BLOOD COMPONENTS Decreasing phosphodiesterase 5A activity contributes to platelet cGMP accumulation during storage of apheresis-derived platelet concentrates Anna Kobsar, Evelyn Putz, Pinar Yilmaz, Elke Weinig, Markus Boeck, and Juergen Koessler

BACKGROUND: Platelet storage lesion (PSL) considerably decreases the quality of platelets (PLTs) in concentrates characterized by a loss of signaling responses to agonists and impaired PLT activation, secretion, and aggregation. To understand the role of inhibitory signaling pathways in the mechanism of PSL, the basal state of the cyclic nucleotide (CN)-dependent signaling systems in stored PLTs was investigated. STUDY DESIGN AND METHODS: Whole blood samples (WB) and apheresis-derived PLT concentrates (APCs) were obtained from healthy volunteers. Washed PLTs were prepared from WB on Day 0 and from APCs on Days 0, 2, and 5. The basal phosphorylation of the vasodilator-stimulated phosphoprotein (VASP) and phosphodiesterase 5A (PDE5A) levels were quantified by Western blot. CN and PDE5A activity were measured by enzyme-linked immunoassay kits. Fibrinogen binding and aggregation were measured in PLT-rich plasma of WB or APC samples. Unpaired t test was used for statistical analysis. RESULTS: Basal VASP phosphorylation levels were comparable in WB and APCs on Day 0. VASP phosphorylation increased significantly during storage of APCs, more pronounced at Ser239 than at Ser157. Similarly, intracellular cGMP, but not cAMP, concentration continuously increased in stored PLTs, whereas PDE5A levels and activity significantly decreased accompanied by diminished thrombin receptor activator peptide 6–induced fibrinogen binding and aggregation. CONCLUSION: Storage of APCs leads to intracellular cGMP accumulation that could be caused by degradation of PDE5A. Enhanced cGMP level supports subsequent cGMP-dependent protein kinase–mediated increase of VASP phosphorylation resulting in reduced fibrinogen binding and aggregation.

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latelet storage lesion (PSL) occurring during platelet (PLT) concentrate production and storage considerably decreases the quality of PLTs used for transfusion. Alterations of activating signal cascades lead to the loss of PLT response to physiologic agonists with impaired integrin activation, secretion, or aggregation.1 Studies of apheresis-derived PLT concentrates (APCs) revealed an accumulation of soluble P-selectin, PLT factor 4,2 CD62P-positive PLTs,3,4 and increasing intracellular Ca2+ levels.5 Flow cytometric analysis of PLTs in stored APCs show decreased expression of surface glycoproteins GPIIb/IIIa and GPIb,6,7 accompanied by the loss of highaffinity thrombin receptors on PLT surface.8,9 Further studies reported that cytoskeleton proteins like talin and kindlin-3, which are key promoters triggering the conformational change of the αIIbβ3 complex, develop early alterations in the first 24 hours of APC storage.4 Besides, an increased abundance of tyrosine-protein kinase Src and LIM domain protein 1 in the cytosolic fraction of stored PLTs were found.4

ABBREVIATIONS: APC(s) = apheresis-derived platelet concentrate(s); CN = cyclic nucleotide; NO = nitric oxide; PDE5A = phosphodiesterase 5A; PKA = cAMP-dependent protein kinase; PKG = cGMP-dependent protein kinase; PRP = platelet-rich plasma; PSL = platelet storage lesion; TRAP-6 = thrombin receptor activator peptide 6; VASP = vasodilator-stimulated phosphoprotein; WB = whole blood. From the Institute of Transfusion Medicine and Haemotherapy, University of Wuerzburg, Wuerzburg, Germany. Address correspondence to: Juergen Koessler, MD, Institute of Transfusion Medicine and Haemotherapy, University of Wuerzburg, Oberduerrbacher Straße 6, D-97080 Wuerzburg, Germany; e-mail: [email protected] Received for publication April 27, 2013; revision received June 12, 2013, and accepted June 12, 2013. doi: 10.1111/trf.12360 TRANSFUSION 2014;54:1008-1014.

cGMP ACCUMULATION IN STORED PLTs

In summary, recent experimental data about stored human PLTs have mostly focused on (pre-)activating mechanisms during production and storage of PLT concentrates. In contrast, alterations of inhibitory signaling pathways have not yet been thoroughly investigated. The most essential endogenous mechanism of PLT inhibition is mediated by cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG) regulated by intracellular cAMP and cGMP concentrations.10-12 The vasodilator-stimulated phosphoprotein (VASP) is one of the major common substrates for both PKA and PKG in PLTs and its phosphorylation is a sign of PLT inhibition.11,12 In this study, we examined these inhibitory PLT pathways and their influence on the functionality of stored PLTs to improve the understanding of the processes contributing to PSL.

