treated with a photochemical process for pathogen inactivation: the SPRINT Trial. Blood 2004;104:1534–41. [46] Slichter SJ, Raife TJ, Davis K, Rheinschmidt M, Buchholz DH, Corash L, et al. Platelets photochemically treated with amotosalen HCl and ultraviolet A light correct prolonged bleeding times in patients with thrombocytopenia. Transfusion 2006;46:731–40. [47] Janetzko K, Cazenave JP, Kluter H, Kientz D, Michel M, Beris P, et al. Therapeutic efficacy and safety of photochemically treated apheresis platelets processed with an optimized integrated set. Transfusion 2005;45:1443–52. [48] Lozano M, Knutson F, Tardivel R, Cid J, Maymo RM, Lof H, et al. A multi-centre study of therapeutic efficacy and safety of platelet components treated with amotosalen and ultraviolet A pathogen inactivation stored for 6 or 7 d prior to transfusion. Br J Haematol 2011;153:393–401. [49] van Rhenen D, Gulliksson H, Cazenave JP, Pamphilon D, Ljungman P, Kluter H, et al. Transfusion of pooled buffy coat platelet components prepared with photochemical pathogen inactivation treatment: the euroSPRITE trial. Blood 2003;101:2426–33. [50] Kerkhoffs JLH, van Putten WLJ, Novotny VMJ, Boekhorst P, Schipperus MR, Zwaginga JJ, et al. Clinical effectiveness of leucoreduced, pooled donor platelet concentrates, stored in plasma or additive solution with and without pathogen reduction. Br J Haematol 2010;150:209–17. [51] Cazenave JP, Follea G, Bardiaux L, Boiron JM, Lafeuillade B, Debost M, et al. A randomized controlled clinical trial evaluating the performance and safety of platelets treated with MIRASOL pathogen reduction technology. Transfusion 2010;50:2362–75. [52] Aebersold R, Mann M. Mass spectrometry–based proteomics. Nature 2003;422:198–207.

