ORIGINAL ARTICLE p38MAPK is involved in apoptosis development in apheresis platelet concentrates after riboflavin and ultraviolet light treatment Zhongming Chen,1,2 Peter Schubert,1,2,3 Brankica Culibrk,1,2 and Dana V. Devine1,2,3

BACKGROUND: Pathogen inactivation (PI) accelerates the platelet (PLT) storage lesion, including apoptotic-like changes. Proteomic studies have shown that phosphorylation levels of several kinases increase in PLTs after riboflavin and UV light (RF-PI) treatment. Inhibition of p38MAPK improved in vitro PLT quality, but the biochemical basis of this kinase’s contribution to PLT damage requires further analysis. STUDY DESIGN AND METHODS: In a pool-and-split design, apheresis PLT concentrates were either treated or kept untreated with or without selected kinase inhibitors. Samples were analyzed throughout 7 days of storage, monitoring in vitro quality variables including phosphatidylserine exposure, degranulation, and glucose metabolism. Changes in the protein expression of Bax, Bak, and Bcl-xL and the activities of caspase-3 and -9 were determined by immunoblot analysis and flow cytometry, respectively. RESULTS: The expression levels of the proapoptotic proteins Bax and Bak, but not the antiapoptotic protein Bcl-xL, were significantly increased after the RF-PI treatment. This trend was reversed in the presence of p38MAPK inhibitor SB203580. As a result of increasing proapoptotic protein levels, caspase-3 and -9 activities were significantly increased in RF-PI treatment during storage compared with control (p < 0.05). Similarly, p38MAPK inhibition significantly reduced these caspase activities compared with vehicle control after RF-PI treatment (p < 0.05). CONCLUSION: These findings revealed that p38MAPK is involved in signaling leading to apoptosis triggered by RF-PI. Elucidation of the biochemical processes influenced by PI is a necessary step in the development of strategies to improve the PLT quality and ameliorate the negative effects of PI treatment.

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ransfusion-associated infectious risk and reduced transfusion efficacy due to decreasing quality of platelets (PLTs) in blood bank storage are still major concerns. The introduction of pathogen inactivation (PI) systems provided a promising, effective, and reliable method to further reduce the potential risk of transfusion-transmitted infections beyond traditional screening and testing strategies.1 PI is based on illumination using ultraviolet light with or without a photosensitizer targeting pathogen DNA or RNA to inhibit their proliferation.2,3 Currently, there are three different methods available on the market based on different system characteristics.4 One of these PI systems uses riboflavin plus UV light (RF-PI; Mirasol, TerumoBCT, Lakewood, CO) to irreversibly damage nucleic acids of infectious agents. While research revealed that this system inactivates most of the blood-borne viruses,5 bacteria,6 and parasites7 and residual white blood cells as well as reduces antigen expression,8 in vitro studies also showed that PI treatment has a negative impact on the PLT quality, ABBREVIATIONS: MLCK = myosin light chain kinase; PD = PD98059; PI = pathogen inactivation; PS = phosphatidylserine; RF-PI = riboflavin/UV and pathogen inactivation; SB = SB203580; Y = Y27632. From the 1Canadian Blood Services; and the 2Centre for Blood Research and 3Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada. Address reprint requests to: Dana Devine, PhD, Canadian Blood Services, UBC Centre for Blood Research, 2350 Health Sciences Mall, Vancouver, BC, Canada V6T 1Z3; e-mail: [email protected] This study was supported in part by a grant from Health Canada and Canadian Blood Services to DVD. ZC is the recipient of a Canadian Blood Services Postdoctoral Fellowship Award. Received for publication June 27, 2014; revision received August 29, 2014, and accepted September 8, 2014. doi: 10.1111/trf.12905 © 2014 AABB TRANSFUSION **;**:**-**. Volume **, ** **

