Transfusion and Apheresis Science 51 (2014) 58–64

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Transfusion and Apheresis Science j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / t r a n s c i

In vitro properties of apheresis platelet during extended storage in plasma treated with anandamide Yunlong Zhuang a, Guijie Ren b,*, Huiyu Li c, Keli Tian b, Yi Zhang a, Wenben Qiao a, Xiangmin Nie a, Yan Liu a, Yonghong Song a, Chuanfu Zhu a a

Blood Center of Shandong Province, Jinan 250014, Shandong Province, China School of Medicine, Shandong University, Jinan 250012, Shandong Province, China c Bio-technology Department, College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China b



Article history: Received 11 February 2013 Received in revised form 7 March 2014 Accepted 24 March 2014 Keywords: Anandamide Extended storage Platelet storage lesions


Background: In China apheresis platelets (PLTs) are stored in plasma for only 5 days, resulting in PLT inventory pressures. Anandamide (ANA) was reported to be a potential agent to inhibit PLT apoptosis. The aim of this study was to evaluate the characteristics of extended storage PLTs in plasma treated with ANA in vitro. Methods: Apheresis PLTs (n = 20) were prepared in plasma treated with ANA, and stored at 22 °C for up to 11 days. On day 1, 3, 5, 7, 9 and 11, PLTs were tested for PLT count, mean PLT volume (MPV), PLT distribution width (PDW), pH, pCO2, pO2, hypotonic shock response (HSR), phosphatidylserine (PS) exposure and soluble P-selectin content. Results: PLTs stored in plasma with/without ANA didn’t show significant differences during the first 5 days of storage. From the 7th day on, PLTs stored in plasma with ANA displayed significantly lower PS expression, soluble P-selectin content and higher HSR scores than those stored in plasma without ANA (P < 0.05), respectively. Conclusion: The extended storage of PLTs in plasma treated with 0.5 μmol/l ANA showed better characteristics of the PLTs, compared with the control group, which was suggested to potentially alleviate the PLT storage lesion. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction Most platelet (PLT) concentrates are collected by apheresis and stored in plasma, and their shelf life is limited to 5 days in China. PLT storage-dependent deleterious changes leading to progressive damage in PLT structure and function are referred to as PLT storage lesions (PSL) which cause loss of PLT viability. The mechanisms responsible for the PSL are not fully understood but are clearly multifactorial. Certain parameters that indicate the decline in the quality of PLTs during storage include increasing expression of P-selectin (CD 62P) and phosphatidylserine (PS) on the PLT membrane. Exposure of PS is typical of nucleated

* Corresponding author. Tel.: +86 531 88382346; fax: +86 531 82969968. E-mail address: [email protected] (G. Ren). 1473-0502/© 2014 Elsevier Ltd. All rights reserved.

cells and points to the fact that apoptotic machinery is present in the PLTs [1]. The PS was described as a target molecule for the recognition and removal of activated or damaged PLTs, and the exposure of PS may represent the terminal loss of PLT function [2]. Mitochondrial-mediated apoptosis of PLTs during storage was reported [3,4]. During storage, the metabolic activity of PLTs continues to consume nutrients and to produce harmful metabolic products. Glycolysis results in a fall in pH and PLT morphology begins to change [5]. The quality of stored PLTs can be assessed by measuring the factors. Anandamide, i.e. N-Arachidonoylethanolamine (ANA or AEA), is an endogenous lipid which belongs to the family of endocannabinoids (eCBs). After it binds to cannabinoid receptors (CB1 and CB2), ANA is taken up into cell by an ANA-membrane transporter and catalyzed by fatty acid amidehydrolase (FAAH). In human PLTs the ANA-membrane

