BLOOD COMPONENTS High-density lipoprotein 3 and apolipoprotein A-I alleviate platelet storage lesion and release of platelet extracellular vesicles Annika Pienimaeki-Roemer,1 Astrid Fischer,1 Maria Tafelmeier,1 Evelyn Orsó,1 Tatiana Konovalova,1 Alfred Böttcher,1 Gerhard Liebisch,1 Armin Reidel,2 and Gerd Schmitz1 BACKGROUND: Stored platelet (PLT) concentrates (PLCs) for transfusion develop a PLT storage lesion (PSL), decreasing PLT viability and function with profound lipidomic changes and PLT extracellular vesicle (PL-EV) release. High-density lipoprotein 3 (HDL3) improves PLT homeostasis through silencing effects on PLT activation in vivo. This prompted us to investigate HDL3 and apolipoprotein A-I (apoA-I) as PSLantagonizing agents. STUDY DESIGN AND METHODS: Healthy donor PLCs were split into low-volume standard PLC storage bags and incubated with native (n)HDL3 or apoA-I from plasma ethanol fractionation (precipitate IV) for 5 days under standard blood banking conditions. Flow cytometry, Born aggregometry, and lipid mass spectrometry were carried out to analyze PL-EV release, PLT aggregation, agonist-induced PLT surface marker expression, and PLT and plasma lipid compositions. RESULTS: Compared to control, added nHDL3 and apoA-I significantly reduced PL-EV release by up to −62% during 5 days, correlating with the added apoA-I concentration. At the lipid level, nHDL3 and apoA-I antagonized PLT lipid loss (+12%) and decreased cholesteryl ester (CE)/free cholesterol (FC) ratios (−69%), whereas in plasma polyunsaturated/saturated CE ratios increased (+3%) and CE 16:0/20:4 ratios decreased (−5%). Administration of nHDL3 increased PLT bis(monoacylglycero)phosphate/ phosphatidylglycerol (+102%) and phosphatidic acid/ lysophosphatidic acid (+255%) ratios and improved thrombin receptor–activating peptide 6–induced PLT aggregation (+5%). CONCLUSION: nHDL3 and apoA-I improve PLT membrane homeostasis and intracellular lipid processing and increase CE efflux, antagonizing PSL-related reduction in PLT viability and function and PL-EV release. We suggest uptake and catabolism of nHDL3 into the PLT open canalicular system. As supplement in PLCs, nHDL3 or apoA-I from Fraction IV of plasma ethanol fractionation have the potential to improve PLC quality to prolong storage.

P

latelet (PLT) concentrates (PLCs), obtained by plateletpheresis aimed for transfusion, undergo a PLT storage lesion (PSL) upon agitation at room temperature in gas-permeable PLT bags

ABBREVIATIONS: ABC = ATP-binding cassette transporter; apoA-I = apolipoprotein A-I; BMP = bis(monoacylglycero)phosphate; CD62P = P-selectin; CE = cholesteryl ester; Cer = ceramide; CL = cardiolipin; FC = free cholesterol; HDL3 = high-density lipoprotein 3; LCAT = lecithin-cholesterol acyltransferase; LPA = lysophosphatidic acid; LPC = lysophosphatidylcholine; MPV = mean platelet volume; MVB = multivesicular body; nHDL3 = native HDL3; PA = phosphatidic acid; PC = phosphatidylcholine; PDW = platelet distribution width; PE P = phosphatidylethanolamine plasmalogen; PG = phosphatidylglycerol; PL = phospholipid; PLC(s) = platelet concentrate(s); P-LCR = platelet large-cell ratio; PL-EV(s) = platelet extracellular vesicle(s); PRP = platelet-rich plasma; PSL = platelet storage lesion; PUFA = polyunsaturated fatty acid; S1P = sphingosine-1-phosphate; SM = sphingomyelin; SPH = sphingosine; SR = scavenger receptor; TRAP-6 = thrombin receptor–activating peptide 6. From the 1Institute for Clinical Chemistry and Laboratory Medicine, Regensburg, Germany; and 2Fresenius Kabi Deutschland GmbH, Bad Homburg, Germany. Address reprint requests to: Gerd Schmitz, Institute for Laboratory Medicine and Transfusion Medicine, University of Regensburg, Franz-Josef-Strauss-Allee 11, D-93053 Regensburg, Germany; e-mail: [email protected]. The study received funding from the European Community’s Seventh Framework Programme (FP7/2007-2013) under grant agreement No: 202272, IP-Project LipidomicNet, and partly from the Federal Ministry of Education and Research under the Project Number FKZ01KU1216J. “The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.” Received for publication November 2, 2013; revision received January 27, 2014, and accepted January 29, 2014. doi: 10.1111/trf.12640 © 2014 AABB TRANSFUSION 2014;54:2301-2314. Volume 54, September 2014 TRANSFUSION

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during the 5-day PLC “shelf life.” The PSL includes changes in metabolism and reduced response to activating stimuli, with decreased PLT extracellular surface antigen expression (CD41, CD61, CD42b) and increased PLT activation markers (CD62P, CD63).1,2 Loss of surface membrane integrity and shape change result in fragmentation and PLT extracellular vesicle (PL-EV) release.3 Reported PSL cellular mechanisms include senescence, shear stress, apoptosis, loss of mitochondrial potential,4 cytoplasmic condensation, and fusion of granules with the plasma membrane.5 Beyond their role in reverse cholesterol transport and cell membrane homeostasis, high-density lipoprotein (HDL) particles exert additional protective effects, including antioxidant function, endothelial cell protection, and anti-inflammatory and antithrombotic effects.6,7 Starting from discoidal apolipoprotein A-I (apoA-I) and phospholipid (PL) complexes, the formation of which involves the major plasma HDL regulator ATP-binding cassette transporter A1 (ABCA1), small spherical pre-β HDL particles are formed, which mature by sequential uptake of cholesterol from peripheral tissues through concerted action of ABCA1, ABCG1, and scavenger receptor (SR)-B1.6 In contrast to ABCA1 and ABCG1, which promote unidirectional cholesterol efflux, SR-B1 binds HDL3 with high affinity,8 mediating bidirectional cholesterol flux.9 In plasma, apoA-I is a cofactor in the esterification process of HDL3-cholesterol by lecithin-cholesterol acyltransferase (LCAT), of which the major product in plasma is cholesteryl ester (CE) 18:2.10 The hydrophobic CEs are deposited in the core of HDL3, giving space on the HDL surface for more polar lipids such as free cholesterol (FC) and PL to increase HDL3 size, with parallel decreased affinity of LCAT to the larger sized HDL2 particles with a higher surface tension.11 As a blood cell with a low phosphatidylcholine (PC)/FC ratio,12 membrane homeostasis of mature and newly released reticulated PLTs critically depends on plasma lipoproteins, which transfer and/or exchange cholesterol, PLs, sphingolipids, and fatty acids from circulating lipoproteins of the surrounding plasma to the PLT cell membrane13 or exchange these lipids with plasma lipoproteins.14 There are many possible mechanisms behind the “anti-PLT” effect of HDL particles observed in numerous in vitro PLT studies and after infusion of reconstituted HDL,15,16 prepared from Fraction IV (FIV) of fractionated plasma ethanol precipitation17 and varying compositions of soybean PLs, showing that low plasma HDL levels inversely correlate with PLT reactivity and vascular disease progression.7 Approximately 3200 HDL3-binding sites have been counted on PLTs, in contrast to approximately 1500 for low-density lipoprotein,18 and there is increasing evidence for PLTs interacting with HDL particles, resulting in a desensitizing and antiatherogenic effect, through lipid 2302