MATERIALS AND METHODS Materials Thrombin receptor activator peptide 6 (TRAP-6) was from BACHEM (Bubendorf, Switzerland). Rabbit monoclonal anti-pan-actin and rabbit polyclonal phosphodiesterase 5A (PDE5A) antibodies were from New England Biolabs GmbH (Frankfurt am Main, Germany). Phospho-VASP Ser239 and phospho-VASP Ser157 antibodies were from Nanotools (Teningen, Germany). Horseradish peroxidase– conjugated goat anti-rabbit and anti-mouse antibodies were from Bio-Rad Laboratories, Inc. (Muenchen, Germany).

Blood collection and apheresis-derived PLT concentrates Venous whole blood samples (WB) and APCs were obtained from seven informed healthy volunteer donors (four male, three female, aged 24-46 years). Peripheral blood was collected in polystyrene tubes containing 3.2% citrate buffer (106 mmol/L trisodium citrate, Sarstedt, Nuembrecht, Germany) before the APC donation. APCs (2.5 × 1011 PLTs in 250 mL of plasma) were collected using an automated blood collection system (Trima Accel, with software Version 5.1 and the Trima Accel LRS PLT, plasma set, CaridianBCT, now TerumoBCT, Lakewood, CO) according to current guidelines and the approval of authorities. The ratio of inlet blood volume to anticoagulant (ACD-A) was 10:1. On Days 0 (2-3 hr after completed apheresis), two and five samples from APCs were taken for analysis under sterile conditions. Analysis of WB samples on Day 0 was started within 1 hour after blood collection. Our studies with human PLTs were approved by the local ethics committee of the University of Wuerzburg.

The study was performed according to our institutional guidelines and to the Declaration of Helsinki.

Preparation of washed PLTs Washed PLTs were prepared as described.11 A total of 3 mmol/L EGTA was added to WB to prevent PLT activation. PLT-rich plasma (PRP) was obtained by centrifugation of WB at 330 × g for 5 minutes. Subsequently, samples of PRP (or APCs after addition of 3 mmol/L EGTA) were centrifuged at 430 × g for 10 minutes. Pelleted PLTs were then washed once in CGS buffer (120 mmol/L sodium chloride, 12.9 mmol/L trisodium citrate, 30 mmol/L d-glucose, pH 6.5) and resuspended in HEPES buffer (150 mmol/L NaCl, 5 mmol/L KCl, 1 mmol/L MgCl2, 10 mmol/L d-glucose, 10 mmol/L HEPES, pH 7.4) to the final appropriate concentration. After resting for 15 minutes in a 37°C water bath, washed PLTs were used for the experiments. Protein concentrations in the samples were measured with BCA protein assay reagent following the manufacturer’s instructions (Thermo Fisher Scientific p/a Perbio Science German, Bonn, Germany).

Western blot analysis PLT lysates containing 2 μg protein were loaded onto the gel, separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred onto nitrocellulose membranes. The membranes were incubated with appropriate primary antibodies overnight at 4°C. For visualization of the signal, goat anti-rabbit or anti-mouse immunoglobulin G conjugated with horseradish peroxidase were used as secondary antibodies, followed by detection with an ECL detection kit (GE Healthcare, Piscataway, NJ). Blots were analyzed densitometrically using computer software (NIH Image J, http://rsbweb .nih.gov/ij/) for uncalibrated optical density.

cAMP and cGMP measurement For cAMP and cGMP determination, the PLT concentration was adjusted to 3 × 108 PLTs/mL. A total of 100 μL of the PLT suspension was used for each measurement. Samples were stopped with 5% trichloroacetic acid. After extraction with ether cAMP and cGMP in washed PLTs were detected by cAMP (enzyme-linked immunoassay) EIA kit and GMP EIA kit, respectively, following the manufacturer’s instructions (Cayman Chemical, Hamburg, Germany).