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which can be detected as PLT activation via protein kinases and apoptosis-like changes. Compared to the well-known mechanism of apoptosis in nucleated cells, which plays a key role in several physiologic processes, the mechanism of apoptosis development in anucleated PLTs is still controversial. Phosphatidylserine (PS) exposure on the PLT surface, identified as an ingestion signal for macrophages, and microparticle release are associated with apoptosis but can also occur upon agonist stimulation.9 It is generally acknowledged that apoptosis is important for clearance of aged PLTs from the circulation.10,11 Seeking to understand the biochemical mechanism, models have been generated to investigate apoptosis development in PLTs treated with different stimuli such as thrombin,12 calcium ionophore,13 and the BH3-only mimics, ABT-737 or ABT-26314 as well as in stored PLTs at 22 or 37°C.15-17 Apoptosis in stored PLTs shares features with nucleated cell apoptosis including PS exposure, caspase-3 activation, and gelsolin cleavage.15,18-20 The treatment of PLT concentrates with PI accelerates apoptosis.21 However, the biochemical mechanism of PLT apoptosis triggered by PI treatment is still poorly understood. We have recently reported protein kinase activation upon RF-PI treatment and demonstrated that the phosphorylation levels of all tested kinases were increased upon RF-PI treatment compared to control samples.22 Among others, p38MAPK and MEK exhibited a greater than twofold increase in activation upon RF-PI treatment. Propelled by this finding, an inhibitor of p38MAPK, SB203580 (SB), was added to PLTs before RF-PI treatment and resulted in a significant reduction of PS exposure and improvement in PLT quality during storage compared to controls without inhibitor. This observation suggests that PS externalization in RF-PI–treated PLTs might be regulated by p38MAPK via an unknown signaling pathway. In the study reported here we test the hypothesis that p38MAPK regulates the apoptotic signaling pathway in PLTs and that this regulation is altered by RF-PI.

MATERIALS AND METHODS

obtained from all healthy volunteers before blood donation. Phlebotomy and plateletpheresis were carried out by the NetCAD development laboratory of Canadian Blood Services (Vancouver, British Columbia, Canada) using an automated blood collection system (Trima Accel, TerumoBCT).

Mirasol treatment and sample preparation The procedure for Mirasol treatment, addition of riboflavin solution to a final concentration of 50 μmol/L followed by UV light illumination, was carried out according to the manufacturer’s protocol (TerumoBCT). For nontreated controls, the same volume of saline (instead of the riboflavin solution) was added to produce a similar PLT count in the units, and units were not illuminated. Samples were collected on Days 1, 5, and 7 of storage and the PLT lysates were prepared as published previously.23

In vitro PLT quality measurement The PLT count and mean PLT volume were measured on a hematology analyzer (ADVIA 120, Siemens, Deerfield, IL). Metabolites (glucose, lactate, and pH) were quantified using a blood gas analyzer (Gem Premier 3000, Instrumentation Laboratories, Bedford, MA). If lactate levels were out of the range of the analyzer, the samples were diluted with phosphate-buffered saline (PBS) and retested. PLT activation was monitored by the expression of P-selectin (CD62P) on the PLT surface using flow cytometry (FACS Canto II, BD Biosciences, Mississauga, Ontario, Canada). Briefly, the PLT sample was diluted with PBS to a count of approximately 200 × 109/L. A 5-μL sample was then incubated for 30 minutes with phycoerythrin-labeled anti-CD62P (Beckman-Coulter, Mississauga, Ontario, Canada). Levels of apoptosis were determined by changes in the exposure of PS on the PLT surface using annexin V binding. Briefly, the PLT sample was diluted with PBS to a count of 100 × 109 PLTs/L, incubated for 30 minutes with annexin V fluorescein isothiocyanate (FITC; BD Biosciences) in a calciumcontaining buffer and analyzed by flow cytometry.

Materials Common chemicals were purchased from Sigma-Aldrich (St Louis, MO) or Fisher Scientific (Ottawa, Ontario, Canada). Inhibitors used were SB (Santa Cruz Biotechnology, Santa Cruz, CA), specific targeting to p38MAPK kinase, PD98059 (PD; Cell Signaling, Danvers, MA), specific targeting to MEK kinase, and Y27632 (Y; SigmaAldrich), specific targeting to ROCK kinase.

