BO LR OI GO ID NCAOLM PA OR NT EI CN LT ES Platelet-derived microparticles induce polymorphonuclear leukocyte-mediated damage of human pulmonary microvascular endothelial cells Ru Feng Xie,1 Ping Hu,2 Zhi Cheng Wang,3 Jie Yang,1 Yi Ming Yang,1 Li Gao,1 Hua Hua Fan,1 and Yong Ming Zhu1

BACKGROUND: Platelets (PLTs) stored at 22°C accumulate microparticles and biologic response modifiers (BRMs) that induce inflammatory reactions in transfusion recipients. However, soluble BRMs are fully diluted in the recipient’s blood circulation. The mechanisms by which BRMs exert their effects have not been elucidated. The objectives of this study were to determine the effect of PLT microparticles (PMPs) on polymorphonuclear leukocyte (PMN)-mediated human pulmonary microvascular endothelial cell (HMVEC) damage and determine the role of soluble CD40 ligand (sCD40L). STUDY DESIGN AND METHODS: PMPs were isolated from apheresis PLT concentrates. We used a two-insult in vitro model of HMVEC damage to investigate the effects of PMP and sCD40L and role of apocynin, an inhibitor of PMN respiratory burst. Their priming activities were measured using hydrogen peroxide production. The expression of intercellular cell adhesion molecule-1 (ICAM-1) and integrin αM (CD11b) were also determined. RESULTS: Lipopolysaccharide (LPS)-activated HMVEC damage and PMN respiratory burst depend on the presence of PMP and the concentration of sCD40L. PMP-induced PMN-mediated HMVEC damage was significantly reduced by apocynin-treated PMNs (p < 0.05). The surface expression of ICAM-1 on HMVEC was increased by LPS stimulation. The expression of CD11b on PMNs was increased by PMP priming. Blocking ICAM-1 with a monoclonal antibody (MoAb) CD54 significantly reduced HMVEC damage (p < 0.05). The treatment of endothelial cells but not PMN with a MoAb targeting CD40 failed to prevent the HMVEC damage caused by PMPs (p > 0.05). CONCLUSION: PMPs carry a concentrated CD40L signal, promote PMN-mediated HMVEC damage, and may affect the development of transfusion-related acute lung injury.

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ransfusion-related acute lung injury (TRALI) is one of the most common transfusionassociated adverse reactions and has a high rate of morbidity and mortality. Although the incidence of antibody-mediated TRALI can be reduced by using plasma from male donors for the production of high-plasma-volume blood components, the residual incidence of non–antibody-mediated TRALI must be addressed.1 Previous studies have demonstrated that the transfusion of stored blood products such as platelet (PLT) concentrates with biologic response modifiers (BRMs) may induce TRALI.2-4 ABBREVIATIONS: A-PLTs = apheresis platelet concentrate; BRM(s) = biologic response modifier(s); fMLP = formyl-Met-Leu-Phe; GPA = glycophorin A; HMVEC(s) = human pulmonary microvascular endothelial cell(s); ICAM-1 = intercellular cell adhesion molecule-1; LPS = lipopolysaccharide; PMP(s) = platelet microparticle(s); sCD40L = soluble CD40 ligand. From the 1Shanghai Blood Center; 2The Institute of Life Science, East China Normal University; and 3Huashan Hospital, Shanghai, China. Address reprint requests to: Ru Feng Xie, Blood Engineering Laboratory, Shanghai Blood Center, Room 701, 1191 Hong Qiao Road, Shanghai 200051, China; e-mail: [email protected] or Yong Ming Zhu, Shanghai Blood Center, Room 501, 1191 Hong Qiao Road, Shanghai 200051, China; e-mail: [email protected] Ru Feng Xie (Shanghai Blood Center) and Ping Hu (Institute of Life Science, East China Normal University) contributed equally to this article. [Correction added on 23 January 2015, after first online publication: equal author contribution noted] This study was supported by the research funds of National Science Foundation of China 81270650 and funds of Shanghai Municipal Health Bureau 20114294. Received for publication July 17, 2014; revision received October 12, 2014, and accepted October 13, 2014. doi: 10.1111/trf.12952 © 2015 AABB TRANSFUSION 2015;55:1051–1057 TRANSFUSION **;**:**-**. Volume **, ** ** TRANSFUSION 1 Volume 55, May 2015 TRANSFUSION 1051