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transporter and FAAH have been previously demonstrated [6]. Moreover, the expressions of CB1 and CB2 receptors have been proven recently in human PLTs [7]. Both CB1 and CB2 are coupled to G 1 /G proteins and can activate the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) pathway [8,9]. PLTs can regulate endogenous eCBs in the periphery by metabolizing them [10,11]. Recently, it was shown that ANA was able to inhibit human PLT apoptosis through CB1dependent PI3K/Akt signal pathway at 37 °C [3]. However, the effect of ANA on the quality of extended storage apheresis PLTs under standard blood bank conditions is poorly documented. Increasing the allowable shelf life of PLTs stored at room temperature for more than 5 days can be a means to relieve PLT inventory pressures [12]. The aim of this study was to evaluate the characteristics of apheresis PLTs in vitro stored for up to 11 days in plasma in the presence of ANA. 2. Materials and methods 2.1. Reagents Chemicals were all analytical pure grade. ANA, dimethyl sulfoxide (DMSO), 3-(4, 5-diethylthiazol-2-yl)-2, 5-diphenyltet-razolium bromide (MTT) were purchased from Sigma Chemical (St. Louis, MO, USA). Annexin V-FITC apoptosis kit was purchased from BioVision Inc (Milpitas, CA, USA). Human soluble P-Selectin ELISA Kit was purchased from R&D Systems China Co. Ltd (Shanghai, China). 2.2. PLT collection, storage, and sampling According to Chinese guidelines, all voluntary donors in the Blood Centre of Shandong Province in China need to fill the questionnaire sheet to exclude high risk behaviors, some diseases and consumption of any medications known to alter PLT functions or antibiotics within the 7 days before PLT donation. The donors also undergo physical examination by doctors before donation. Apheresis PLTs were collected into plasma using the Fenwal Amicus Separator System (Baxter Healthcare Crop., Deerfield, IL, USA) with anticoagulant citrate dextrose solution formula A (ACD-A), with whole blood: ACD-A is 9.5:1.0. All samples were tested after obtaining the donors’ informed consent. PLT concentrations per bag were more than 2.5 × 1011. After mixing three donors’ PLTs and splitting them into seven groups (four repeats/ group), different working concentrations of ANA (0.1, 0.5, 1, 5, 10 and 50 μmol/l) were added to the six groups, respectively, with another group without ANA as the control. Those samples were stored for 7 days at 22–24 °C and then the PLT viabilities were measured. The most effective concentration of ANA was chosen to perform all subsequent experiments. Samples (n = 20) (120 ml/each) were taken and split into two parts (60 ml/each). ANA was added to one part as the experimental group, and the other part without ANA served as the control group. All samples were stored in a PL-2410 container (Amicus Apheresis kit, Baxter Healthcare Crop.) on a flatbed shaker (50–60 cycles/min) at 22– 24 °C for up to 11 days. The samples were taken on the 1st, 3rd, 5th, 7th, 9th and 11th day under sterile conditions.


2.3. Measurement of variables in vitro PLT viability was evaluated by measuring the mitochondrial reduction of MTT to formazan, according to the manufacturer’s protocol. PLT count, mean PLT volume (MPV) and PLT distribution width (PDW) were determined by automated blood cell counter (Mindray BC-3000 plus, Mindray Crop., Shenzhen, China) in a 1:10 dilution in PBS. pH, pO2 and pCO2 were measured immediately at 37 °C with a blood gas analyzer (ABL 725, Radiometer Medical ApS, Copenhagen, Denmark) after sampling. The pH(37 °C) was subsequently converted to pH(22 °C) using a given formula. The hypotonic shock response (HSR) was measured with the aggregometer by addition of distilled water to the PLT suspension and measurement of the change in light transmittance [2]. For measurement of PS exposure, PLTs (3 × 106) were diluted in 100 μl of annexin V binding buffer and stained with 5 μl of annexin V – fluorescein isothiocyanate (FITC; Biolegend, San Diego, CA, USA). The tubes were incubated at room temperature in dark for 15 min. Then the PLTs were diluted and analyzed with Partec PAS flow cytometry (PARTEC, Nuremberg, Germany). Enzyme-linked immunosorbent assay kit for soluble P-selectin was used according to the manufacturer’s instructions (R&D Systems Inc., Minneapolis, MN, USA).