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exchange.14 Earlier data showed protein expression of ABCA1 in PLTs;19,20 however, molecular evidence for interaction of HDL particles with ABCA1 in PLTs is missing. Flow cytometry showed that apoA-I directly binds PLTs21 and PLTs express several ABC-transporters,22 affecting PLT transport processes to granules, signal lipid release, and lipid homeostasis.23 Human PLT HDL3 effects can also be mediated by PLT αIIbβ3-integrin (CD41/CD61).24 PLTs express SR-B1,25 which upon HDL3 binding modulates PLT reactivity.25,26 The apoA-I–SR-B1– endothelial nitric oxide synthase axis is antithrombotic in mice:27 The SR-B1 cascade can also be initiated by binding of HDL-bound sphingosine-1-phosphate (S1P) to S1P receptor 2/3.28 PLTs express S1P receptor 2, which is regulated in Type 2 diabetes29 inhibiting PLT aggregation.30 Parallel to selective lipid uptake, a cholesterol “diffusion model” along the cholesterol concentration gradient31 and endocytosis and retroendocytosis of holo-HDL particles via SR-B1, SR-BII, CD36, clathrin-coated pits, and caveolae exist.32 Lipid-poor apoA-I binds the cell surface ATP synthase (ecto-F1-ATPase) β-chain to generate adenosine diphosphate (ADP) that binds the purinergic receptor P2Y13, inducing HDL endocytosis and cholesterol efflux.32 PLTs express at least P2Y1 and P2Y13 G-protein receptors, which regulate the PLT aggregation response.33 Recently we reported profound lipidomic changes during PLC storage, with loss of total lipid and increased lysophosphatidylcholine (LPC)/PC and CE/FC ratios, especially plasmatic LCAT-derived CE 18:2 (linoleate).34 This reflects LCAT substrate consumption and suggests uptake and trapping of plasma lipids and lipoproteins into the PLT open canalicular system,34 large enough in diameter (70-160 nm) to incorporate plasma lipoprotein particles and which increase during storage to 25% of PLT total volume.35 Increased ceramide (Cer)34 suggests reversion of sphingolipid metabolism from S1P toward Cer,36 but not a “salvage pathway,” generating Cer from cell membrane sphingomyelin (SM).37 The above lipidomic changes reduce plasma membrane integrity, increasing PL-EVs release into the surrounding plasma, possibly explaining loss of PLT lipids during storage.36 Other authors also showed increased PL-EVs in stored PLCs.3,38 Due to their procoagulant characteristics39 and high Cer36 and LPC content,36,40 increased PL-EVs may show unwanted effects in patients when transfused. Increased PL-EV levels in vivo have been reported in Type 2 diabetes,41 stroke,42 acute coronary syndromes,43 and vascular disease,44 rendering activated and senescent PLTs and PL-EVs pivotal players in inflammation and atherosclerotic remodeling.45 To improve the conditions for prolonged PLT viability and PLC shelf life during in vitro PLC storage, there are ongoing developments to improve PLC storage bag materials46,47 and PLT additive solution formulations,48 to

HDL3/ApoA-I IN PLT STORAGE LESION

reduce storage-associated mechanical stress, optimize aerobic metabolism, and decrease PLT activation. Based on this, we here extended the well-known beneficial HDL3 effect on PLTs toward in vitro analysis of stored PLCs, showing antagonizing effects on the PSL by stabilization of PLT lipid changes and lipid homeostasis, reducing PL-EV release and improving PLT function. Presented data may be a starting point for the development of a new generation of PLC additives based on human apoA-I from plasma ethanol fractionation17 or as reconstituted HDL49 to improve PLT viability and function to prolong PLC storage time.

grated chemistry system (Dimension Vista 1500 Intelligent Lab System; Siemens Healthcare Diagnostics Inc., Tarrytown, NY). On the day of apheresis, freshly isolated nHDL3 was added to PLT-rich plasma (PRP) to double (2:1) or equal (1:1) the HDL3 cholesterol concentration in PLC plasma. Alternatively, identical concentrations of nHDL3 and apoA-I were added to double the PLC plasma apoA-I concentration. Added PBS in similar volumes served as control. Total incubation volumes were 60 mL for each mini-PLT bag. The incubations were stored 5 days under standard blood banking conditions. On Days 0, 3, and 5, samples for analysis were withdrawn with sterile docking systems.

MATERIALS AND METHODS PLT donors, plateletpheresis, and sampling of volume-reduced PLCs PLCs were collected on an automated blood collection system (Trima Accel, TerumoBCT, Lakewood, CO) from healthy normolipidemic volunteers as described.36 The PLCs were rested undisturbed for 1 hour at ambient temperature to allow dissociation of any PLT microaggregates. Analyzed PLCs fulfilled all criteria for transfusion acceptance. Using a sterile tube sealing device, PLCs were equally split into previously described and validated airpermeable plateletpheresis bag systems (Compoflex 4V-transferbag F 730, 4 × 150 mL, Fresenius-Kabi, Bad Homburg, Germany).36

Preparation of HDL3 and apoA-I from human plasma Plasma from normolipidemic fasting donors of the apoE3/E3 genotype was recalcified, and the plasmatic coagulation system was induced to obtain serum. Lipoproteins were sequentially removed by ultracentrifugation by adjusting to their respective density (HDL3, 1210 g/ cm3) with respective amount of KBr (density, 2.74 g/cm3). Native HDL3 (nHDL3) was dialyzed against Ca2+- and Mg2+free phosphate-buffered saline (PBS) with and without added ethylenediaminetetraacetic acid (1.12 g/L), sterile filtered, and immediately added to split PLC units. Virus-inactivated human apoA-I was isolated by cold ethanol precipitation from precipitate IV, obtained from plasma fractionation according to Kistler and Nitschmann.49-52 The detailed isolation procedure is described by Lerch and colleagues.49 Approximately 1 kg of apoA-I from 700 kg of filtered precipitate IV was yielded (30% recovery).