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According to a two-insult model,5 TRALI is the result of two separate clinical events. The first insult is the patient’s clinical condition, which may lead to the recruitment of polymorphonuclear leukocytes (PMNs) to the activated pulmonary microvascular endothelium. The second insult is the subsequent transfusion of antibodies or BRMs from blood components that activate the adherent PMNs. This activation results in PMN-mediated destruction of endothelial cells, capillary leakage, and acute lung injury. Therefore, investigations of the roles of PMNs and priming and activation factors present in blood components are important for elucidating the pathophysiologic mechanisms of vascular endothelial cell damage. Cardo and coworkers6 reported in 2008 that microparticles in stored red blood cells (RBCs) were able to prime neutrophil respiratory burst. However, this response was abrogated by leukoreduction at collection. Khan and colleagues4 reported in 2006 that soluble CD40 ligand (sCD40L) accumulated during PLT storage and promoted the PMN-mediated cytotoxicity of human pulmonary microvascular endothelial cells (HMVECs). Furthermore, Kaufman and coworkers7 indicated that the level of membrane-bound CD40L in PLT concentrates increased during 5 days of storage. Our recent study demonstrated that PLT-derived microparticles (PMPs) carried concentrated CD40L signal. These microparticles effectively prime formyl-Met-Leu-Phe (fMLP)-activated PMN respiration burst.8 However, the effects of PMPs from PLT concentrates on endothelial cells are unknown. In this study, we examined the effects of PMPs isolated from apheresis PLT concentrates (A-PLTs) on lipopolysaccharide (LPS)-activated HMVEC damage mediated by PMN.We also evaluated the correlation between PMPs and CD40L.

MATERIALS AND METHODS A-PLT preparation and PMP isolation A-PLTs were prepared with a mobile collection system (MCS+ 9000, Haemonetics Corp., Braintree, MA) at the Shanghai Blood Center. The day after preparation was defined as Day 1. The A-PLTs were collected into gaspermeable storage bags with ACD-A and stored on a horizontal agitator at 22 ± 2°C. The samples were drawn by a sterile tubing welder (Terumo BCT, Lakewood, CO) during storage. The PMP isolation was performed as described previously, with modifications.8 In brief, PLT-poor plasma was separated by centrifugation of the A-PLTs at 1500 × g for 20 minutes. The plasma was then centrifuged at 20,000 × g for 1 hour. The PMP pellet was washed with phosphatebuffered saline (PBS; pH 7.2, Gibco, Life Technologies, Grand Island, NY) with a 0.3% citric acid (Sigma, St Louis, MO) additive and was resuspended in PBS to make a 10-fold concentrated solution. The amount of PMPs 1052 TRANSFUSION Volume 55,**May 2015 2 TRANSFUSION Volume **, **

and sCD40L carried by PMPs were assayed as described previously.8

Flow cytometry The presence or absence of specific markers for blood cells on PMPs was detected by flow cytometry (FACSCalibur, Becton Dickinson, Franklin Lakes, NJ). For white blood cell (WBC)-specific antigen labeling, 10 μL of isolated PMPs was incubated with 10 μL of mouse antihuman CD41a-phycoerythrim (PE; immunoglobulin [Ig]G1, Clone HIP8, eBioscience, Affymetrix Co., Santa Clara, CA) and 10 μL of mouse anti-human CD45fluorescein isothiocyanate (FITC; BD Biosciences, San Jose, CA) in 100 μL of PBS for 30 minutes at room temperature. The mixture was diluted to 600 μL with PBS before flow cytometry analysis. The isotype antibodies were used as controls. For RBC-specific antigen labeling, 10 μL of isolated PMPs was incubated with 10 μL of purified mouse anti-human glycophorin A (GPA) combined with mouse anti-human CD41a-PE in 100 μL of PBS for 30 minutes at room temperature (anti-GPA was supplied by International Blood Group Reference Laboratory, Lot 698L). The PMPs were then washed with PBS by centrifugation at 20,000 × g for 1 hour. The PMPs were resuspended in 100 μL of PBS with 10 μL of goat anti-mouse IgG (H+L)FITC (Jackson ImmunoResearch Laboratories, West Grove, PA) secondary antibody. The mixture was incubated for an additional 30 minutes at room temperature and then diluted to 600 μL with PBS before flow cytometry analysis. The isotype antibody was used as a control. The expression of intercellular cell adhesion molecule-1 (ICAM-1; CD54) on LPS-activated HMVECs and the surface expression of CD11b on PMP primed PMNs were also determined. For HMVEC and PMN labeling, 1 × 106 cells were incubated with 10 μL of mouse antihuman CD54-PE (BD Biosciences) or 10 μL of mouse antihuman CD11b-PE (BD Biosciences) in 100 μL of PBS for 30 minutes at room temperature. The cells were diluted to 600 μL with PBS before flow cytometry analysis.