PDE5A activity measurement PLT PDE5A activity was measured using the PDE5A enzyme assay kit from Biomol GmbH (Hamburg, Germany) with fluorescence-labeled cGMP on a multilabel Volume 54, April 2014

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plate reader (Victor 1420, Perkin Elmer, Waltham, MA) following the manufacturer’s instructions.

Flow cytometry analysis

PLT aggregation PLT aggregation was measured using an aggregometer (APACT 4004, LabiTec, Ahrensburg, Germany). PRP or material from stored APCs (diluted with plasma to PRP PLT concentration) were stimulated with 5 μmol/L TRAP-6. Aggregation was measured for 5 minutes under continuous stirring at 1000 rpm and 37°C.

Statistical analysis All experiments were performed at least in triplicate and data shown are means ± standard error of the mean (SEM). The n values refer to the number of experiments, each made with different blood donors. Differences between groups were analyzed by unpaired t test. p values of less than 0.05 were considered significant.

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Ten microliters of PRP or material from stored APCs (diluted with plasma to achieve a PLT concentration comparable to that in PRP) were incubated with 10 μL of mouse anti-fibrinogen fluorescein isothiocyanate (FITC)conjugated antibody and then stimulated for 2 minutes with 5 μmol/L TRAP-6. Samples were stopped with 0.1% formaldehyde, diluted with 400 μL of PBS-5 mmol/L glucose-0.5% bovine serum albumin and subsequently analyzed on a flow cytometer (FACSCalibur, Becton Dickinson, Franklin Lakes, NJ) using computer software (CELLQuest, Version 6.0, Becton Dickinson). The PLT population was identified by its forward and side scatter distribution and 20,000 events were analyzed for mean fluorescence.

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Fig. 1. The basal VASP phosphorylation increases in stored human PLTs. Quiescent washed human PLTs (1 × 108/mL) from WB and from APC on Days 0, 2, and 5 were lysed with Laemmli buffer and analyzed on Western blot for VASP Ser239 (A) and VASP Ser157 (B) phosphorylation. After scanning, bands were quantified by the Image J program. VASP phosphorylation in the samples was normalized to actin control. Blots are representative of six independent experiments. Results are means ± SEM; n = 6; *p < 0.05 compared to WB PLTs and PLTs of APCs on Day 0.

RESULTS Basal PLT VASP phosphorylation increases during APC storage Western blot analysis of basal VASP phosphorylation at Ser239 and at Ser157 did not show significant differences in washed PLTs derived from WB and in washed PLTs derived from APCs on Day 0 (Fig. 1). However, during APC storage, there was a significant increase of the basal VASP phosphorylation at Ser239 by approximately 87% on Day 2 and almost by fivefold on Day 5 compared to Day 0 (Fig. 1A). The changes of basal VASP phosphorylation at Ser157 were weaker than at Ser,239 but showed the same tendency with an increase of 15% on Day 2 and of 74% on Day 5 in the course of APC storage (Fig. 1B).

Intracellular cGMP levels increase in stored PLTs As VASP phosphorylation in PLTs is regulated by cAMPand cGMP-dependent protein kinase activities, intracellu1010

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lar cAMP and cGMP levels were measured. There were no significant differences in the basal cGMP and cAMP concentrations between washed PLTs of WB and APCs on Day 0 (Fig. 2). During storage, cGMP levels continually increased until Day 5 when they reached a significant elevation of 56% compared to Day 0 (Fig. 2A). In contrast, cAMP values were unchanged during the investigated period of time (Fig. 2B).

