A signaling pathway contributing to platelet storage lesion development: targeting PI3-kinase–dependent Rap1 activation slows storage-induced platelet deterioration

BACKGROUND: The term platelet storage lesion (PSL) describes the structural and biochemical changes in platelets (PLTs) during storage. These are typified by alterations of morphologic features and PLT metabolism leading to reduced functionality and hence reduced viability for transfusion. While the manifestations of the storage lesion are well characterized, the biochemical pathways involved in the initiation of this process are unknown.
STUDY DESIGN AND METHODS: A complementary proteomic approach has recently been applied to analyze changes in the PLT proteome during storage. By employing stringent proteomic criteria, 12 proteins were identified as significantly and consistently changing in relative concentration over a 7-day storage period. Microscopy, Western blot analysis, flow cytometry, and PLT functionality analyses were used to unravel the involvement of a subset of these 12 proteins, which are connected through integrin signaling in one potential signaling pathway underlying storage lesion development.
RESULTS: Microscopic analysis revealed changes in localization of glycoprotein IIIa, Rap1, and talin during storage. Rap1 activation was observed to correlate with expression of the PLT activation marker CD62P. PLTs incubated for 7 days with the PI3-kinase inhibitor LY294002 showed diminished Rap1 activation as well as a moderate reduction in integrin αIIbβ3 activation and release of α-granules. Furthermore, this inhibitor seemed to improve PLT integrity and quality during storage as several in vitro probes showed a deceleration of PLT activation.
CONCLUSION: These results provide the first evidence for a signaling pathway mediating PSL in which PI3-kinase–dependent Rap1 activation leads to integrin αIIbβ3 activation and PLT degranulation.     

The platelet storage lesion

The continuous increase in the demand for platelet transfusion has necessitated the need to establish standards for determining the quality of platelets during storage. Bacterial contamination of platelet products and deleterious changes in structure and function referred to as the platelet storage lesion (PSL), have restricted the platelet shelf life to 5 days. The PSL and platelet health variables have been well studied and documented. The precise correlation between in vitro assays and in vivo platelet recovery and survival is yet to be established. This review presents an overview of the current understanding of PSL and the novel approaches being developed to negate the storage lesion.     

Platelet Storage Lesions: What More Do We Know Now?

Platelet concentrate (PC) transfusions are a lifesaving adjunct to control and prevent bleeding in cancer, hematologic, surgical, and trauma patients. Platelet concentrate availability and safety are limited by the development of platelet storage lesions (PSLs) and risk of bacterial contamination. Platelet storage lesions are a series of biochemical, structural, and functional changes that occur from blood collection to transfusion. Understanding of PSLs is key for devising interventions that prolong PC shelf life to improve PC access and wastage. This article will review advancements in clinical and mechanistic PSL research. In brief, exposure to artificial surfaces and high centrifugation forces during PC preparation initiate PSLs by causing platelet activation, fragmentation, and biochemical release. During room temperature storage, enhanced glycolysis and reduced mitochondrial function lead to glucose depletion, lactate accumulation, and product acidification. Impaired adenosine triphosphate generation reduces platelet capacity to perform energetically demanding processes such as hypotonic stress responses and activation/aggregation. Storage-induced alterations in platelet surface proteins such as thrombin receptors and glycoproteins decrease platelet aggregation. During storage, there is an accumulation of immunoactive proteins such as leukocyte-derive cytokines (tumor necrosis factor α, interleukin (IL) 1α, IL-6, IL-8) and soluble CD40 ligand which can participate in transfusion-related acute lung injury and nonhemolytic transfusion reactions. Storage-induced microparticles have been linked to enhanced platelet aggregation and immune system modulation. Clinically, stored PCs have been correlated with reduced corrected count increment, posttransfusion platelet recovery, and survival across multiple meta-analyses. Fresh PC transfusions have been associated with superior platelet function in vivo; however, these differences were abrogated after a period of circulation. There is currently insufficient evidence to discern the effect of PSLs on transfusion safety. Various bag and storage media changes have been proposed to reduce glycolysis and platelet activation during room temperature storage. Moreover, cryopreservation and cold storage have been proposed as potential methods to prolong PC shelf life by reducing platelet metabolism and bacterial proliferation. However, further work is required to elucidate and manage the PSLs specific to these storage protocols before its implementation in blood banks.     