2.4. Bacterial testing At the end of days 7, 9 and 11 of storage, 7 ml (the recommended volume from the BacT/Alert manufacturer) of PLT concentrate was withdrawn from each bag under strict aseptic technique and inoculated into aerobic and anaerobic bottles, respectively. The culture bottles were placed in the BacT/Alert 3D incubator for a maximum of 10 days or until they were flagged as positive, which was monitored by the BacT/Alert system (bioMerieux Canada Inc., Quebec, Canada).

2.5. Statistical analysis Data were expressed as mean ±SD. Results were compared using a paired t test for experiments. Correlations between different parameters measured on day 11 of storage were performed using Pearson correlation (SPSS16.0, Chicago, IL, USA). A P value less than 0.05 was considered to be a significant difference.

3. Results 3.1. ANA concentration and PLT viability In this study, we investigated the effect of the ANA concentration (from 0.1 to 50 μmol/l) on PLT viability. After 7 days of storage, the most effective concentration of ANA was 0.5 μmol/l, which showed significantly increasing PLT survival (about 1.7 fold more than controls) (Fig. 1). Thus, we chose 0.5 μmol/l ANA to perform all subsequent experiments.


Y. Zhuang et al./Transfusion and Apheresis Science 51 (2014) 58–64

3.4. Soluble P-selectin, PS and HSR

Fig. 1. PLT viability in the presence of ANA. PLTs were incubated at 22– 24 °C for 7 days, in the absence (control) or in the presence of increasing concentrations (0.1–50 μmol/l) of ANA. Values are reported as fold over control (arbitrarily set to 1). The data represent the mean ± SD of at least three independent experiments. *P < 0.05 indicates statistical significance vs control.

3.2. PLT count, MPV and PDW Apheresis PLTs were stored at 22–24 °C for up to 11 days in plasma with/without 0.5 μmol/l ANA, and the characteristics of the PLTs were detected on the 1st, 3rd, 5th, 7th, 9th and 11th day of storage (Fig. 2). PLT counts dropped slightly in the two groups during the storage period. There were no significantly changes in the two groups until day 11 of storage, the PLT counts of the experimental group dropped slowly, compared with the control group (P < 0.05) (Fig. 2a). MPV was on the rise during the storage period: the values of the experimental group rose from 6.9 ± 0.4 fl to 8.1 ± 0.6 fl while the control group rose from 6.8 ± 0.5 fl to 8.9 ± 0.5 fl, with a significant difference between the two groups on the 11th day of storage (Fig. 2b). PDW rose progressively during storage; interestingly, the PDW of the experiment group (16.4 ± 0.5%) had better characteristics compared with the control group (17.0 ± 0.7%) on the 11 th day of storage (P < 0.05) (Fig. 2c).

3.3. pH, pO2 and pCO2 The pH (22 °C) value of the experimental group decreased slightly slower than those in the control group during 11 days of storage: the experimental group decreased from 7.4 ± 0.1 to 6.4 ± 0.1 while the control group decreased from 7.4 ± 0.1 to 6.1 ± 0.1, with significant differences between the two groups on the 7th, 9th and 11th day of storage (Fig. 2d), respectively. The pO2 during storage increased while pCO2 decreased (Fig. 2e, f). There were no significant differences of the pO2, whereas the pCO2 showed a significant difference on day 11 between the experiment and control groups.