PDE5A level and activity continually decrease in stored PLTs The removal of PLT cGMP is regulated by PDE5A, a specific cGMP-degrading enzyme.13-15 Western blot analysis showed that the level of intracellular PDE5A was similar in PLTs of WB and in APCs on Day 0 (Fig. 3A). On Days 2 and 5, the PDE5A levels were decreased by 30 and 40%, respectively (Fig. 3A), indicating that increased cGMP

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Fig. 2. The basal cGMP, but not cAMP, concentration increases in stored human PLTs. Washed human PLTs (3 × 108/mL) from WB and from APCs on Days 0, 2, and 5 were stopped with 5% trichloroacetic acid. After extraction with ether, the contents of CNs cGMP (A) and cAMP (B) were measured with the cGMP or the cAMP EIA kit according to manufacturer’s instructions. Results are presented as fold accumulation compared to CN levels in PLTs of APCs on Day 0 (mean ± SEM; n = 7; *p < 0.05 compared to WB PLTs and PLTs of APCs on Day 0).

concentrations in stored PLTs were caused by reduced cGMP degradation due to lower PDE5A activity. Direct measurement of PDE5A activity demonstrated that on Day 2 of APC storage, the enzyme activity was significantly decreased by 17% compared to PLTs from WB, whereas it was decreased by 23% on Day 5 (Fig. 3B).

TRAP-6–induced fibrinogen binding and PLT aggregation decrease in stored PLTs Fibrinogen binding and aggregation were measured in freshly prepared PRP and stored APCs to prove the influ-

Fig. 3. The level (A) and activity (B) of PLT PDE5A decrease during storage of APCs. (A) Quiescent washed human PLTs (1 × 108/mL) from WB and from APCs on Days 0, 2, and 5 were lysed with Laemmli buffer and analyzed on Western blot for total PDE5A. After scanning, bands were quantified by the Image J program. PDE5A levels in the samples were normalized to actin control. Blots are representative of seven independent experiments (mean ± SEM; n = 7; *p < 0.05 compared to WB PLTs and PLTs of APCs on Day 0). (B) Quiescent washed human PLTs (1.5 × 109 PLTs in 30 μL of HEPES buffer) from WB and from APCs on Days 0, 2, and 5 were lysed with 250 μL of lysis buffer according to PDE5A enzyme assay kit instructions. The activity of PDE5A was assessed in 5 μg of PLT lysate. Results are presented as fold stimulation compared to PDE5A activity in freshly isolated washed PLTs from WB (mean ± SEM; n = 6; *p < 0.05, compared to PRP from WB).

ence of increased cyclic nucleotide (CN) levels and enhanced VASP phosphorylation on PLT function. Mean fluorescence of TRAP-6–stimulated fibrinogen binding decreased by approximately 14 and 23.5% in APCs on Days 2 and 5, respectively (Fig. 4A). Basal fibrinogen binding in freshly prepared PRP (mean fluorescence intensity [MFI], 16.4 ± 2.4) was comparable to stored APCs (MFI, 16.5 ± 3.7 on Day 0, 18.4 ± 3.4 on Day 2, and 19.2 ± 2.3 on Day 5). Volume 54, April 2014

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Fig. 4. TRAP-6–induced fibrinogen binding and aggregation decrease in stored PLTs. (A) PRP or material from stored APCs (diluted with plasma to match PRP PLT concentrations) were incubated with equal volumes of mouse anti-fibrinogen FITCconjugated antibody and then stimulated for 2 minutes with 5 μmol/L TRAP-6. Samples were stopped with formaldehyde and analyzed by flow cytometry (mean ± SEM; n = 5; *p < 0.05). (B) The mean values of 5 μmol/L TRAP-6–induced aggregation (final aggregation after 5 min) in PRP and in APCs on Days 0, 2, or 5 are shown (mean ± SEM; n = 5; *p < 0.05, compared to PRP from WB). [Correction added after online publication 02-Aug-2013: PLR on axis has been updated to PLT.]

Accordingly, TRAP-6–induced aggregation decreased from 84.6 ± 2.8% (PRP) and 86.5 ± 4.0% (APC on Day 0) to 69.3 ± 9.5% (APC on Day 2) and to 44.6 ± 10.1% (APC on Day 5; Fig. 4B).