[53] Walsh GM, Rogalski JC, Klockenbusch C, Kast J. Mass spectrometry–based proteomics in biomedical research: emerging technologies and future strategies. Expert Rev Mol Med 2010;12:1–28 e30. [54] Walther TC, Mann M. Mass spectrometry–based proteomics in cell biology. J Cell Biol 2010;190:491–500. [55] Senis Y, Garcia A. Platelet proteomics: state of the art and future perspective. Methods Mol Biol 2012;788:367–99. [56] Wright B, Stanley RG, Kaiser WJ, Gibbins JM. The integration of proteomics and systems approaches to map regulatory mechanisms underpinning platelet function. Proteomics Clin Appl 2013;7:144–54. [57] Di Michele M, Van Geet C, Freson K. Recent advances in platelet proteomics. Expert Rev Proteomics 2012;9:451–66. [58] Holly SP, Parise LV. Big science for small cells: systems approaches for platelets. Curr Drug Targets 2011;12:1859–70. [59] Zufferey A, Fontana P, Reny JL, Nolli S, Sanchez JC. Platelet proteomics. Mass Spectrom Rev 2012;31:331–51. [60] Zufferey A, Ibberson M, Reny J-L, Xenarios I, Sanchez J-C, Fontana P. Unraveling modulators of platelet reactivity in cardiovascular patients using omics strategies: towards a network biology paradigm. Transl Proteomics 2013;1:25–37. [61] Corthals GL, Wasinger VC, Hochstrasser DF, Sanchez JC. The dynamic range of protein expression: a challenge for proteomic research. Electrophoresis 2000;21:1104–15. [62] Hsieh SY, Chen RK, Pan YH, Lee HL. Systematical evaluation of the effects of sample collection procedures on low-molecular-weight serum/plasma proteome profiling. Proteomics 2006;6:3189–98. [63] Delobel J, Rubin O, Prudent M, Crettaz D, Tissot JD, Lion N. Biomarker analysis of stored blood products: emphasis on pre-analytical issues. Int J Mol Sci 2010;11:4601–17. [64] Boschetti E, Righetti PG. The ProteoMiner in the proteomic arena: a non-depleting tool for discovering low-abundance species. J Proteomics 2008;71:255–64. [65] Egidi MG, Rinalducci S, Marrocco C, Vaglio S, Zolla L. Proteomic analysis of plasma derived from platelet buffy coats during storage at room temperature. An application of ProteoMiner (TM) technology. Platelets 2011;22:252–69. [66] Taylor CF, Paton NW, Lilley KS, Binz PA, Julian RK, Jones AR, et al. The minimum information about a proteomics experiment (MIAPE). Nat Biotechnol 2007;25:887–93. [67] Zhang YY, Fonslow BR, Shan B, Baek MC, Yates JR. Protein analysis by shotgun/ bottom-up proteomics. Chem Rev 2013;113:2343–94. [68] Maurer-Spurej E, Chipperfield K. Past and future approaches to assess the quality of platelets for transfusion. Transfus Med Rev 2007;21:295–306. [69] Thiele T, Steil L, Gebhard S, Scharf C, Hammer E, Brigulla M, et al. Profiling of alterations in platelet proteins during storage of platelet concentrates. Transfusion 2007;47:1221–33. [70] Thon JN, Schubert P, Duguay M, Serrano K, Lin S, Kast J, et al. Comprehensive proteomic analysis of protein changes during platelet storage requires complementary proteomic approaches. Transfusion 2008;48:425–35. [71] Thiele T, Iuga C, Janetzky S, Schwertz H, Gesell Salazar M, Furll B, et al. Early storage lesions in apheresis platelets are induced by the activation of the integrin alphaIIbbeta (3) and focal adhesion signaling pathways. J Proteomics 2012;76:297–315. [72] Thiele T, Sablewski A, Iuga C, Bakchoul T, Bente A, Gorg S, et al. Profiling alterations in platelets induced by Amotosalen/UVA pathogen reduction and gamma irradiation—a LC-ESI-MS/MS-based proteomics approach. Blood Transfus 2012;10(Suppl. 2):s63–70. [73] Prudent M, Crettaz D, Delobel J, Tissot JD, Lion N. Proteomic analysis of Intercepttreated platelets. J Proteomics 2012;76:316–28. [74] Marrocco C, D'Alessandro A, Girelli G, Zolla L. Proteomic analysis of platelets treated with gamma irradiation versus a commercial photochemical pathogen reduction technology. Transfusion 2013;53:1808–20. [75] Schubert P, Karwal S, Devine DV. Dynamics of signal transduction triggered in platelet by riboflavin/UV treatment: relocalization of GTPases enhances platelet function. Transfusion 2012;52(Suppl S3):19A. [76] Schubert P, Culibrk B, Coupland D, Scammell K, Issa-Gyongyossy MI, Devine DV. Riboflavin and ultraviolet light treatment potentiates vasodilator-stimulated phosphoprotein Ser-239 phosphorylation in platelet concentrates during storage. Transfusion 2012;52:397–408. [77] Glenister KM, Payne KA, Sparrow RL. Proteomic analysis of supernatant from pooled buffy-coat platelet concentrates throughout 7-day storage. Transfusion 2008;48:99–107. [78] Schubert P, Culibrk B, Karwal S, Slichter SJ, Devine DV. Optimization of platelet concentrate quality: application of proteomic technologies to donor management. J Proteomics 2012;76:329–36. [79] Schubert P, Culibrk B, Goodrich RP, Devine DV. Changes in the protein profiling triggered by riboflavin/UV treatment using quantitative proteomics: increase in cytoskeletal protein expression. Transfusion 2012;52(Suppl S3):19A–20A. [80] Li JZ, Lockerbie O, de Korte D, Rice J, McLean R, Goodrich RP. Evaluation of platelet mitochondria integrity after treatment with Mirasol pathogen reduction technology. Transfusion 2005;45:920–6. [81] Picker SM, Steisel A, Gathof BS. Cell integrity and mitochondrial function after Mirasol-PRT treatment for pathogen reduction of apheresis-derived platelets: results of a three-arm in vitro study. Transfus Apher Sci 2009;40:79–85. [82] Ruane PH, Edrich R, Gampp D, Keil SD, Leonard RL, Goodrich RP. Photochemical inactivation of selected viruses and bacteria in platelet concentrates using riboflavin and light. Transfusion 2004;44:877–85. [83] Liumbruno G, D'Alessandro A, Grazzini G, Zolla L. Blood-related proteomics. J Proteomics 2010;73:483–507. [84] Reid S, Johnson L, Woodland N, Marks DC. Pathogen reduction treatment of buffy coat platelet concentrates in additive solution induces proapoptotic signaling. Transfusion 2012;52:2094–103.