Analysis of PLT variables On Days 0 and 5, aliquots of PLC samples were determined for PLT count (×109/L), mean PLT volume (MPV; fL), PLT distribution width (PDW; fL), and PLT large cell ratio (P-LCR; %) on a standard hematology analyzer (XE5000, Sysmex, Norderstedt, Germany).

PL-EV analysis by flow cytometry The ratios of PL-EVs (of greater than 500 nm) to PLTs (in %) were analyzed by flow cytometry (Navios, Beckman Coulter, Krefeld, Germany) as described.36 The micro-flow cytometer (Model A-50, Apogee Flow Systems, Hertfordshire, UK) scatter size was calibrated by a silica bead mix (110- to 1300-nm beads; refractive index 1.42), containing two fluorescently labeled (excitation at 488 nm) bead populations (110 and 500 nm; refractive index 1.59; Apogee Flow Systems). Staining of 100 μL of 1:100 diluted PRP was carried out as above.36 After 500 μL of PBS was added, CD61-positive events/μL were analyzed by gating for PLTs (2-3 μm) and 180 to 300 nm (size = S) and 300 to 590 nm (size = M) PL-EVs.

PLT aggregation PLT aggregation after 5 days was analyzed by Born aggregometry (APACT 4 S Plus, Rolf Greiner BioChemica GmbH, Flacht, Germany). PRP was centrifuged at 1500 × g for 15 minutes at room temperature to generate PLT-poor plasma for use as blank. PRP, diluted 1:1 with PLT-poor plasma, was stimulated by agonists at 37°C, at the following final concentrations: ristocetin (1.5 mg/mL), arachidonic acid (0.5 mg/mL), thrombin receptor–activating peptide-6 (TRAP-6; 0.1 mmol/L), or ADP (20 μmol/L). Maximal aggregation for each sample was determined and compared to control.

Addition of HDL3 and apoA-I and PLT-rich plasma storage

Analysis of PLT surface marker expression upon agonist stimulation

ApoA-I (mg/dL) and HDL3 were quantitated automatically according to the manufacturer on an automated inte-

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cytometry (FACSCanto II, FACSDiva software, Becton Dickinson, Franklin Lakes, NJ). PLT activation was induced by 10 μmol/L TRAP-6 (SFLLRN, BACHEM Biochemica GmbH, Heidelberg, Germany) or by 5 μmol/L ADP (DiaMed Diagnostika Deutschland GmbH, München, Germany) for 10 minutes at room temperature. PLT activation was stopped by adding 2 mL of Ca2+- and Mg2+-deficient PBS. Activated PLTs were analyzed immediately. PLTs were identified through their CD41 (glycoprotein IIb/IIIa subunit) positivity. The following conjugated monoclonal antibodies were applied: CD62P– fluorescein isothiocyanate (FITC; Clone CLB-Thromb/6, IOT/Beckman Coulter), CD41-R–phycoerythrin (R-PE Clone P2, IOT/Beckman Coulter), PAC-1–FITC (Clone PAC-1, Becton Dickinson), and CD36–R-PE (Clone CB38, BD Biosciences-Pharmingen, Heidelberg, Germany).

Lipid mass spectrometry PLTs and plasma were isolated by gel filtration and centrifugation, respectively, and prepared as described.34 Glycerophospholipids and cholesterol were extracted according to Bligh and Dyer.53 Sphingolipids (except for Cer and SM) and lysophosphatidic acid (LPA) were extracted by butanolic extraction.54 Lipids were processed and quantified by electrospray ionization tandem mass spectrometry as described,55-57 whereas phosphatidylethanolamine plasmalogens (PE P) were analyzed according to Zemski Berry.58 Minor sphingolipids and glycerophospholipids including cardiolipin (CL) and bis(monoacylglycero)phosphate (BMP) were analyzed by hydrophilic interaction as described.59-61 Deisotoping and data analysis were performed by self-programmed computer spreadsheet macros (Excel, Microsoft Corp., Redmond, WA).56 Lipid species were annotated according to the recently published proposal for shorthand notation of lipid structures that are derived from mass spectrometry.62 Glycerophospholipid species (except PE P) were assigned based on the assumption that only fatty acids with an even number of carbon atoms are present. SM species annotation is based on the assumption that a sphingoid base with two hydroxyl groups is present.

Statistical analysis and data presentation Analyses were performed with computer software (SPSS 19.0 for Windows, IBM Corp., Armonk, NY). Data were tested for normal distribution by Shapiro-Wilk test and expressed as mean ± SD. Statistical significance was determined by paired t test, with p values less than 0.05, less than 0.01, and less than 0.001. Correlation analyses were performed by bivariate correlation analysis to calculate Pearson’s correlation coefficient (R) and significance value of correlation. 2304

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RESULTS Analysis of PLT variables Analysis of PLT count and variables reflecting PLT size heterogeneity, MPV, PDW, and P-LCR revealed that all observed changes were within accepted reference ranges for stored PLCs (Table 1). However, small but significantly decreased PLT counts and PDWs during 5 days were seen in control (PLT count, −3%, p < 0.05; PDW, −6%, p < 0.05) and upon added nHDL3 (PLT count, −4%, p < 0.01; PDW, −5%, p < 0.05) (Table 1).