Apheresis PLT concentrates preparation This study was approved by the research ethics board of Canadian Blood Services and informed consent was 2

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Inhibitor study and specificity analyses To determine which kinases are involved in PLT apoptotic signaling after RF-PI treatment and subsequent storage, three selected kinases were targeted by the use of specific inhibitors. Double apheresis PLT concentrates were pooled and split into six small illumination bags (TerumoBCT) with a volume of 69 ± 1 mL and a concentration of 1121 × 109 ± 118 × 109 PLTs/L. Unit 1 served as an untreated control; Unit 2 served as a RF-PI–treated control; Unit 3 served as a vehicle control with RF-PI treatment containing the equivalent amount of the solvent

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(dimethylsulfoxide [DMSO]); and Units 4, 5, and 6 were spiked under sterile conditions with final concentration of 10 μmol/L of the inhibitors SB (targeting p38MAPK kinase), PD (targeting MEK kinase), and Y (targeting ROCK kinase) before the RF-PI treatment, respectively. The inhibitor stock solutions (SB and PD in DMSO, Y in PBS) were diluted in PBS and sterile-filtered before spiking. Units 2 to 6 were subjected to the RF-PI treatment 1 hour after adding inhibitors. Under sterile conditions, PLT samples were drawn immediately after RF-PI treatment on Day 1, defined as the day of donation, and on Days 5 and 7 of storage, followed by subsequent analyses of in vitro quality measures and PLT lysis preparation.

Immunoblot analyses For protein analyses, washing and lysis procedures were performed as described previously.23 PLT lysates were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and blotted onto nitrocellulose membranes (Bio-Rad, Mississauga, Ontario, Canada). Membranes were probed with primary antibodies against Bax, Bak, Bcl-xL, cleaved caspase-3, cleaved caspase-9 (Cell Signaling) and actin (Sigma-Aldrich) followed by labeling with their respective secondary antibodies (Licor, Lincoln, NE). Protein band intensities were quantified by densitometry using the imaging analysis software on a bioimaging system (Odyssey and LI-COR, respectively, Licor, Lincoln, NE).

Flow assay for active caspase-3 and -9 The activation states of caspase-3 and -9 were determined by cell-penetrating FITC-conjugated probes FITC-DEVEFMK and FITC-LEHD-FMK (Biovision, Milpitas, CA), which bind irreversibly to active caspase-3 and -9, respectively. The PLT samples were diluted with Buffer A (1 mmol/L MgCl2, 5.6 mmol/L glucose, 0.1% bovine serum albumin, and 10 mmol/L HEPES in PBS, pH 7.4) to PLT count of 200 × 109 PLTs/L in total volume of 50 μL. Four microliters of FITC-DEVE-FMK or FITC-LEHD-FMK, diluted 1:10 in Buffer A, were then added and incubated for 25 minutes in dark at 37°C. PLT samples diluted to a total volume of 1 mL with Buffer A were analyzed on a flow cytometer (FACSCanto II, BD Biosciences). In parallel, a fresh prepared PLT-rich plasma sample was run to set a gate for analysis.

Statistical analysis Statistical analyses were performed using two-way analysis of variance with repeated measures, and Bonferroni post hoc analyses were conducted using computer software (GraphPad Prism 5, GraphPad, Inc.,

San Diego, CA). A p value of less than 0.05 was considered significant.

RESULTS In vitro measurements of PLT quality: during storage, after RF-PI treatment with and without kinase inhibitors The in vitro quality results obtained from units containing the SB inhibitor were comparable to the data from a previous study that we carried out in full-sized storage bags.24 Alterations in PLT metabolism were monitored by pH, glucose consumption, and production of lactate during storage. As shown in Table 1, acceleration of PLT metabolism after RF-PI was demonstrated by increased glucose consumption, which led to increased lactate production and a concomitant decrease in pH in RF-PI–treated units with or without DMSO compared with the untreated control. In the other study arms, the PD and Y inhibitors did not have a noticeable effect on PLT metabolism compared to the RF-PI–treated samples without inhibitors or to the vehicle control. However, the SB inhibitor showed a significant deceleration of metabolism indicated by a reduced glucose consumption, and consequently lower lactate production resulting in a decreased pH compared to the treated DMSO control. Flow cytometry analyses showed that the SB inhibitor mediated a significant reduction of CD62P expression compared to the vehicle control sample with treatment during storage. Again, the treated units in presence of PD or Y inhibitor did not show a significant reduction of CD62P expression compared to RF-PI treatment without inhibitor. The annexin V binding assay was used to monitor development of PLT apoptosis. As shown in Table 1, annexin V binding to the PLT surface increased in untreated and treated samples during storage and further increased upon RF-PI treatment. The SB inhibitor–treated units, but not the PD- or Y-treated units, demonstrated a significant reduction of PS exposure at Day 7 compared to treated units without inhibitor.