There was an increase in soluble P-selectin content during the storage of the PLTs. However, no significant differences were observed between the experimental and control groups until the 7th, 9th and 11th day of storage (75.1 ± 6.4 ng/ ml vs 90.4 ± 8.9 ng/ml; 81.6 ± 6.1 ng/ml vs 117.6 ± 8.0 ng/ ml; 103.6 ± 7.9 ng/ml vs 144.7 ± 7.8 ng/ml; P < 0.05) (Fig. 2g). The rates of positive PLT PS rose more slowly in the experimental group than those in the control group during storage (Fig. 2h). The rates of positive PS in the experimental group on the 7th, 9th and 11th day of storage (8.1 ± 1.4%, 13.2 ± 1.8% and 22.6 ± 1.8%) were less than those (13.0 ± 2.5%, 25.6 ± 2.0% and 40.5 ± 2.0%) in the control group (P < 0.05). The PLT HSR decreased more slowly over the storage period in the experimental group than those in the control group (Fig. 2i). The HSR in the experiment group on the 7th, 9th and 11th day of storage were 68.6 ± 6.2%, 58.4 ± 5.2% and 45.7 ± 4.4%, respectively, while those in the control group were 61.1 ± 6.5%, 48.9 ± 4.6% and 30.3 ± 3.9%, respectively, with significant differences between the two groups. 3.5. Correlation among PLT character parameters In the experimental group, significant positive correlations were found between pH and pCO2 and HSR while the pH correlated inversely with PS and P-selectin (P < 0.05) (Table 1); the pCO2 showed significant inverse correlations with PS and P-selectin, and were positively correlated with the HSR; there were significant inversely correlations of the HSR with PS and P-selectin, whereas PS and P-selectin showed positive correlations in the experimental group (P < 0.05). The PLT parameters examined in the control group were similar to those in the experiment group. 3.6. Bacterial testing Bacterial contamination was not detected in any of the units. 4. Discussions The eCB system plays a key role in the pathophysiology of the central nervous system [13] and peripheral tissues [14]. When administrated in vivo, ANA induces antinociception, hypothermia, hypomotility and catalepsy [15–18]. ANA may also be involved in the control of pain initiation [19] or sleep induction [20] as well as being involved in immune [10,21] and cardiovascular [11,14] or reproductive functions [22,23]. eCBs regulate survival and death in a cell type specific manner and depending on the receptors engaged [24,25]. Remarkably, eCBs are able to modulate, through CB1 receptors, the PI3K/Akt pathway, to inhibit the release of cytochrome c and the expression of caspase-3 and caspase-9, which serves as a pivotal antiapoptotic signal [3]. In this study, we found that the most efficient concentration of ANA for increasing apheresis PLT (stored in plasma at blood bank conditions) viability was 0.5 μmol/l while higher concentrations of ANA gradually decreased PLT

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Fig. 2. Mean values of PLT count, PDW, MPV, pH, pCO2, pO2, HSR, PS and P-selectin made of apheresis PLT stored in plasma with presence of ANA (●) or absence of ANA (○) for up to 11 days. *P < 0.05 indicates statistical significance vs control on the same day of storage.


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Table 1 Correlation of PLT character parameters in PLTs treated with or without (bold) ANA and stored at 22 °C for 11 days. PLT count PLT count MPV PDW pH pO2 pCO2 P-selectin PS HSR

−0.25 −0.18 0.21 0.43 0.38 0.12 −0.31 0.46










−0.21 0.68

0.23 −0.59 −0.80

0.38 0.37 0.63 0.48

0.39 0.43 −0.48 0.81* 0.69

0.14 0.56 0.64* −0.83* 0.60 −0.73*

−0.28 0.59 0.56 −039* 0.46 −0.64* 0.78*

0.48 −0.59 −0.67* 0.49* 0.71 0.44* −0.65* −0.56*

0.76 −0.65 0.36 0.48 0.59 0.62 −0.70

−0.77 0.61 −0.59 0.68* 0.54 −0.74*

0.47 0.85* −0.86* −0.23* 0.73*

0.65 0.58 0.42 0.56

−0.79* −0.68* 0.63*

0.71* −0.78*


R2 values are from Pearson correlations. Figures in bold indicate correlations among PLT parameters examined in the control group. * P < 0.05 indicates statistically significant correlations.