Suppression of PL-EV release by apoA-I and nHDL3 Added nHDL3 corresponding 2:1 to the nHDL3 cholesterol concentration present in PLC plasma (a mean of 2.1 mg/mL added apoA-I above control) significantly reduced larger than 500 nm PL-EV/PLT ratio on Day 3 (−60%, p < 0.01) and it remained significantly lower compared to control on Days 4 (−64%, p < 0.01) and 5 (−62%, p < 0.001; Fig. 1A). Added nHDL3 corresponding to 1:1 of that present in PLC plasma (a mean of 1.3 mg/mL added apoA-I above control) reduced larger than 500 nm PL-EV/ PLT ratio by −33% (p < 0.05) on Day 3, by −34% (p < 0.05) on Day 4, and by −33% (p < 0.01) on Day 5 (Fig. 1B). Identical additions of nHDL3 or apoA-I to yield 1.0 mg more apoA-I/mL than in PLC plasma showed that only apoA-I, but not nHDL3, significantly inhibited the release of PL-EVs larger than 500 nm (−13%, p < 0.05; Fig. 1C) and smaller than 500 nm (−25%, p < 0.05; Fig. 1D). Correlation analysis of added apoA-I or nHDL3 from each experiment with the extent of suppression of released PL-EVs larger than 500 nm after 5 days revealed a highly significant negative correlation (R = 0.63, p < 0.001) for added apoA-I (mg/dL) to the extent of PL-EV release suppression (compared to respective control) on Day 5 (Fig. 2).

Effects of apoA-I and nHDL3 on agonist-induced PLT aggregation and membrane surface marker expression Whereas apoA-I did not show significant effects on agonist-induced PLT aggregation, added nHDL3 significantly increased maximal aggregation upon TRAP-6 (+4%, p < 0.05) stimulation and slightly upon arachidonic acid and ADP stimulation, compared to control (Table 2). Analysis of mean fluorescence intensities (MFIs) of spontaneous (basal) CD62P expression for 5 days showed a nonsignificant increase of CD62P externalization in all samples, with no significant differences between samples in percentage increase to basal upon agonist stimulation (Table S1, available as supporting information in the online version of this paper). Compared to control, added

ApoA-I D5 1015 ± 128 8.4 ± 0.5 8.5 ± 0.9 14.5 ± 3.4

Percent change −3 1 0 +6

apoA-I showed elevated absolute MFIs of basal (p < 0.05) and ADP-stimulated (p < 0.05) CD62P expression on Day 0, whereas added nHDL3 showed significantly lower (p < 0.05) basal CD62P expression than added apoA-I (Table S1). On Day 5 added apoA-I showed significantly increased basal (p < 0.01), ADP- (p < 0.01) and TRAP-6– stimulated (p < 0.05) CD62P expression, compared to control (Table S1).

nHDL3

D0 1050 ± 141 8.6 ± 0.5 9.2 ± 0.8 15.7 ± 3.4

D5 1020 ± 148 8.5 ± 0.5 8.7 ± 0.8 14.9 ± 3.6

Percent change −4‡ 0 −5† −5

D0 1049 ± 142 8.3 ± 0.5 8.6 ± 0.7 13.7 ± 3.2

Effect of apoA-I and nHDL3 on PLT and plasma lipid composition

PLT (×109/L) MPV (fL) PDW (fL) P-LCR (%)

* PBS addition in similar volumes served as control. † p < 0.05, compared to Day 0 within one sample. ‡ p < 0.01, compared to Day 0 within one sample.

Percent change −3† −2 −6† −7 Control

D5 1003 ± 128 8.6 ± 0.5 8.9 ± 0.9 15.5 ± 3.4 D0 1030 ± 138 8.7 ± 0.5 9.5 ± 0.9 16.6 ± 3.5 Variable

TABLE 1. PLT variables on Day 0 (D0) and Day 5 (D5) in control, upon addition of nHDL3 or apoA-I to double the apoA-I content present in PLT concentrate plasma*

HDL3/ApoA-I IN PLT STORAGE LESION

Added nHDL3 increased total plasma lipid by +41% (p < 0.01) on Day 0 and by +43% (p < 0.01) on Day 5, compared to control. This was not detected upon added apoA-I, corresponding to the fact that added nHDL3 is lipidated, whereas apoA-I is not. Added nHDL3 and apoA-I showed beneficial effects on plasma CE species distribution. After 5 days added nHDL3 showed increased polyunsaturated fatty acid (PUFA) CE/saturated CE species (+3%, p < 0.001; Fig. 3A) and decreased (−5%, p < 0.05) CE 16:0/CE 20:4 ratios (Fig. 3B), compared to control. Similar effects, which did not reach significances, were detected by addition of apoA-I. Plasma lipid ratios confirmed plasmatic LCAT activity during storage,34 which upon added nHDL3 seemed to be accelerated, showing decreased FC (−25%, p < 0.01) and increased LPC (+59%, p < 0.05; Fig. 4B), including the majority of LPC lipid species (Fig. S1, available as supporting information in the online version of this paper), as well as increased Day 5/Day 0 CE/FC (p < 0.01), CE/PC (p < 0.01), and LPC/PC (p < 0.01; Fig. 5B) ratios, compared to control. Agreeing on the fact that nHDL3, but not lipid-free apoA-I, contains LCAT63 needed for cholesteryl esterification, added apoA-I did not result in plasma changes above control for FC or LPC (Fig. S2, available as supporting information in the online version of this paper), nor did plasma CE/FC, CE/PC, FC/PC, or LPC/PC ratios change (Table S2B, available as supporting information in the online version of this paper). Added nHDL3 and apoA-I antagonized PLT total lipid loss by +12% and +26%, respectively, compared to respective controls. Added nHDL3 decreased Day 5/Day 0 CEs by −55% (p < 0.05) (Fig. 4A), CE/FC ratio by −69% (p < 0.05), and CE/PC ratio by −62% (p < 0.05) (Fig. 5A), compared to control. Similar effects were observed by apoA-I, with significantly decreased CE/FC and CE/PC ratios on Day 5, compared to control (Table S2A). However, in contrast to apoA-I (Table S4, available as supporting information in the online version of this paper), added nHDL3 elevated the absolute amount of all CE species on Day 0 (Table S3, available as supporting information in the online version of this paper), with most significant increases of the most prevalent nHDL3 species 18:2 and 18:1 (p < 0.01; Fig. 6A). Volume 54, September 2014 TRANSFUSION

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A

B

0.7

0.4

0.6 0.3

PL-EVs/PLTs

PL-EVs/PLTs

0.5 0.4 0.3 0.2

0.2

0.1

0.0 Day 0

**

Day 3

Day 4

*** Day 0

0.20

D 3000000

0.16

2500000

*

0.12

**

0.0 Day 5

PL-EVs/µL

PL-EVs/PLTs

C

**

*

*

0.1

0.08

Day 3

Day 4

Day 5

*

2000000 1500000 1000000

0.04 500000 0.00

0

Control

nHDL3

ApoA-I

Control

nHDL3

ApoA-I

Fig. 1. PLCs were stored at standard blood banking conditions for 5 days and treated either by nHDL3 at concentrations in ratios 2:1 (A) or 1:1 (B) of the HDL3-cholesterol present in donor PLC plasma or with concentrations that doubled the apoA-I concentration present in PLC plasma (C and D). The same volumes of PBS additions served as controls. CD61-positive PL-EVs larger than 500 nm (A-C) and smaller than 500 nm (D) were analyzed on Days 0, 3, 4, and 5. Mean ± SD of PL-EV/PLT ratios were calculated for control (○) and for added nHDL3 (●; A, B) or shown as bars for Day 0 (□) and Day 5 (■; C, D). In D, absolute amounts of PL-EVs/μL are shown. Significances are indicated as asterisks for each time point between control and nHDL3 or apoA-I addition.