Apoptotic events triggered by RF-PI treatment To assess apoptotic events triggered by RF-PI treatment in apheresis PLT concentrates, Western blot and flow cytometry analyses were used to study expression levels of Bcl-2 family proteins and the activity of caspases, respectively. The immunoblot analyses showed no significant changes in the protein expression level of antiapoptotic protein Bcl-xL in both study arms during storage (Fig. 1A, white and black bars). However, expression levels of two proapoptotic proteins, Bax and Bak, significantly increased in RF-PI treatment samples on Day 7 compared to untreated control (Figs. 1B and 1C, white and black bars). Similarly, flow cytometry analyses showed that the Volume **, ** **

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p < 0.05 d

p < 0.05 compared to Day 5 vehicle controls; c

p < 0.001 compared to Day 7 vehicle controls; b

tration of the inhibitors was 10 μmol/L. a p < 0.001 compared to Day 5 vehicle controls; compared to Day 7 vehicle controls; e p < 0.05 compared to Day 7 vehicle controls.

* Sample analyses occurred 1 day after production (Day 1) and after 5 and 7 days of storage. Results are displayed as means of three independent replicates and the respective SD. The final concen-

8.1 ± 5.7 7.26 ± 0.03b 11.7 ± 2.3 12.5 ± 1.4b 24.7 ± 6.3d 9.1 ± 5.4e 11.6 ± 6.8 7.03 ± 0.06 9.6 ± 2.2 16.0 ± 1.1 56.8 ± 10.5 11.2 ± 5.8 11.8 ± 3.6 6.98 ± 0.15 8.9 ± 3.0 15.2 ± 0.8 63.8 ± 8.8 14.0 ± 8.0 7.36 ± 0.01 16.6 ± 1.6 2.9 ± 0.2 22.1 ± 17.1 6.7 ± 3.0 7.33 ± 0.02a 13.2 ± 1.9 9.2 ± 0.9a 22.9 ± 5.3c 7.32 ± 0.03 16.6 ± 1.9 3.4 ± 0.1 41.1 ± 6.0 10.5 ± 10.3 7.19 ± 0.03 11.7 ± 1.9 11.9 ± 0.5 51.5 ± 0.3 7.32 ± 0.02 16.5 ± 1.9 3.3 ± 0.1 41.1 ± 8.0 12.6 ± 10.9 7.18 ± 0.03 12.0 ± 1.9 12.1 ± 0.9 49.4 ± 5.3

7.6 ± 3.2e 12.9 ± 7.3 12.7 ± 5.9 7.2 ± 2.9 7.20 ± 0.04b 11.3 ± 2.1 13.9 ± 0.5b 50.1 ± 7.2 10.8 ± 6.5 7.04 ± 0.06 9.8 ± 2.4 16.1 ± 1.0 59.9 ± 2.6 9.8 ± 4.1 7.03 ± 0.04 9.6 ± 2.0 15.9 ± 0.9 56.4 ± 2.8 7.35 ± 0.04 17.0 ± 1.9 3.0 ± 0.1 31.2 ± 15.1 7.3 ± 3.7 7.30 ± 0.03a 13.1 ± 1.9 10.0 ± 0.3a 33.4 ± 10.3 7.32 ± 0.02 16.8 ± 1.8 3.4 ± 0.1 36.1 ± 8.2 7.9 ± 4.8 7.20 ± 0.04 11.9 ± 1.8 12.0 ± 0.4 51.7 ± 2.9 7.32 ± 0.02 16.3 ± 1.6 3.3 ± 0.1 36.0 ± 5.1 15.9 ± 11.8 7.19 ± 0.03 11.9 ± 1.9 11.7 ± 0.3 52.4 ± 1.3

pH pH pH Treatment

4

Control RF-PI Vehicle (RB/UV) SB (RB/UV) PD (RB/UV) Y (RB/UV)

Day 7

Glucose (mmol/L) Annexin V CD62P (%) (%) Day 5

Lactate (mmol/L) Glucose (mmol/L) Annexin V (%) CD62P (%) Lactate (mmol/L) Glucose (mmol/L)

Day 1

TABLE 1. Summary of routine PLT quality measures using a variety of in vitro assays*

Lactate (mmol/L)

CD62P (%)

Annexin V (%)

CHEN ET AL.

caspase-3 and -9 activities were also increased in untreated and treated samples during storage (Figs. 1D and 1E, white and black bars), and their activities are significantly increased in RF-PI treatment compared to control. To verify the results obtained from the flow cytometry assay, immunoblot analyses were used to assess the degree of cleaved caspase-3 and -9 during storage by monitoring the 17- and 37-kDa bands, respectively. The results also demonstrated that these caspase activities were significantly increased in treated samples compared to the untreated control, which is in agreement with the flow cytometry data (Fig. 1G and 1F, white and black bars).