DISCUSSION The understanding of alterations in PLT signaling during storage is fundamental for the identification of novel approaches to avoid or to reduce storage lesions. In our study, we investigated the basal condition of inhibitory PLT cAMP- and cGMP-signaling pathways in stored APCs. 1012

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An elevation of cGMP and cAMP levels in PLTs induces VASP phosphorylation and consecutively PLT inhibition. Storage lesions are usually attributed to increased (pre-) activation of PLTs in the artificial milieu of PLT concentrates.2,4,6 Therefore, we expected to find decreased levels of basal PLT VASP phosphorylation in APCs under storage. However, in contrast to that assumption, our experimental data show a continuous and significant increase of basal VASP phosphorylation at both Ser239 (fivefold compared to Day 0) and Ser157 (almost twofold compared to Day 0) over 5 days of storage (Fig. 1). A growing intensity of VASP phosphorylation at Ser239 and at Ser157 was also found in a recently published proteomics study with stored APCs.16 The accumulation of phosho-VASP in PLTs is usually a result of CN-dependent activation of PKA and PKG.10-12 Both of these kinases are able to phosphorylate VASP at Ser157 and Ser239; however, PKA phosphorylates VASP preferentially at Ser157 and consecutively at Ser239, whereas PKG prefers Ser239 and thereafter Ser157.10 To reveal the mechanism facilitating VASP phosphorylation in stored PLTs, the intracellular levels of cAMP and cGMP were assessed. The concentration of PLT cAMP did not significantly change during storage of APCs (Fig. 2B). In contrast, cGMP levels continuously increased and were elevated by 50% on Day 5 of storage (Fig. 2A). This indicates that increased VASP phosphorylation was initiated by cGMP-dependent PKG activation. Besides, basal VASP phosphorylation at Ser239 was much more pronounced than at Ser157, also providing evidence for PKG activation rather than PKA activation. The alterations were time dependent with an ongoing increase of cGMP levels and VASP phosphorylation during storage. Soluble guanylyl cyclase is responsible for cGMP production in PLTs after stimulation with nitric oxide (NO).17 Earlier studies demonstrated that human PLTs do not contain NO synthases—neither endothelial nor inducible NO synthases or their mRNA.18 That means that the increase of intracellular cGMP concentration in stored PLTs might not result from de novo cGMP synthesis. Instead, alterations in the feedback regulation of cGMP levels could be causative. A negative feedback regulation of cGMP in PLTs is provided by specific PDE-mediated degradation. Human PLTs express three different PDEs: PDE2A, PDE3A, and PDE5A,13 but only PDE5A specifically degrades cGMP.13-15 Our Western blot data showed a progressive reduction of PDE5A in PLTs under storage resulting in a significant and almost twofold decrease of PLT PDE5A on Day 5 (Fig. 3A). These data correspond to observed changes of PDE5A activity with a continuous decrease by 17 and 23% on Days 2 and 5, respectively (Fig. 3B). Obviously, PLTs are exposed to an accelerated catabolism of PLT PDE5A during storage, resulting in decreased PDE5A activity and consecutively in the accumulation of intracellular cGMP and enhanced VASP phosphorylation.

cGMP ACCUMULATION IN STORED PLTs

Recently, studies showed that levels of PLT thrombin receptors, PAR-1 and PAR-4, do not begin to decrease before 5 days of storage9 so that we used TRAP-6, an activator of thrombin PAR-1 receptor, for testing PLT activation over the time of storage. TRAP-6–induced fibrinogen binding and PLT aggregation permanently decreased during storage of APCs (Fig. 4), indicating that the increase of VASP phosphorylation is accompanied by a reduction of PLT reactivity. These observations are in accordance to previous studies, which showed that phosphorylated VASP keeps PLT GPIIb/IIIa in the resting conformation supporting the inhibition of fibrinogen binding, adhesion, and aggregation.19,20 It should be noted that our study has some limitations. Numerous morphologic and biochemical changes appear during PLT storage that could additionally influence cGMP levels and PLT function.3,4,6 Furthermore, the study was performed with apheresis-derived PLT concentrates. It would be of interest to determine whether the observed alterations are also reproducible in pooled PLT concentrates as a general phenomenon of storage lesion. In summary, the study investigated the role of CN-dependent inhibitory signaling pathways in stored PLTs. In conclusion of our results, production and storage of APCs might disturb an intrinsic regulation of PLT PDE5A turnover, resulting in its degradation, followed by cGMP accumulation and increased basal VASP phosphorylation, eventually contributing to functional impairment of stored PLTs. Further studies are required to elucidate the molecular mechanisms affecting PDE5A degradation as well as the functional and clinical significance of these alterations.