The PLT and nHDL3 particle CE species compositions showed highly significant positive correlations, which on Day 0 were higher upon added nHDL3 (R = 0.99; p < 0.0001) than in control (R = 0.98; p < 0.0001; Fig. 6A). PLT storage induced increased PLT and plasma LPA levels.36 Added nHDL3 decreased PLT Day 5/Day 0 LPA by −47% (p < 0.05), with significantly reduced LPA 20:4 (p < 0.05; Fig. S3, available as supporting information in the online version of this paper) and LPA/PC ratio (p < 0.05) during 5 days, compared to control (Fig. 5A). In contrast, plasma Day 5/Day 0 LPA increased above control by +221% (p < 0.05) upon added nHDL3 (Fig. 4B). Added apoA-I showed opposite effects, with slightly increased PLT Day 5/Day 0 LPA and significantly decreased (p < 0.05) plasma LPA by −26% (Fig. S2), with major decrease (p < 0.05) in LPA 20:4 (−30%). 2306

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During 5 days added nHDL3 increased PLT phosphatidic acid (PA)/LPA ratio by +78% (p < 0.01; Table 3A) and antagonized significantly decreased BMP/phosphatidylglycerol (PG) and CL/PA ratios observed in control, with higher BMP/PG than in control already on Day 0 (Table 3A). Added ApoA-I did not show these PLT effects (Table S2A). Added nHDL3 reduced plasma Cer (−9%, p < 0.05), hexosylceramide (−21%, p < 0.05), S1P d18:1 (−62%, p < 0.05), S1P d18:0 (−100%, p < 0.05), and ceramide1-phosphate (−44%, p < 0.05; Fig. 4B), reflected as decreased Cer/SM (p < 0.05) on Days 0 and 5 (p < 0.01) and as decreased (p < 0.05) S1P d18:1/Cer and S1P d18:0/Cer on Day 5 (Table 3B). Added apoA-I showed similar plasma effects, except for S1P d18:1, which slightly increased compared to control (Fig. S2B),

HDL3/ApoA-I IN PLT STORAGE LESION

PL-EV/PLT reduction on Day 5

80

R = 0.63 p = 0.0009

60 40 20 0 –20 –40 –60 –80 0.0

0.5

1.0

1.5

2.0

2.5

Added apoA-I (mg/mL) Fig. 2. Pearson’s linear correlation to compare the effect of added apoA-I (mg/mL) to the effect on PL-EV release during 5 days PLC storage. The y-axis indicates the reduction (in %) of PL-EV/PLT ratio on Day 5 upon added nHDL3 and apoA-I, compared to respective control. The x-axis shows added apoA-I (mg/mL). Correlation coefficient R and significance p values are shown.

also detected as increased Day 5 S1P d18:1/Cer ratio (Table S2B).

DISCUSSION Our novel approach using combined lipidomic and PLT quality testing revealed potential mechanisms of how PLT lipid homeostasis is dependent on plasma HDL components to maintain PLT function and reduce PL-EV release, connecting the known antiatherogenic effect of HDL3 to PL-EV suppression. Interestingly, the effect on PL-EV release positively correlated with apoA-I concentration and nHDL3 was only able to suppress PL-EV release at higher than 1.3 mg/mL added nHDL3-apoA-I, whereas apoA-I was already effective at lower concentrations, also suppressing smaller than 500 nm PL-EV release, which might represent the majority of PL-EVs,64 including exosomes from intracellular multivesicular bodies (MVBs).65 The differences in action on PL-EV release between nHDL3 and apoA-I might rely on diverse mechanisms of communication with PLTs or might reflect the fact that purified apoA-I is a more constant formulation, while nHDL3 from human plasma donors shows significant donor-dependent variation in lipid composition. Added apoA-I also stabilized small, but significant, changes in PLT variables observed in control and upon nHDL3 addition. ApoA-I rapidly forms complexes with PLs and FC from cell membranes or from intact plasma lipoproteins to form lipid-poor pre-β-particles and plasma HDL3 can be converted back to lipid-poor particles by the action of CE transfer protein, LCAT, or PL transfer protein.66 Thus, an intermediate particle population between apoA-I

and HDL3, like pre-β-HDL might represent the PL-EV– suppressing molecule. CE from HDL3 can be exchanged to low-density lipoprotein or triacylglycerol-rich lipoproteins for triacylglycerols by CE transfer protein, increasing HDL3 size.11,66 Added nHDL3 and apoA-I resulted in an increased plasma particle mean diameter after 5 days (Fig. S4), suggesting lipoprotein metabolism of added nHDL3/apoA-I toward less dense particles. Added nHDL3 elevated plasma lipid content, whereas added lipid-free apoA-I did not. HDL3, but not apoA-I, is enriched in LCAT activity.11 Consequently, only nHDL3 increased plasma CE/FC and significantly up regulated plasma LPC species, resulting from LCAT-dependent PLA2 activity67 toward the sn-2 position of PC to form LPC. The same explanation might be the reason why only nHDL3, but not ApoA-I, showed significant beneficial effects on plasma CE distribution during 5 days decreasing plasma CE 16:0/20:4 and increasing PUFA CE/saturated CE ratios. These ratios correlate with atherogenic risk in vertebrates, including humans,68 and suggest interaction between nHDL3 and PLTs during storage. Added nHDL3 and apoA-I improved PLT lipid status, agreeing with studies showing that PLT cholesterol content depends on the surrounding lipid composition13 and where FC diffuses between cell membranes and plasma lipoproteins along a concentration gradient.31 Added nHDL3 also decreased plasma Cer and slightly increased PLT sphingosine (SPH) d18:1, suggesting increased plasma neutral ceramidase activity, converting Cer to SPH d18:1 at the plasmatic side of the cell membrane to incorporate SPH d18:1 into the plasma membrane.69 Cellular endocytosis of HDL was already suggested in 1974.70 In macrophages, “retroendocytosis” was shown to excrete modified HDL particles, yielding cholesterol efflux.71 In vitro, a rapid (minutes) retroendocytosis pool and a slow-turnover pool presented by MVBs exist for HDL.32 MVBs are precursors of exosomes or can be metabolized within lysosomes.72 HDL catabolism is limited to certain cells or states where extracellular cholesterol is limited73 or where the cholesterol demand is increased, like in metabolically active growth phases.74 Thus, an endocytotic mechanism for added and endogenic plasma nHDL3 into PLTs seems logical, since in vitro PLC storage represents a situation of decreased extracellular cholesterol supply. Endocytosis might occur via the open canalicular system, a PLT-specific endomembraneous system of numerous plasma cell membrane invaginations, serving a role in the exchange of substances between PLT intracellular stores and blood plasma35,75 and which fuses with intracellular lipid membranes like α-granules.35 Thus, added nHDL3 might also beneficially affect intracellular membrane homeostasis. In macrophages a similar capturing of lipoproteins into tubular compartments was reported.76 The fact that added nHDL3, but not apoA-I, immediately raised PLT CEs, especially Volume 54, September 2014 TRANSFUSION