Inhibitors modulate PLT apoptotic events upon RF-PI treatment Previous studies in our laboratory revealed that phosphorylation levels of a variety of kinases are elevated after RF-PI treatment. To assess their involvement in the development of apoptosis triggered by RF-PI treatment, the effect of inhibitors SB, PD, and Y administered to PLT concentrates before RF-PI treatment was investigated. Immunoblot analyses showed that the increased expression levels of proteins Bax and Bak triggered by the RF-PI treatment were significantly reduced in the samples with the SB inhibitor compared to vehicle control, but not in treated units with the PD or Y inhibitor (Figs. 1B and 1C) even when the concentration of the PD or Y inhibitors was increased to 20 μmol/L (data not shown). Furthermore, there was no significant difference in the protein level of the antiapoptotic Bcl-xL throughout storage with or without inhibitors (Fig. 1A). The assay using flow cytometry revealed that the SB inhibitor significantly reduced the caspase-3 and -9 activities induced by RF-PI treatment during storage (Figs. 1D and 1E). However, the PD or Y inhibitor did not show a significant difference compared to vehicle controls. To verify these results, the cleavage activity of the two caspases were assessed by monitoring their cleaved products appearing as 17- and 37-kDa bands derived from caspase-3 and -9, respectively (Figs. 1F and 1G). The results showed that the SB inhibitor significantly reduced these cleavage activities compared to vehicle controls. The PD or Y inhibitor did not show significant reduction of caspase-3 and -9 activities in flow cytometry and immunoblot analyses even when the concentration of the PD or Y inhibitors was increased to 20 μmol/L (data not shown).

Verification of the specificity of selected inhibitors To verify the specificity of the inhibitors, the phosphorylation profiles of p38MAPK, ERK (downstream of MEK kinase), and myosin light chain kinase (MLCK, downstream of ROCK kinase) were analyzed in RF-PI–treated units with and without inhibitors. Representative sets of

APOPTOSIS IN AP/PC AFTER PI

Fig. 1. Flow cytometry and Western blot analyses for Bcl-2 family protein expression levels and caspase activities. Representative sets of immunoblot analyses showing the protein expression levels of antiapoptotic protein Bcl-xL (A), proapoptotic proteins Bax (B) and Bak (C), the caspase-3–cleaved band (17 kDa, D), and the caspase-9 cleaved band (37 kDa, E) and flow cytometry analyses displaying the caspase-3 (F) and caspase-9 (G) activities showing apheresis PLTs kept untreated (□) or treated with RF-PI without inhibitor (■), with vehicle of DMSO ( ), with p38MAPK inhibitor SB ( ), with MEK inhibitor PD ( ), or with ROCK inhibitor Y ( ) on Days 1, 5, and 7 during blood banking storage. A representative actin blot is shown in the inset as loading control. *Significance (p < 0.05) of three independent experimental sets.

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immunoblots for the total amounts of p38MAPK (Fig. 2A), ERK (Fig. 2C), and MLCK (Fig. 2E) did not show a significant change in untreated control and treated units with or without inhibitors, respectively. However, the phosphorylation levels of these kinases were significantly decreased in the respective units with the inhibitors SB, PD, and Y after treatment throughout storage (Figs. 2B, 2D, and 2F).