viability. To some extent these results were similar to the reports previously described [3,26], although the PLTs were incubated at 37 °C in their studies. Previous studies also showed that millimole concentrations of ANA were able to activate human PLTs [6,16], but these levels are much higher than those in the blood [27]. Indeed, ANA was present in plasma in nanomolar concentration [27,28]. Nonetheless, ANA can be released locally by macrophages and endothelial cells, and its local concentration may be higher [11]. So it has been speculated that the anti-apoptotic action of ANA can also occur in vivo [3]. Recently, Signorello et al. reported that low concentrations of ANA contributed to extending PLT survival, through PI3K/Akt pathway activation, stimulating endothelial nitric oxide synthase activity and increasing nitric oxide (NO) levels in human PLTs [26]. Increasing the NO level in human PLTs could be one of many beneficial effects produced by ANA during ischemic conditions [29], as ANA produces vasodilatation in the arterial system [30]. Moreover ANA, by stimulating NO elevation, can limit the amplification of PLT thrombus formation in vivo [31]. This was the first in vitro study to describe the characteristics of apheresis PLTs treated with or without ANA and stored for up to 11 days in plasma at 22–24 °C. The results indicated that ANA-treated PLTs had better characteristics in PLT count, MPV, PDW and pCO2, especially on day 11 of storage. Interestingly, the decreasing pH value fell significantly slower in the experimental group from day 7 of storage on than those in the control group, which suggested a reduction in glucose consumption and lactate production in the experimental group. This may have contributed to the facts that ANA inhibited PLT apoptosis and therefore reduced the metabolic rate of the PLTs. The inverse correlations of pH with PS and P-selectin suggested that the decreasing of pH might be one of the factors having a deleterious effect on the characteristics of PLTs. Apoptosis is regulated by caspases, a group of cysteine proteases, and is characterized by PS expression on the outer leaflet of the plasma membrane. The PS externalization was considered as an apoptotic marker. PS exposure in stored PLTs was inhibited by decreasing the release of cytochrome c and the inactivation of caspase-3, and blocking these procedures could attenuate PLT apoptosis [32,33]. Intriguingly, ANA inhibited the cytochrome c releasing and the caspase-3 expressions of PLTs stored in plasma at 37 °C [3].

Therefore, it is conceivable that ANA may decrease the PLT PS exposure. Our results in this study were in accord with the inferences mentioned above, which suggested that ANA might improve the characteristic of stored PLTs by reducing the positive expression of PS. Unfortunately, we did not incorporated multiple assays to characterize apoptosis of the PLTs including mitochondrial membrane potential, Decoy cell death receptor 2, caspases, etc. Further confirmatory tests are needed. The PSL leads to an increase of P-selectin expression, which is a typical PLT activation maker [34]. In this study, ANA-treated PLTs showed less release of soluble P-selectin during storage, which suggested ANA inhibited PLT activation to some extent. Exposure of PS and P-selectin on the PLT plasma membrane reflects two fundamental PLT processes: apoptosis and activation, respectively [35]. The significant positive correlation between PS and P-selectin in our study suggested the concurrent events of PLT apoptosis and activation existed in the stored PLTs. The ability to predict the likely in vivo recovery and survival of specific PLTs would provide a valuable adjunct to current transfusion practice, potentially improving clinical outcome, while simultaneously providing more effective inventory control. The superior in vitro results of PLTs stored in the plasma paralleled the in vivo results. Substantially better results were found for maintenance of hypotonic shock response and/or extent of shape change, two in vitro parameters that previously have been shown to correlate highly with in vivo recovery and survival [36–38]. The HSR measures PLT energy reserves and integrity under stress. AuBuchon et al. reported that the in vitro tests of HSR for stored PLTs in plasma on day 5 and 7 were 52 ± 15% and 43 ± 20%, respectively, and in vivo recoveries declined with the two additional days of storage from 54.4 ± 13.6% to 48.7 ± 15.0%, and survivals decreased from 6.7 ± 1.0 to 5.4 ± 1.7 days [39]. The means for HSR in vitro were 43.6 ± 14.7% for PLT stored up to 7 days in plasma, and these PLTs mean recoveries and survivals in the in vivo studies were 36.6% ± 10.7% and 4.5 ± 1.3 days, respectively [36]. Compared with our HSR results for PLTs treated by ANA on day 11 (Fig. 2i), it was speculated that our in vivo recovery and survival results might be similar to those above on day 7 [36,39]. PLTs stored in plasma with ANA might maintain PLT energy reserves and integrity. However, it cannot be excluded that this was due to the differences in collection, storage systems and different experimental conditions. In