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TABLE 2. Born aggregometry showing mean ± SD maximal aggregation response (in %) upon TRAP-6 (0.1 mmol/L), ristocetin (1.5 mg/mL), arachidonic acid (0.5 mg/mL), or ADP (20 μmol/L) stimulation after 5 days of PLC storage* Addition Control (PBS) nHDL3 apoA-I

TRAP-6 (0.1 mmol/L) 84 ± 3 88 ± 7‡ 82 ± 4

Ristocetin (1.5 mg/mL) 81 ± 4 81 ± 3 78 ± 10

Arachidonic acid (0.5 mg/mL) 82 ± 7 88 ± 9 83 ± 6

ADP (20 μmol/L)† 15 ± 1 30 ± 5 11 ± 0

* On Day 0, nHDL3 or apoA-I was added in concentrations that doubled the apoA-I concentration present in PLC plasma. Control = PBS addition to fill up the incubation volume. † Aggregation response to ADP was not detected in all experiments. ‡ p < 0.05, compared to control.

B

1.2

*

1.1

**

10.0 9.6

CE 16:0/20:4

PUFA/saturated CE-species

A 10.4

9.2 8.8 8.5 0.8

1.0 0.9 0.8 0.7 0.1 0.0

0.0 Control

nHDL3

Control

nHDL3

Fig. 3. (A) PUFA CE/saturated CE and (B) CE 16:0/20:4 ratios in PLC plasma upon added nHDL3 in concentrations that doubled the HDL3-cholesterol level in PLC plasma. Added PBS in similar volumes served as control. Shown are ratios for Day 0 (□) and Day 5 (■). Asterisks indicate significances between control and added nHDL3.

the CE18:2 (the most prevalent CE in HDL3), and that we observed high correlation of PLT and nHDL3 CE species compositions immediately upon nHDL3 addition, supports endocytosis of HDL by PLTs and indicates the dependence of PLT lipid composition on plasma lipoproteins.13,14 nHDL3 and apoA-I antagonized increased PLT CE/FC ratio during 5 days. ApoA-I and HDL3 promote cellular cholesterol efflux both by fast, specific energy-dependent and by slow passive diffusion-type processes.77 Cholesterol efflux from cells to HDL3 correlate with SR-B1 expression levels.78 However, binding of HDL3 to SR-B1 is not the major determinant for efflux; instead a reorganization of lipid domains and rafts in the plasma membrane seems to be more important,9,78 possibly explaining the tendency toward higher spontaneous expression of PLT surface markers upon added nHDL3 and apoA-I. According to the diffusion model and PLT microdomain reorganization, high surrounding cholesterol is rapidly inserted into the PLT membrane, improving sensitivity, as PLT membrane cholesterol contributes to PLT reactivity.16 In contrast, low surrounding cholesterol extracts PLT membrane cholesterol, reducing sensitivity to agonists.79 Short incubation of washed PLTs with HDL3 2308

TRANSFUSION Volume 54, September 2014

and ADP improved PLT aggregation.80 Here, added nHDL3 together with TRAP-6 stimulation increased PLT aggregation after 5 days, which is an important finding for stored PLCs, which are known to lose ability to respond to stimuli during storage.1,81 Activated PLTs release the minor sphingolipid S1P into surrounding plasma,82 also detected during PLC storage.36 Added nHDL3 decreased plasma S1P d18:1 after 5 days, possibly explaining improved aggregability upon stimulation. Added nHDL3 increased plasma LPC, which can be converted to the lysolipid PLT-activating factor that binds PLT-activating factor receptor.83 Alternatively, plasma autotaxin converts LPC to the strong PLT agonist LPA, potentiating PLT aggregation via the thrombin receptor.84 Accordingly, added apoA-I had no effects on plasma LPC and decreased plasma LPA, with no effect on PLT aggregation. Added nHDL3 elevated PLT PA/LPA ratio during storage, whereas apoA-I did not. This agrees with the fact that nHDL3 provides lipids, for example, fatty acid residues to PLTs, which by cellular LPA acyl transferase are transferred to the LPA C-3 OH group to produce PA, which induces a membrane curvature change beneficial for vesicle fission and fusion processes.85 Also the conical

B

A

FC CE SM dhSM Cer LPC PC PS PE PI PC O PE P HexCer Hex2Cer SPH d18:1 SPH d18:0 SPC S1P d18:1 S1P d18:0 LPA PA

**

HDL3/ApoA-I IN PLT STORAGE LESION

FC

*

CE SM dhSM

*

Cer

*

LPC PC PE PI PC O PE P

*

HexCer Hex2Cer SPH d18:1 SPC

*

S1P d18:1

*

*

S1P d18:0

PG BMP

*

C1P

0

2

4 Day 5/Day 0

*

LPA

CL

0

6

2

4

6

8

10

Day 5/Day 0

Fig. 4. Lipid mass spectrometry of PLCs treated with nHDL3 at concentrations doubling the HDL3-cholesterol level in PLC plasma. Shown are Day 5/Day 0 ratios for each lipid class upon added nHDL3 (■) or similar volumes of PBS (control; □) for (A) PLTs and (B) plasma. Asterisks indicate significant differences between control and nHDL3-addition. dhSM = dihydroSM; PS = phosphatidylserine; PI = phosphatidylinositol; PC O = PC-plasmalogen; HexCer = hexosylCer; Hex2Cer = di-hexosylCer (most likely lactosylCer); SPC = sphingosylphosphorylcholine.