DISCUSSION While the application of PI to blood products should provide a significant improvement in the safety profile of PLT concentrates, it also results in a reduction in PLT in vitro quality highlighted by accelerated PLT storage lesion development and decreased transfusion outcome.25-27 The investigation of the biochemical mechanisms involved in the PLT deterioration upon PI treatment might provide helpful insights to design strategies to ameliorate this technology. RF-PI treatment targets the RNA or DNA of pathogens; however, as PLTs contain mRNA this illumination might also impact PLT function.28-31 The procedure creates a similar situation to cells challenged by stress, triggering the activation of a variety of protein kinases, called stressactivated kinases, such as p38MAPK, ERK, and c-JUN.32-34 Immunoblot analyses in this study using the RF-PI system monitored the phosphorylated levels of protein kinases and showed that p38MAPK, ERK, and MLCK were significantly activated in PLTs during storage after RF-PI treatment. The p38MAPK and ERK activation upon RF-PI treatment is in agreement with our previous work.22 In addition, this is the first time that the MLCK activation has been reported in RF-PI–treated PLTs during storage, further expanding our knowledge of kinase activation profiles in PLTs triggered by RF-PI treatment. However MLCK phosphorylation levels in other PI systems and its connection to PLT function needs to be analyzed further. The development of apoptosis has been described in stored PLTs.15,35 There is an ongoing debate whether PSL and apoptosis development are linked.36 Studies by Li and colleagues18 suggested that PLT activation does not impact caspase activation and therefore apoptosis; however, more recent studies have shown that thrombin triggers activation of Bcl-2 family proteins.12 Finally, a recent study has shown that the inhibition of caspase-3 leads to improved PLT function throughout storage.37 Further investigations are necessary to solve this puzzle. In this study, we investigated the development of apoptosis events in apheresis PLT concentrates by comparing untreated PLTs with RF-PI–treated PLTs throughout storage. Immunoblot analyses showed that the proapoptotic proteins Bax and Bak significantly increased in the treated sample compared to control on Day 7, but not the antiapoptotic protein Bcl-xL. As a result of 6

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increased expression of proapoptotic proteins, caspase-3 and -9 activities were also significantly increased after RF-PI treatment. From this observation we concluded that PLT apoptosis occurred in apheresis PLT concentrates during storage and was significantly increased by RF-PI treatment during storage. This finding is consistent with a recent study showing increased expression of proapoptotic proteins Bax and Bak and caspase-3 activity as well as gelsolin and ROCK cleavage in PLT concentrates produced using the buffy coat method and stored in additive solution treated by RF-PI.21 In addition, apoptosis development in RF-PI–treated PLTs during storage is also in agreement with the classic PLT apoptosis model, with increased expression of proapoptotic Bax and Bak proteins, caspse-3 activation, and PS exposure documented in PLTs stimulated with the strong agonist thrombin.12 p38MAPKs are activated by stress stimuli, such as UV irradiation, heat shock, and osmotic shock in mammalian cells and might mediate tumor cell apoptosis after photostimulation.38,39 Some studies have reported that p38MAPK is phosphorylated by PLT activation using agonists, such as collagen,40,41 thrombin,42 and ADP.43 This study and our previous findings identified p38MAPK as a key player in PLT function, which is activated upon RF-PI treatment. This is demonstrated by the finding that inhibition of this kinase by SB before illumination resulted in a reduction of metabolic activity, alpha granule release, and PS externalization. Not many kinases are known to be involved in apoptosis, especially in PLTs. However, the kinase Akt is known to suppress apoptosis development in mammalian cells upon phosphorylation or activation.44 However, to our knowledge this concept is not described in PLTs, so it can only be subject to speculation. The initial signaling trigger of PI treatment that activates p38MAPK is unknown, but even if multiple pathways are activated leading to activation and inhibition of apoptosis development, the p38MAPK cascade seems to be dominant. An in vivo study with normal subjects evaluating the efficacy of the RF-PI system has shown a significant reduction in both recovery and survival.45 However, it was concluded as these capabilities are not dissimilar to licensed PLT components, RF-PI–treated PLTs have sufficient hemostatic capacity to support patients with thrombocytopenia. A subsequent clinical trial revealed a significant lower corrected count increment (CCI) for the treated PLT units compared to the untreated control units, but the study failed to show noninferiority of illuminated units over control on predefined CCI criteria.46 Collectively, these data suggest that while biochemical processes including apoptosis are affected by PI treatment, their impairment does not translate into significant clinical impact at least in nonbleeding patients with hypoproliferative thrombocytopenia. Whether these reductions in in vivo characteristics are due to the development of apoptosis is unclear, but studies in mice have