PMN respiratory burst detection The priming activity of PMP for fMLP (Sigma-Aldrich, St Louis, MO)-activated PMN respiratory burst was measured as described previously.7 To determine the activity of sCD40L, human sCD40L, and 0.5 mg/mL cross-linking (Miltenyi Biotec, Inc., Auburn, CA) antibody were incubated together for 30 minutes before cell application. The inhibition of NADPH oxidase was performed by incubating PMNs with 300 to 1200 μmol/L apocynin (SigmaAldrich) for 15 minutes at 37°C before loading the indicator dye dihydrorhodamine 123 (Sigma-Aldrich).

PMN-mediated HMVEC damage assay HMVECs (PriCells, Wuhan Biomedical Technology Co., Ltd., Wuhan, Hubei, China) were grown to more than 90%

PLT MICROPARTICLES MICROPARTICLES INDUCE INDUCE HMVEC HMVEC DAMAGE DAMAGE PLT

TABLE 1. PMP counts and sCD40L concentration in plasma and PMPs from A-PLTs after 3 days of storage sCD40L (pg/mg protein) Sample 1 2 3 4 5 Mean ± SD

PMP count (PMP/μL)

Plasma sCD40L (pg/mL)

Plasma

PMP

120,762 138,253 275,109 105,674 70,816 142,123 ± 78,347

1605.31 2294.00 3107.54 3849.00 2205.77 2162.32 ± 873.97

33.12 52.16 40.93 81.46 34.53 48.44 ± 19.93

4449.18 4151.99 9530.53 8482.84 4020.71 6127.05 ± 2659.24

confluence in 96-well plates. By modifying the method of Wyman and colleagues,9 cells were treated with 2 to 2000 ng/mL LPS from Salmonella enteritidis (LPS, SigmaAldrich) for 6 hours. The cells were detached with 0.05% trypsin-EDTA (Gibco). The expression of ICAM-1 on HMVEC was assayed by flow cytometry. PMNs were added to 200 ng/mL LPS-treated cells at a 10:1 effector cell-totarget cell ratio. After settling, the PMNs were exposed to PMPs, sCD40L, or buffer for 30 minutes. The numbers of viable cells were counted over a 1-mm2 surface area by fluorescence microscopy after staining with a cell imaging kit (LIVE/DEAD, Life Technologies Corporation, Eugene, OR). The inhibition of NADPH oxidase in PMNs was performed by incubating PMNs with 300 to 1200 μmol/L apocynin for 15 minutes at 37°C before adding them to HMVECs. To determine the role of ICAM-1 on HMVECs, 1 million cells were incubated with 0.5 μg of purified mouse anti-human CD54 (BioLegend, Inc., San Diego, CA) in a 100-μL volume at 37°C for 30 minutes before the addition of PMNs. Purified mouse anti-human CD40 was used to block CD40 (BioLegend, Inc.) expressed on HMVECs according to the methods described.

Statistical analysis All of the data are presented as the mean ± SD. The p values between the groups were calculated with the paired t test. A p value of less than 0.05 was defined as a significant difference. Computer software (SPSS 17.0, SPSS, Inc., Chicago, IL) was used for the statistical analysis.

stored for 3 days are presented in Table 1. The average amount of sCD40L per microgram of protein for PMPs was 68-fold greater than that for plasma.

PMN respiratory burst The expression of CD11b on PMNs exposed to 5% to 30% PMP isolated from A-PLTs stored for 3 days increased in a dose-dependent manner (Fig. 2). The results showed that PMPs primed fMLP-activated PMN respiratory burst significantly starting at a PMP ratio of 10% (Fig. 3A), which was equivalent to a sCD40L concentration of 5 μg/mL (Fig. 3B).