12

B

Day 5/Day 0

10

14 12

*

* **

10

8

Day 5/Day 0

A

*

6

*

4

**

6 4

2

*

8

**

2

0

0 Control

nHDL3

Control

nHDL3

Fig. 5. Change in lipid class ratios during 5 days of PLC storage (Day 5/Day 0) upon added nHDL3 in concentrations that doubled the HDL3-cholesterol level in PLC plasma or upon added PBS (control) in similar volumes for (A) PLTs and (B) plasma. Asterisks indicate significances between control and nHDL3 addition. ( ) FC/PC; (■) CE/FC; ( ) CE/PC; (□) LPC/PC; ( ) LPA/PC.

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2309

B

40 18:2

30

R = 0.99 p = 0.001

20 18:1 10

16:0 20:4

R = 0.98 p = 0.001

0

0

5000

Day 5 PLT CE-species content (nmol/mg)

A

Day 0 PLT CE-species content (nmol/mg)

PIENIMAEKI-ROEMER ET AL.

40 18:2

30

R = 0.99 p = 0.001 R = 0.99 p = 0.001

20 18:1 10

16:0 20:4

0

0

10000 15000 20000 25000 30000

5000

10000 15000 20000 25000 30000

nHDL3 CE-species content (µmol/mg)

nHDL3 CE-species content (µmol/mg)

Fig. 6. Linear Pearson’s correlation of CE species content (μmol/mg) in isolated nHDL3 (x-axis) to mean CE species content (nmol/ mg) in PLTs on (A) Day 0 and (B) Day 5 upon added nHDL3 (●) that doubled the HDL3-cholesterol level in PLC plasma or PBS (○, control) in similar volumes. Correlation coefficients (R) and significances (p) for each condition are shown. The most prevalent nHDL3 species are indicated.

TABLE 3. Lipid class ratios on Day 0 (D0) and Day 5 (D5) in (A) PLTs and (B) plasma upon added nHDL3 to double the nHDL3 cholesterol level in PLC plasma* Ratio A. PLTs CE/FC CE/PC FC/PC LPC/PC LPA/PC LPA/LPC PA/LPA CL/PA BMP/PG Cer/SM Cer/PC S1P d18:1/Cer PS/PC PE P/PE B. Plasma CE/FC CE/PC FC/PC LPC/PC LPA/PC LPA/LPC Cer/SM Cer/PC S1P d18:1/Cer S1P d18:0/Cer C1P/Cer PE P/PE

D0

Control D5

Change in %

D0

nHDL3 D5

Change in %

0.2 0.4 2 0.02 0.0006 0.03 13 1.26 0.012 0.06 0.02 0.07 0.6 1.7

0.8 1.2 1.6 0.021 0.0012 0.06 16 0.56 0.004 0.1 0.04 0.01 0.6 1.8

+300a +84a −21a +5 +78 +100 +22 −55a −65b +67c +56a −83 −11 +3

0.5d 0.9d 1.8 0.024 0.0008 0.03 11 1.25 0.005d 0.06 0.02 0.06 0.5 1.8

0.8 1.1 1.4e 0.018 0.0007 0.05 20 0.68 0.006 0.1 0.04 0.01 0.6 1.8

+67 +4 −22a −25 −8 +54 +78b −45 +37 +72c +77c −85a +13 0

7.2 3.8 0.54 0.2 0.001 0.005 0.055 0.006 0.2 0.1343 0.007 0.4

8.9 4.6 0.53 0.3 0.001 0.003 0.055 0.007 0.3 0.1685 0.013 0.4

+24a +21b −3 +41a +5 −27 0 +20b +56a +25 +85a +4

7.9 3.2d 0.41e 0.1 0.001 0.004d 0.047d 0.005d 0.2 0.1484 0.012d 0.4

14.2e 4.6 0.33e 0.3 0.003e 0.009 0.044e 0.006e 0.2d 0.0003d 0.012 0.5

+79b +42c −19b +152c +528 +143 −6 +20b −29a −100b +7 +5a

* Added PBS in similar volumes served as control. a p < 0.05, b p < 0.01, c p < 0.001, significant change in ratio within the group; d p < 0.05, e p < 0.01; significant difference in ratio on Day 0 or Day 5 between control and upon added nHDL3.

shaped MVB curvature and late endosome marker BMP86 and the mitochondrial stability marker CL87 induce membrane curvature and form domains of membrane contact platforms.88 Cellular BMP content correlates to cellular FC, regulating cholesterol transport89 and lysosomal processing of lipids.72 In Niemann-Pick disease, 2310

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added BMP reversed the cholesterol accumulating phenotype.90 Added nHDL3 increased PG, BMP, and CL content, especially PG to BMP conversion, suggesting improved intracellular PLT cholesterol homeostasis and cellular (lysosomal) lipid processing. Increased PA/LPA, BMP, and CL might thus be beneficial for the aging PLT to maintain

HDL3/ApoA-I IN PLT STORAGE LESION

vital intracellular fission–fusion and vesicle trafficking processes to improve intracellular vesicular transport and recycling, especially important for PLT-specific granules, which fuse with endocytic vesicles and MVBs.91 For the first time we presented data on how plasma apoA-I and HDL3 might beneficially affect PLT lipid homeostasis, PLT function, and viability, reducing PL-EV release during PLC storage. Our data may provide insight into the in vivo mechanisms of PLT senescence and mechanisms of how apoA-I and HDL exert their antiatherogenic effects by improving PLT homeostasis and suppression of PL-EV release. Primarily, our study shows the possibility of apoA-I from FIV of plasma ethanol fractionation, as a substituent to reduce the PSL with better PLT viability and/or prolonged shelf life. ACKNOWLEDGMENTS

9. de La Llera-Moya M, Rothblat GH, Connelly MA, et al. Scavenger receptor BI (SR-BI) mediates free cholesterol flux independently of HDL tethering to the cell surface. J Lipid Res 1999;40:575-80. 10. Liu M, Bagdade JD, Subbaiah PV. Specificity of lecithin : cholesterol acyltransferase and atherogenic risk: comparative studies on the plasma composition and in vitro synthesis of cholesteryl esters in 14 vertebrate species. J Lipid Res 1995;36:1813-24. 11. Borggreve SE, de Vries R, Dullaart RPF. Alterations in highdensity lipoprotein metabolism and reverse cholesterol transport in insulin resistance and type 2 diabetes mellitus: role of lipolytic enzymes, lecithin : cholesterol acyltransferase and lipid transfer proteins. Eur J Clin Invest 2003;33:1051-69. 12. Leidl K, Liebisch G, Richter D, et al. Mass spectrometric analysis of lipid species of human circulating blood cells. Biochim Biophys Acta 2008;1781:655-64.