M. Prudent et al. / Transfusion Medicine Reviews 28 (2014) 72–83 [85] Schubert P, Coupland D, Culibrk B, Goodrich RP, Devine DV. Riboflavin and ultraviolet light treatment of platelets triggers p38MAPK signaling: inhibition significantly improves in vitro platelet quality after pathogen reduction treatment. Transfusion 2013;53:3164–73. [86] Wu WW, Wang GH, Baek SJ, Shen RF. Comparative study of three proteomic quantitative methods, DIGE, cICAT, and iTRAQ, using 2D gel- or LC-MALDI TOF/ TOF. J Proteome Res 2006;5:651–8. [87] Swiss-2DPAGE. http://world-2dpage.expasy.org/swiss-2dpage/viewer. [88] Duan XB, Kelsen SG, Merali S. Proteomic analysis of oxidative stress-responsive proteins in human pneumocytes: insight into the regulation of DJ-1 expression. J Proteome Res 2008;7:4955–61. [89] Gu L, Cui T, Fan CX, Zhao HY, Zhao CL, Lu LL, et al. Involvement of ERK1/2 signaling pathway in DJ-1-induced neuroprotection against oxidative stress. Biochem Biophys Res Commun 2009;383:469–74. [90] Kamp F, Exner N, Lutz AK, Wender N, Hegermann J, Brunner B, et al. Inhibition of mitochondrial fusion by alpha-synuclein is rescued by PINK1, Parkin and DJ-1. Embo J 2010;29:3571–89. [91] Xiong H, Wang DL, Chen LN, Choo YS, Ma H, Tang CY, et al. Parkin, PINK1, and DJ1 form a ubiquitin E3 ligase complex promoting unfolded protein degradation. J Clin Invest 2009;119:650–60. [92] Zhou WB, Bercury K, Cummiskey J, Luong N, Lebin J, Freed CR. Phenylbutyrate upregulates the DJ-1 protein and protects neurons in cell culture and in animal models of parkinson disease. J Biol Chem 2011;286:14941–51. [93] Xu XM, Lin H, Maple J, Bjorkblom B, Alves G, Larsen JP, et al. The Arabidopsis DJ1a protein confers stress protection through cytosolic SOD activation. J Cell Sci 2010;123:1644–51. [94] Herrero E, de la Torre-Ruiz MA. Monothiol glutaredoxins: a common domain for multiple functions. Cell Mol Life Sci 2007;64:1518–30. [95] Johansson C, Roos AK, Montano SJ, Sengupta R, Filippakopoulos P, Guo K, et al. The crystal structure of human GLRX5: iron-sulfur cluster co-ordination, tetrameric assembly and monomer activity. Biochem J 2011;433:303–11. [96] Burch PT, Burch JW. Alterations in glutathione during storage of human-platelet concentrates. Transfusion 1987;27:342–6. [97] Jin JG, Kunapuli SP. Coactivation of two different G protein–coupled receptors is essential for ADP-induced platelet aggregation. Proc Natl Acad Sci U S A 1998;95: 8070–4. [98] Larson MK, Chen H, Kahn ML, Taylor AM, Fabre JE, Mortensen RM, et al. Identification of P2Y(12)-dependent and -independent mechanisms of glycoprotein VI-mediated Rap1 activation in platelets. Blood 2003;101:1409–15. [99] Offermanns S. Activation of platelet function through G protein–coupled receptors. Circ Res 2006;99:1293–304. [100] Woulfe D, Yang J, Brass L. ADP and platelets: the end of the beginning. J Clin Invest 2001;107:1503–5. [101] Broos K, De Meyer SF, Feys HB, Vanhoorelbeke K, Deckmyn H. Blood platelet biochemistry. Thromb Res 2012;129:245–9. [102] Ohlmann P, Laugwitz KL, Nurnberg B, Spicher K, Schultz G, Cazenave JP, et al. The human platelet ADP receptor activates G(i2) proteins. Biochem J 1995;312:775–9. [103] Malik M, Shukla A, Amin P, Niedelman W, Lee J, Jividen K, et al. S-Nitrosylation regulates nuclear translocation of chloride intracellular channel protein CLIC4. J Biol Chem 2010;285:23818–28. [104] Palmfeldt J, Vang S, Stenbroen V, Pavlou E, Baycheva M, Buchal G, et al. Proteomics reveals that redox regulation is disrupted in patients with ethylmalonic encephalopathy. J Proteome Res 2011;10:2389–96. [105] Fernandez-Salas E, Sagar M, Cheng C, Yuspa SH, Weinberg WC. p53 and tumor necrosis factor alpha regulate the expression of a mitochondrial chloride channel protein. J Biol Chem 1999;274:36488–97. [106] Singh H, Cousin MA, Ashley RH. Functional reconstitution of mammalian 'chloride intracellular channels' CLIC1, CLIC4 and CLIC5 reveals differential regulation by cytoskeletal actin. Febs J 2007;274:6306–16. [107] Hammer E, Goritzka M, Ameling S, Darm K, Steil L, Klingel K, et al. Characterization of the human myocardial proteome in inflammatory dilated cardiomyopathy by label-free quantitative shotgun proteomics of heart biopsies. J Proteome Res 2011;10:2161–71. [108] Kauskot A, Di Michele M, Loyen S, Freson K, Verhamme P, Hoylaerts MF. A novel mechanism of sustained platelet alphaIIbbeta3 activation via PEAR1. Blood 2012;119:4056–65. [109] Stone MJ, Chuang S, Hou X, Shoham M, Zhu JZ. Tyrosine sulfation: an increasingly recognised post-translational modification of secreted proteins. N Biotechnol 2009;25:299–317. [110] McFarland J, Menitove J, Kagen L, Braine H, Kickler T, Ness P, et al. Leukocyte reduction and ultraviolet B irradiation of platelets to prevent alloimmunization and refractoriness to platelet transfusions. N Engl J Med 1997;337:1861–9.