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fact, the stored PLT had a total average lifespan greater than it would have had in the normal circulation and factors in the transfusion recipient promote recovery from any senescence-based storage lesion [40]. Catani et al. reported that ANA might represent an exciting therapeutic target for the inhibition of PLT demise in thrombocytopenias [3]. Clinical studies to determine the in vivo survival and hemostatic capabilities of stored PLTs with ANA are needed to determine whether our findings are relevant to the practice of transfusion medicine. 5. Conclusion This study investigated the effect of ANA on properties of apheresis PLT stored in plasma for up to 11 days. PLTs stored in plasma with the presence of 0.5 μmol/l ANA showed better characteristics such as pH, HSR, PS and P-selectin, compared with the control under standard blood bank conditions. This was suggested to potentially alleviate the PSL in the experimental group during extended storage of PLTs. However, more targeted studies would be required to confirm these events as well as the PLT recovery and survival in vivo. Acknowledgements This work was supported by Shandong Province Young and Middle-Aged Scientists Research Awards Fund (BS2011SW044), Medicine and Health Science and Technology Development Planning Projects of Shandong Province (2013WS0170), the Natural Science Foundation of Shandong province (ZR2009DM040 and ZR2009CM068), and Shandong Province Young and Middle-Aged Scientists Research Awards Fund (2007BS02006), the Natural Scientific Innovative Foundation of Shandong University (2009TS119). References [1] Brown SB, Clarke MC, Magowan L, Sanderson H, Savill J. Constitutive death of platelets leading to scavenger receptor-mediated phagocytosis. A caspase-independent cell clearance program. J Biol Chem 2000;275:5987–96. [2] Cookson P, Sutherland J, Turner C, Bashir S, Wiltshire M, Hancock V, et al. Platelet apoptosis and activation in platelet concentrates stored for up to 12 days in plasma or additive solution. Transfus Med 2010;20:392–402. [3] Catani MV, Gasperi V, Evangelista D, Finazzi Agrò A, Avigliano L, Maccarrone M. Anandamide extends platelets survival through CB1-dependent Akt signaling. Cell Mol Life Sci 2010;67:601–10. [4] Mason KD, Carpinelli MR, Fletcher JI, Collinge JE, Hilton AA, Ellis S, et al. Programmed anuclear cell death delimits platelet life span. Cell 2007;128:1173–86. [5] Shrivastava M. The platelet storage lesion. Transfus Apher Sci 2009;41(2):105–13. [6] Maccarrone M, Bari M, Menichelli A, Del Principe D, Agrò AF. Anandamide activates human platelets through a pathway independent of the arachidonate cascade. FEBS Lett 1999;447:277–82. [7] Catani MV, Gasperi V, Catanzaro G, Baldassarri S, Bertoni A, Sinigaglia F, et al. Human platelets express authentic CB1 and CB2 receptors. Curr Neurovasc Res 2010;7:311–8. [8] Gómez del Pulgar T, Velasco G, Guzmán M. The CB1 cannabinoid receptor is coupled to the activation of protein kinase B/Akt. Biochem J 2000;347(Pt2):369–73. [9] Liu J, Gao B, Mirshahi F, Sanyal AJ, Khanolkar AD, Makriyannis A, et al. Functional CB1 cannabinoid receptors in human vascular endothelial cells. Biochem J 2000;346(Pt3):835–40.


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In vitro properties of apheresis platelet during extended storage in plasma treated with anandamide.

In China apheresis platelets (PLTs) are stored in plasma for only 5 days, resulting in PLT inventory pressures. Anandamide (ANA) was reported to be a ...
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