Activation of HMVECs and PMN-mediated HMVEC damage The surface expression of ICAM-1 increased on endothelial cells after treating HMVECs with 2 to 2000 ng/mL LPS (Fig. 4). Neither PMNs nor PMPs alone caused significant damage to HMVECs treated with 200 ng LPS. However, PMNs with 20% PMPs caused significant endothelial cell damage compared with the PMN control (Fig. 5A, p < 0.05). Meanwhile, sCD40L also caused significant HMVEC damage at a concentration of 25 μg/mL (Fig. 5B, p < 0.05).

RESULTS PMPs isolated from A-PLTs Microparticles isolated from PLT concentrates were derived from PLTs based on their expression of glycoprotein GpIIb (CD41). Neither GPA nor CD45, which are specific markers for RBCs and WBCs, respectively, were found on the microparticles (Fig. 1). The PMP counts and soluble CD40L concentration for plasma or PMPs isolated from A-PLTs

Fig. 1. Identification of PMPs by flow cytometry. PMPs were dual labeled with mouse anti-human CD41a-PE and GPA-FITC or CD45.

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Fig. 2. CD11b expression on PMNs. PMNs were primed with 5% to 30% PMP (vol/vol) and then labeled with mouse antihuman CD11b-PE. The peaks from left to right are the buffer control, 5% PMP-treated PMNs, and 30% PMP-treated PMNs.

Inhibition of PMN-mediated HMVEC damage Treating PMNs with apocynin, which is an inhibitor of NADPH oxidase, at a concentration of 1200 μmol/L inhibited PMP-primed PMN respiratory burst completely (Fig. 6, p < 0.05). Apocynin-treated PMNs also caused significantly less HMVEC damage in the presence of PMPs (Fig. 7, p < 0.05). Blocking ICAM-1 on HMVECs with a monoclonal antibody (MoAb) to CD54 may decrease PMN-mediated HMVEC damage induced by PMPs (Fig. 8, p < 0.05). However, blocking CD40 on HMVECs but not PMN failed to prevent endothelial cell damage (Fig. 9).

DISCUSSION The concentration of blood cell–derived microparticles may increase during storage.10-12 In a previous study, we reported that PMP concentration increased during 5 days of storage at 22°C.7 This study showed that sCD40L was concentrated in PLT-derived microvesicles. The concentration of sCD40L per microgram protein was approximately 68-fold greater than that in plasma. This result is consistent with the result of Sprague in mouse experiments.13 Urner and colleagues14 demonstrated in 2012 that PLT concentrates are more potent than FFP at triggering inflammatory reactions in endothelial cells. This result most likely goes along with the lipid content of the blood components. We have demonstrated for the first time that PMPs carry membrane-bound sCD40L and induce HMVEC damage. The damage induction was also dose dependent. In our experiments, 20% PMP, which was equivalent to 5.22 ng/mL sCD40L, was added to endothelial cells. The most efficient concentration of recombinant 1054 TRANSFUSION Volume 55,**May 2015 4 TRANSFUSION Volume **, **

Fig. 3. Priming activity of isolated PMP (A) and recombinant sCD40L (B). Oxidation of the indicator dye dihydrorhodamine 123 to fluorescent rhodamine 123 in PMNs was measured by flow cytometry. The histogram shows geometrical median fluorescence intensity (MFI) of fMLP (1 μmol/L)-activated PMNs that were primed with 5% to 30% PMP (A) and 0.2 to 5 μg/mL sCD40L (B) (n = 5). *p < 0.05 was defined as a significant difference with the fMLP control.

sCD40L was 25 μg/mL. These data suggested that sCD40L carried by PMPs were more efficient in inducing HMVEC damage. CD40 is the receptor of CD40L and is expressed on the surface of monocytes, macrophages, endothelial cells, and PLTs. CD40 expression was also recently identified on neutrophils.4 The CD40–CD40L interaction results in neutrophil respiratory burst, which is implicated in inflammation, atherogenesis, and thrombosis.15,16 Our results showed that PMPs induced HMVEC damage and that the damage was not prevented by treating the endothelial cells with a MoAb targeting CD40 before the PMNs were added. These results suggested that PMPs carried sCD40L, which interacted with CD40 on PMNs and induced a respiratory burst, thus mediating HMVEC cytotoxicity. To examine the effect of PMNs on HMVEC damage, we used the NADPH oxidase inhibitor apocynin to treat PMNs