83

[111] Qiagen. http://www.qiagen.com/Products/Genes%20and%20Pathways/Pathway %20Central/. [Accessed: January - March 2013]. [112] Servier. http://www.servier.fr/servier-medical-art. [113] Schmidt VA, Scudder L, Devoe CE, Bernards A, Cupit LD, Bahou WF. IQGAP2 functions as a GTP dependent effector protein in thrombin-induced platelet cytoskeletal reorganization. Blood 2003;101:3021–8. [114] Holbrook LM, Watkins NA, Simmonds AD, Jones CI, Ouwehand WH, Gibbins JM. Platelets release novel thiol isomerase enzymes which are recruited to the cell surface following activation. Br J Haematol 2010;148:627–37. [115] Shattil SJ, Kim C, Ginsberg MH. The final steps of integrin activation: the end game. Nat Rev Mol Cell Biol 2010;11:288–300. [116] Canobbio I, Reineri S, Sinigaglia F, Balduini C, Torti M. A role for p38 MAP kinase in platelet activation by von Willebrand factor. Thromb Haemost 2004;91:102–10. [117] Kramer RM, Roberts EF, Strifler BA, Johnstone EM. Thrombin induces activation of p38 MAP kinase in human platelets. J Biol Chem 1995;270:27395–8. [118] Kramer RM, Roberts EF, Um SL, BorschHaubold AG, Watson SP, Fisher MJ, et al. p38 Mitogen-activated protein kinase phosphorylates cytosolic phospholipase A(2) (cPLA(2)) in thrombin-stimulated platelets - Evidence that proline-directed phosphorylation is not required for mobilization of arachidonic acid by cPLA(2). J Biol Chem 1996;271:27723–9. [119] Yacoub D, Theoret JF, Villeneuve L, Abou-Saleh H, Mourad W, Allen BG, et al. Essential role of protein kinase C delta in platelet signaling alpha(IIb)beta(3) activation, and thromboxane A(2) release. J Biol Chem 2006;281:30024–35. [120] Flevaris P, Li ZY, Zhang GY, Zheng Y, Liu JL, Du XP. Two distinct roles of mitogenactivated protein kinases in platelets and a novel Rac1-MAPK-dependent integrin outside-in retractile signaling pathway. Blood 2009;113:893–901. [121] Benz PM, Blume C, Seifert S, Wilhelm S, Waschke J, Schuh K, et al. Differential VASP phosphorylation controls remodeling of the actin cytoskeleton. J Cell Sci 2009;122:3954–65. [122] Benz PM, Blume C, Moebius J, Oschatz C, Schuh K, Sickmann A, et al. Cytoskeleton assembly at endothelial cell-cell contacts is regulated by alpha II-spectrin-VASP complexes. J Cell Biol 2008;180:205–19. [123] Wentworth JKI, Pula G, Poole AW. Vasodilator-stimulated phosphoprotein (VASP) is phosphorylated on Ser(157) by protein kinase C-dependent and -independent mechanisms in thrombin-stimulated human platelets. Biochem J 2006;393:555–64. [124] Polanowska-Grabowska R, Gear ARL. Heat-shock proteins and platelet function. Platelets 2000;11:6–22. [125] Aslan JE, McCarty OJT. Rho GTPases in platelet function. J Thromb Haemost 2013;11:35–46. [126] Franke B, Akkerman JWN, Bos JL. Rapid Ca2 +-mediated activation of Rap1 in human platelets. Embo J 1997;16:252–9. [127] Schubert P, Thon JN, Walsh GM, Chen CH, Moore ED, Devine DV, et al. A signaling pathway contributing to platelet storage lesion development: targeting PI3kinase-dependent Rap1 activation slows storage-induced platelet deterioration. Transfusion 2009;49:1944–55. [128] Baig A, Bao XK, Wolf M, Haslam RJ. The platelet protein kinase C substrate pleckstrin binds directly to SDPR protein. Platelets 2009;20:446–57. [129] Lian LR, Wang YF, Flick M, Choi J, Scott EW, Degen J, et al. Loss of pleckstrin defines a novel pathway for PKC-mediated exocytosis. Blood 2009;113:3577–84. [130] Sunahara RK, Tesmer JJG, Gilman AG, Sprang SR. Crystal structure of the adenylyl cyclase activator G(S alpha). Science 1997;278:1943–7. [131] Guidetti GF, Lova P, Bernardi B, Campus F, Baldanzi G, Graziani A, et al. The G(i)coupled P2Y12 receptor regulates diacylglycerol-mediated signaling in human platelets. J Biol Chem 2008;283:28795–805. [132] Wu CY, Dedhar S. Integrin-linked kinase (ILK) and its interactors: a new paradigm for the coupling of extracellular matrix to actin cytoskeleton and signaling complexes. J Cell Biol 2001;155:505–10. [133] Delcommenne M, Tan C, Gray V, Rue L, Woodgett J, Dedhar S. Phosphoinositide3-OH kinase-dependent regulation of glycogen synthase kinase 3 and protein kinase B/AKT by the integrin-linked kinase. Proc Natl Acad Sci U S A 1998;95: 11211–6. [134] Pasquet JM, Noury M, Nurden AT. Evidence that the platelet integrin alpha IIb beta 3 is regulated by the integrin-linked kinase, ILK, in a PI3-kinase dependent pathway. Thromb Haemost 2002;88:115–22. [135] Tu YZ, Li FG, Wu CY. Nck-2, a novel Src homology2/3-containing adaptor protein that interacts with the LIM-only protein PINCH and components of growth factor receptor kinase-signaling pathways. Mol Biol Cell 1998;9:3367–82. [136] Picker SM, Oustianskaia L, Schneider V, Gathof BS. Functional characteristics of apheresis-derived platelets treated with ultraviolet light combined with either amotosalen-HCl (S-59) or riboflavin (vitamin B-2) for pathogen-reduction. Vox Sang 2009;97:26–33.