3. Holme S, Sweeney JD, Sawyer S, Elfath MD. The expression of p-selectin during collection, processing, and storage of platelet concentrates: relationship to loss of in vivo viability. Transfusion 1997;37:12-7. 4. Thiele T, Luga C, Janetzky S, Schwertz H, Gesell Salazar M, Furll B, Volker U, Greinacher A, Steil L. Early storage lesions in apheresis platelets are induced by the activation of the integrin alphaII(b)beta(3) and focal adhesion signaling pathways. J Proteomics 2012;76 Spec No.: 297-315. 5. Feinberg H, Sarin MM, Batka EA, Porter CR, Miripol JE, Stewart M. Platelet storage: changes in cytosolic Ca2+ actin polymerization and shape. Blood 1988;72:766-9. 6. Bock M, Gawaz MP, Dietzler A, Heim MU, Mempel W. Single-donor platelet concentrates: changes of surface glycoproteins during storage. Haemostasis 1994;24:230-5. 7. Wang C, Mody M, Herst R, Sher G, Freedman J. Flow cytometric analysis of platelet function in stored platelet concentrates. Transfus Sci 1999;20:129-39. 8. Lozano ML, Rivera J, Gonzalez-Conejero R, Moraleda JM, Vicente V. Loss of high-affinity thrombin receptors during platelet concentrate storage impairs the reactivity of platelets to thrombin. Transfusion 1997;37:368-75. 9. Schlagenhauf A, Kozma N, Leschnik B, Wagner T, Muntean W. Thrombin receptor levels in platelet concentrates during storage and their impact on platelet functionality. Transfusion 2012;52:1253-9. 10. Butt E, Abel K, Krieger M, Palm D, Hoppe V, Hoppe J, Walter U. cAMP- and cGMP-dependent protein kinase phosphorylation sites of the focal adhesion vasodilatorstimulated phosphoprotein (VASP) in vitro and in intact human platelets. J Biol Chem 1994;269:14509-17. 11. Schwarz UR, Geiger J, Walter U, Eigenthaler M. Flow cytometry analysis of intracellular VASP phosphorylation

ACKNOWLEDGMENTS The authors thank their colleagues of the technical staff of the Institute of Transfusion Medicine and Haemotherapy for donation management and preparation of PLT concentrates. They also thank their colleagues of the Department of Endocrinology and the Department of Nephrology, Internal Medicine 1, University Clinic of Wuerzburg, for technical support.

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13. CONFLICT OF INTEREST The authors declare that they have no conflicts of interest relevant to the manuscript submitted to TRANSFUSION.

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phosphoprotein Ser-239 phosphorylation in platelet concentrates during storage. Transfusion 2012;52:397-408. 17. Friebe A, Koesling D. The function of NO-sensitive guanylyl cyclase: what we can learn from genetic mouse models. Nitric Oxide 2009;21:149-56. 18. Gambaryan S, Kobsar A, Hartmann S, Birschmann I, Kuhlencordt PJ, Muller-Esterl W, Lohmann SM, Walter U.

in cGMP- and cAMP-mediated inhibition of agonistinduced platelet aggregation, but is dispensable for smooth muscle function. EMBO J 1999;18:37-48. 20. Massberg S, Gruner S, Konrad I, Garcia Arguinzonis MI, Eigenthaler M, Hemler K, Kersting J, Schulz C, Muller I, Besta F, Nieswandt B, Heinzmann U, Walter U, Gawaz M.

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19. Aszodi A, Pfeifer A, Ahmad M, Glauner M, Zhou XH, Ny L, Andersson KE, Kehrel B, Offermanns S, Fassler R. The

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Decreasing phosphodiesterase 5A activity contributes to platelet cGMP accumulation during storage of apheresis-derived platelet concentrates.

Platelet storage lesion (PSL) considerably decreases the quality of platelets (PLTs) in concentrates characterized by a loss of signaling responses to...
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