PLT MICROPARTICLES MICROPARTICLES INDUCE INDUCE HMVEC HMVEC DAMAGE DAMAGE PLT

Fig. 4. CD54 expression on HMVECs. HMVECs were grown to at least 90% confluence in 96-well plates. The cells were treated with 2 to 200 ng/mL LPS for 6 hours and were detached with 0.05% trypsin-EDTA. The cells were labeled with mouse anti-human CD54-PE. The peaks from left to right are the buffer control, 2 ng/mL LPS, and 200 ng/mL LPS.

Fig. 6. PMN respiratory burst is inhibited by apocynin. NADPH oxidase was inhibited by incubating PMNs with 300 to 1200 μmol/L apocynin for 15 minutes at 37°C before fMLP activation (n = 5). *p < 0.05 was defined as a significant difference with the fMLP control.

Fig. 7. HMVEC damage is inhibited by apocynin. NADPH oxidase was inhibited by incubating PMNs with 300 to 1200 μmol/L apocynin for 15 minutes at 37°C before the PMNs were added to HMVECs (n = 5). *p < 0.05 was defined as a significant difference with the PMP-induced control.

Fig. 5. HMVEC damage induced by PMP (A) and recombinant sCD40L (B). HMVECs were induced with 200 ng/mL LPS for 6 hours. PMNs were added at a 10:1 effector cell-to-target cell ratio. After settling, the PMNs were exposed to PMPs, recombinant sCD40L,or buffer for 30 minutes. The numbers of viable cells were counted over a 1-mm2 surface area by fluorescent microscopy (n = 5). *p < 0.05 was defined as a significant difference with the PMN control.

before culturing the cells with LPS-activated endothelial cells. This treatment inhibited the PMN respiratory burst and HMVEC damage. Interestingly, the dose of either sCD40L or PMPs used to induce HMVEC damage was higher than the dose needed to prevent PMN respiratory burst. The mechanisms regulating these results require further investigation. The PMN-mediated damage of endothelial cells requires firm adhesion between neutrophil and endothelial cells.9 It has been reported that microparticles from 42-day-stored RBCs primed PMNs and resulted in the up regulation of the adhesive molecular receptor CD11b.6 In this study, surface expression of CD11b was increased on PMPs primed PMNs. Additionally, ICAM-1 expression on HMVECs was increased by LPS. PMP-induced HMVEC damage was eliminated by blocking ICAM-1 with a MoAb targeting CD54. Volume 55, May 2015 TRANSFUSION 1055 Volume **, ** ** TRANSFUSION 5

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washing stored RBCs and PLTs may prevent the onset of TRALI in vivo.19 However, additional data are required to support these clinical practices. In conclusion, PMPs carry concentrated CD40L signal, promote PMN-mediated HMVEC damage, and may be related to TRALI. Whether membrane lipid–bound sCD40L more efficiently induces inflammatory reactions through transfusion requires further investigation. ACKNOWLEDGMENT We thank the Shanghai Jiaotong University School of Medicine for the PMP counting. Fig. 8. HMVEC damage is inhibited by blocking ICAM-1 on

CONFLICT OF INTEREST

HMVECs. To determine the role of ICAM-1 on HMVECs, one million cells were treated with 0.5 μg of purified mouse anti-

The authors have disclosed no conflicts of interest.

human CD54 in a 100-μL volume at 37°C for 30 minutes before PMN addition (n = 5). *p < 0.05 was defined as a significant difference with the PMN control. ( ) IgG1; ( ) anti-CD54.