We thank the staff of the transfusion medicine department for plateletpheresis collection and the technical assistants responsible for lipoprotein preparation, lipid mass spectrometry, flow cytometry, and coagulation. We thank CSL Behring AG for providing the apoA-I supplement and Dr Peter Lerch for reading the manuscript.

13. Schick BP, Schick PK. Cholesterol exchange in platelets, erythrocytes and megakaryocytes. Biochim Biophys Acta 1985;833:281-90. 14. Siegel-Axel D, Daub K, Seizer P, et al. Platelet lipoprotein interplay: trigger of foam cell formation and driver of atherosclerosis. Cardiovasc Res 2008;78:8-17. 15. Lerch PG, Spycher MO, Doran JE. Reconstituted high

CONFLICT OF INTEREST The authors report no conflicts of interest.

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88. Orso E, Grandl M, Schmitz G. Oxidized LDL-induced endolysosomal phospholipidosis and enzymatically modified LDL-induced foam cell formation determine specific lipid species modulation in human macrophages. Chem Phys Lipids 2011;164:479-87. 89. Kobayashi T, Beuchat MH, Lindsay M, et al. Late endosomal membranes rich in lysobisphosphatidic acid regulate cholesterol transport. Nat Cell Biol 1999;1:113-8. 90. Chevallier J, Chamoun Z, Jiang G, et al. Lysobisphosphatidic acid controls endosomal cholesterol levels. J Biol Chem 2008;283:27871-80. 91. Heijnen HF, Debili N, Vainchencker W, et al. Multivesicular bodies are an intermediate stage in the formation of platelet alpha-granules. Blood 1998;91:2313-25.

SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s Web site: Fig. S1. Lipid mass spectrometry of platelet concentrates (PLCs) with added nHDL3 at concentrations to double the HDL3-cholesterol level in PLC-plasma. As control, PBS in similar volumes were added. Shown are significant delta differences (in %) in Day 5/Day 0 ratios of plasma lipidspecies between added nHDL3 and control. Asterisks indicate significances * = p < 0.05, ** = p < 0.01, *** = p < 0.001. Cer = ceramide, Hex2Cer = di-hexosylceramide (most likely lactosylceramide), LPC = lysophosphatidylcholine, PC = phosphatidylcholine, PE = phosphatidylethanolamine, PC O,PC-plasmalogen, PE P = PE-plasmalogen. Fig. 2S. Lipid mass spectrometry of platelet concentrates (PLCs) with added ApoA-I at concentrations to double the ApoA-I level in PLC-plasma. As control, PBS in similar volumes were added. Shown are Day 5/Day 0 ratios of A) platelet and B) plasma lipid classes. Asterisks indicate significances in Day 5/Day 0 ratio between added ApoA-I and control, with * = p < 0.05, ** = p < 0.01. FC = free cholesterol, CE = cholesteryl ester, SM = sphingomyelin, dhSM = dihydroSM, Cer = ceramide, LPC = lysophosphatidylcholine, PC = phosphatidylcholine, PS = phosphatidylserine, PE = phosphatidylethanolamine, PI = phosphatidylinositol, PC-O = PC-plasmalogen, PE P = PE-plasmalogen, HexCer = hexosylCer, Hex2Cer = di-hexosylCer (most likely lactosylceramide), SPH = sphingosine, SPH t18:0 = phytosphingosine, SPC = sphingosylphosphorylcholine, S1P = sphingosine-1-phosphate, LPA = lysophosphatidic acid, LPG = lysophosphatidylglycerol, PA = phosphatidic acid, PG = phosphatidylglycerol, BMP = bis(monoacylglycero)phosphate, CL = cardiolipin. Fig. S3. Lipid mass spectrometry of platelet concentrates (PLCs) with added nHDL3 at concentrations to double the HDL3-cholesterol level in PLC-plasma. As control, PBS in

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similar volumes were added. Shown are absolute levels (nmol/mg cell protein) of platelet lysophosphatidic acid (LPA)-species on Day 0 and Day 5. * = p < 0.05. Fig. S4. Nanoparticle Tracking Analysis (NTA) of platelet concentrates (PLCs) with added nHDL3 or ApoA-I to double the ApoA-I level in PLC plasma. Added PBS in similar volumes served as control. Shown are mean ± SD particle size (nm) on Day 0 and Day 5 from 6 individual experiments. Table S1. Flow cytometric expression analysis of CD62P, CD41 and CD61 without stimulation (Basal) or upon TRAP-6 (10 μM) or ADP (5 μM) stimulation during 5 days platelet concentrate (PLC) storage. Mean fluorescence intensities (MFI) are shown. Changes are indicated as % of basal (100%). * = p < 0.05, ** = p < 0.01, compared to control. † = p < 0.05, compared to added apoA-I. Native (n)HDL3 or apoA-I were added in concentrations to double the apoA-I level in PLC plasma. Added PBS in a similar volume served as control. Table S2. Lipid class ratios on Day 0 (D0) and Day 5 (D5) in A) platelets and B) plasma upon added apoA-I to double the apoA-I concentration in PLC-plasma. Added PBS in similar volumes served as control. *, **, *** = Significant change in ratio within the group, † (p < 0.05), †† (p < 0.01) = significant difference in ratio on Day 5 between control and upon added apoA-I. CE = cholesteryl ester, FC = free cholesterol, PC = phosphatidylcholine, LPC = lysophosphatidylcholine, LPA = lysophosphatidic acid, PA = phosphatidic acid, CL = cardiolipin, Cer = ceramide, SM = sphingomyelin, S1P = sphingosine-1phosphate, PS = phosphatidylserine, PE P = PE-plasmalogen, PE = phosphatidylethanolamine. Table S3. Lipid mass spectrometry data showing absolute amounts (nmol/mg cell protein) of platelet cholesteryl ester (CE)-species on Day 0 (D0) and Day 5 (D5) upon added native (n)HDL3 in a concentration that doubled the HDL3-cholesterol in platelet concentrate plasma. Control = PBS added in a similar volume as nHDL3. Shown are also changes in % from D0 to D5. * = p < 0.05, ** = p < 0.01 for D0-D5 change for respective sample, † = p < 0.05, †† = p < 0.01 between Control and nHDL3 on Day 0. b.d. = below detection limit (all replicates