International interlaboratory study comparing single organism 16S rRNA gene sequencing data: Beyond consensus sequence comparisons.

Technologies for Proteome-Wide Discovery of Extracellular Host-Pathogen Interactions.

Differential proteome and secretome analysis during rice-pathogen interaction.

Global Proteome Changes in Liver Tissue 6 Weeks after FOLFOX Treatment of Colorectal Cancer Liver Metastases.

Whither platelets after TAVR?

The changes of Proteome in MG-63 cells after induced by calcitonin gene-related peptide.

Thrombin induced surface changes of human platelets.

Drought-Induced Leaf Proteome Changes in Switchgrass Seedlings.

Bicarbonate Induced Redox Proteome Changes in Arabidopsis Suspension Cells.

Schistosome infections induce significant changes in the host biliary proteome.

Proteome changes induced by laser in diabetic retinopathy.

Plant pathogen nanodiagnostic techniques: forthcoming changes?

Metabolome and proteome changes with aging in Caenorhabditis elegans.

Brain Proteome Changes Induced by Olfactory Learning in Drosophila.

The in planta proteome of wild type strains of the fire blight pathogen, Erwinia amylovora.

Proteome-wide analysis of lysine acetylation in the plant pathogen Botrytis cinerea.

A global Staphylococcus aureus proteome resource applied to the in vivo characterization of host-pathogen interactions.

The potential role of platelets in the consensus molecular subtypes of colorectal cancer.

Sirtuin Inhibition Induces Apoptosis-like Changes in Platelets and Thrombocytopenia.

SUBAcon: a consensus algorithm for unifying the subcellular localization data of the Arabidopsis proteome.

Proteome Analysis of Pathogen-Responsive Proteins from Apple Leaves Induced by the Alternaria Blotch Alternaria alternata.

Proteome-wide lysine acetylation profiling of the human pathogen Mycobacterium tuberculosis.

Profiling the secretome and extracellular proteome of the potato late blight pathogen Phytophthora infestans.

Proteome data from a host-pathogen interaction study with Staphylococcus aureus and human lung epithelial cells.