APOPTOSIS IN AP/PC AFTER PI

Fig. 2. Immunoblot analyses of selected protein kinases targeted by specific inhibitors. Representative sets of Western blot analyses displaying the relative protein levels of p38MAPK (A), phosphorylation of p38MAPK (P-p38, B), ERK (downstream of MEK kinase, C), phosphorylated ERK (P-ERK, D), MLCK (downstream of ROCK kinase, E), and phosphorylated MLCK (P-MLCK, F) showing apheresis PLTs kept untreated (□) or treated with RF-PI without inhibitor (■), with vehicle of DMSO ( ), with p38MAPK inhibitor SB ( ), with MEK inhibitor PD ( ), or with ROCK inhibitor Y ( ) on Days 1, 5, and 7 during blood banking storage. A representative actin blot is shown in the inset as loading control. *Significance (p < 0.05) of three independent experimental sets.

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demonstrated that PLTs treated with the p38MAPK inhibitor SB used in this study exhibit improved in vivo recovery and survival after transfusion by inhibition of shedding of glycoprotein receptors GPIb-alpha and GPV.47 The flow cytometry and immunoblot analyses showed that the apoptotic events were effectively inhibited in RF-PI–treated PLTs in the presence of the SB inhibitor compared to the vehicle control, including the expression of proteins Bax and Bak and caspase-3 and -9 activities. However, PD or Y inhibitor treatment did not result in a significant difference in PLT quality and development of PLT apoptosis compared to vehicle even when the concentration of the PD or Y inhibitor was doubled. This finding indicates that p38MAPK, but not MEK or ROCK kinase, is involved in development of PLT apoptosis triggered by PI during storage. The link between p38MAPK and apoptosis in stored PLTs is further supported by a study using a different p38MAPK inhibitor, SB202190, which showed that p38MAPK is essential in PLT apoptosis induced by ABT737, but not in PLTs with apoptotic-like events induced by a strong agonist mixture of thrombin plus convulxin, although p38MAPK was activated in both models.48 As the antiapoptotic protein Bcl-xL is essential in the regulation of the PLT life span in vivo,10 ABT-737 is a chemical drug mimic of BH3-only proteins, which triggers Bax- and Bakmediated apoptosis against antiapoptotic proteins such as Bcl-2, Bcl-xL, and Mcl-1.49 Our results showed that RF-PI– triggered PLT apoptosis seems to be regulated by p38MAPK through activation of PLTs to synthesize the proapoptotic proteins Bax and Bak, which is similar to ABT-737–induced cell apoptosis. Although as anucleate cells it was initially believed that PLTs cannot synthesize proteins, we now know that PLTs synthesize quite a number of proteins. After thrombin activation, PLTs synthesize the Bcl-3 protein, which can be blocked by the translation inhibitor rapamycin.50 This finding was further supported by a research reported that PLTs synthesize plasminogen activator inhibitor-1 upon thrombin stimulation and the synthesis can be inhibited by puromycin.51 However, the biologic mechanism involved in the increase of protein level of the proapoptotic proteins Bax and Bak and reverse by p38MAPK inhibitor, SB, after RF-PI treatment during storage needs further investigations. In summary, we have further defined that p38MAPK plays an essential role in PSL upon RF-PI treatment during storage. These findings not only identified p38MAP-kinase as a signaling protein in the apoptotic pathway, but also further expanded our understanding of the molecular mechanisms triggered by PI treatment by the identification of protein targets to hopefully improve the PLT quality. ACKNOWLEDGMENTS The authors thank the Canadian Blood Services (CBS) Development Laboratory (NetCAD) for providing processed blood PLT 8

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concentrates and Dr Geraldine Walsh for critical reading of the manuscript. The views expressed herein do not necessarily represent the view of the federal government of Canada. ZC is the recipient of a Canadian Blood Services Postdoctoral Fellowship award. ZC performed the research, analyzed the data, and wrote the manuscript; PS designed the research, analyzed the data, and wrote the manuscript; BC performed the research; and DVD interpreted the data and wrote the manuscript.

CONFLICT OF INTEREST The authors have no conflict to declare. Reagents, disposables, and the Mirasol instrumentation were provided by TerumoBCT. TerumoBCT was not involved in the study design and provided no editorial control over the research or manuscript.

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p38MAPK is involved in apoptosis development in apheresis platelet concentrates after riboflavin and ultraviolet light treatment.

Pathogen inactivation (PI) accelerates the platelet (PLT) storage lesion, including apoptotic-like changes. Proteomic studies have shown that phosphor...
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