REFERENCES 1. Vlaar AP, Juffermans NP. Transfusion related acute lung injury: a clinical review. Lancet 2013;382:984-94. 2. Middelburg RA, Borkent-Raven BA, Janssen MP, et al. Storage time of blood products and transfusion-related acute lung injury. Transfusion 2012;52:658-67. 3. Tung JP, Fraser JF, Nataatmadja M, et al. Age of blood and recipient factors determine the severity of transfusionrelated acute lung injury. Crit Care 2012;16:R19. 4. Khan SY, Kelher MR, Heal JM, et al. Soluble CD40 ligand accumulates in stored blood components, primes neutrophils through CD40, and is a potential cofactor in the development of transfusion-related acute lung injury. Blood 2006;108:2455-62. 5. Silliman CC, Boshkov LK, Mehdizadehkashi Z, et al. Transfusion-related acute lung injury: epidemiology and a

Fig. 9. Effect of anti-CD40 treatment on HMVEC damage. To determine the effect of CD40 expressed on endothelial cells, HMVECs were treated with excessive antibody for blocking CD40 expressed on those cells completely (isotype IgG1 as control) before PMNs were added. (n = 5). #p > 0.05 was defined as no significant difference with the PMN control. ( ) IgG1; ( ) anti-CD54.

Several attempts have been made to remove microparticles and other BRMs in stored blood components. The removal of microparticles may prevent complications in patients at risk for developing TRALI. Wozniak and colleagues17 recently reported that the removal of phosphatidylserine-positive microparticles from stored RBC units by washing reduced inflammation in a swine model of human TRALI. Tanaka and coworkers18 revealed that cellulose beads were effective in removing three types of BRMs including sCD40L, RANTES, and transforming growth factor-β. Preclinical studies have shown that 1056 TRANSFUSION Volume 55,**May 2015 6 TRANSFUSION Volume **, **

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prospective analysis of etiologic factors. Blood 2003;101: 454-62. Cardo LJ, Wilder D, Salata J. Neutrophil priming, caused by cell membranes and microvesicles in packed red blood cell units, is abrogated by leukocyte depletion at collection. Transfus Apher Sci 2008;38:117-25. Kaufman J, Spinelli SL, Schuntz E, et al. Release of biologically active CD154 during collection and storage of platelet concentrates prepared for transfusion. J Thromb Haemost 2007;5:788-96. Xie RF, Hu P, Li W, et al. The effect of platelet-derived microparticles in stored apheresis platelet concentrates on polymorphonuclear leucocyte respiratory burst. Vox Sang 2014;106:234-41. Wyman TH, Bjornsen AJ, Elzi DJ, et al. A two-insult in vitro model of PMN-mediated pulmonary endothelial damage: requirements for adherence and chemokine release. Am J Physiol Cell Physiol 2002;283:C1592-603. Rank A, Nieuwland R, Liebhardt S, et al. Apheresis platelet concentrates contain platelet-derived and endothelial cellderived microparticles. Vox Sang 2011;100:179-86.

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11. Jy W, Ricci M, Shariatmadar S, et al. Microparticles in stored red blood cells as potential mediators of transfusion complications. Transfusion 2011;51:886-93. 12. George JN, Pickett EB, Heinz R. Platelet membrane

protein kinase C zeda (PKCf) in neutrophils: implications for neutrophil-platelet interactions and neutrophil oxidative burst. PLoS ONE 2013;8:64631. 17. Wozniak MJ, Patel NN, Abidoye A, et al. Abstract 14891:

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tion of adaptive immunity: unique delivery of CD154 signal by platelet-derived membrane vesicles. Blood 2008; 111:5028-36. 14. Urner M, Herrmann IK, Buddeberg F, et al. Effects of blood products on inflammatory response in endothelial cells in vitro. PLoS ONE 2012;7:e33403. 15. Vanichakarn P, Blair P, Wu C, et al. Neutrophil CD40 enhances platelet mediated inflammation. Thromb Res 2008;122:346-58. 16. Jin R, Yu S, Song Z, et al. Soluble CD40 ligand stimulates

2013;128:A14891. 18. Tanaka S, Hayashi T, Tani Y, et al. Removal by adsorbent beads of biological response modifiers released from platelets, accumulated during storage, and potentially associated with platelet transfusion reactions. Transfusion 2010; 50:1096-105. 19. Vlaar AP, Hofstra JJ, Kulik W, et al. Supernatant of stored platelets causes lung inflammation and coagulopathy in a novel in vivo transfusion model. Blood 2010;116: 1360-8.

CD40-dependent activation of the b2 integrin Mac-1 and

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Platelet-derived microparticles induce polymorphonuclear leukocyte-mediated damage of human pulmonary microvascular endothelial cells.

Platelets (PLTs) stored at 22°C accumulate microparticles and biologic response modifiers (BRMs) that induce inflammatory reactions in transfusion rec...
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