N-acetylcysteine reduce the stress induced by cold storage of platelets: A potential way to extend shelf life of platelets

The room temperature storage used for platelets worldwide leads to platelet storage lesion (PSL) and risk of bacterial growth, limiting platelet shelf life and safety in transfusion. Thus, there is a need for an alternative storage method that can serve as effective temperature storage for platelet concentrates (PCs). In the previous investigation, we have shown that N-acetylcysteine (NAC) is a potential candidate for an additive solution to retain platelet characteristics during cold storage for up to 5 days. However, the study partially describes the efficacy and has drawbacks to address. Here, we used the apheresis platelet product with 50 mM NAC and stored up to 10 days under refrigerated condition (4 ± 1 °C). Stored platelet concentrates were analyzed for critical parameters such as platelet activation, annexin V binding, sialic acid, reactive oxygen species (ROS), neuraminidase activity, and in vivo efficacy using Prkdc^scid mice. Investigation observations revealed that PCs with NAC showed reduced platelet activation, annexin V binding, ROS production, and sialic acid levels. in vivo recovery of PCs showed similar recovery rates stored PCs irrespective of treatment or storage condition. However, on the tenth day after 24 h, recovery in room temperature stored concentrates was about 32 %, whereas in NAC treated refrigerated concentrates, it stands at 47 %. These observations indicate that NAC addition protects refrigerated concentrates during long-term storage retaining the platelet integrity. The study also suggests that extending PC storage beyond 10 days is practically accomplishable with efficacy similar to room temperature (RT) stored PCs.     

Assessment of apheresis platelets during 5 days of storage: A National Cancer Institute,Cairo University experience

BackgroundPlatelet transfusion therapy is widely used to prevent hemorrhage in patients with thrombocytopenia and platelet disorders. The platelet concentrate (PC) quality is affected by increased storage time, as reflected in the decreased number of platelets, morphological changes, and impaired functions. This study aimed to analyze the impact of 5 days storage on platelets count and the expression of CD63, and Annexin V as activation markers during PC storage.MethodsFifty PCs collected from single donors were tested for platelet count on days 0, 3, and 5 using a Sysmex blood counter. CD61, CD63, and Annexin V expression was analyzed by a multicolor Navios flow cytometer.ResultsThere was a significant decrease in platelet count during 5 days of storage. There was a direct relationship between storage time and degree of platelet activation. CD63 had almost double increased expression on day 5 than day 3. Annexin V showed significantly increased expression on day 3 with minor differences between days 3 and 5.ConclusionAccording to standard blood bank conditions, PC stored for 5 days showed a degree of in vitro activation as evidenced by CD63 and Annexin V expression, may lead to reduced therapeutic efficacy. Flow cytometry monitoring platelet activation in PC offers a better understanding of the changes during PC storage and may help improve platelet products.     

Physiology of cold-stored platelets

Platelet transfusions are a life-saving medical intervention used for the treatment of thrombocytopenia or hemorrhage. Extensive research has gone into trying to understand how to store platelets prior to the transfusion event. Much has been learned about storage bag materials, synthetic solutions, and how temperature impacts platelet viability and function. While room temperature storage of platelets preserves 24-hour in vivo platelet recovery and survival there is a greater risk for bacterial growth. Therefore, cold storage of platelets has become attractive due to the reduction in potential bacterial proliferation and the maintenance of platelet function beyond 5 days of storage. Cold stored platelets, however, have their own set of challenges. Cold stored platelets become activated through several mechanisms. The morphological and molecular changes that occur due to cold exposure enhance their ability to participate in the hemostatic process at the cost of rapid clearance from circulation. This review focuses on the underlying mechanisms leading to cold platelet activation and the receptor modifications involved in platelet clearance.     

Quality assessment of platelet concentrates supplemented with second-messenger effectors

BACKGROUND: While reducing the potential for bacterial contamination, the storage of platelet concentrates (PCs) at refrigerated temperatures is not routine, because of the induction of the so-called platelet storage lesion. As the modulation of second-messenger levels might help to overcome this drawback, a quality assessment of PCs treated with a mixture of second-messengers effectors known as ThromboSol was performed. STUDY DESIGN AND METHODS: The PCs were supplemented with ThromboSol or phosphate-buffered saline, and stored in parallel at 22 degrees C with continuous agitation or at 4 degrees C. At 1, 5, and 9 days, an in vitro quality assessment of the PCs was performed, including measurement of cell number, metabolic and integrity markers, platelet surface expression of glycoproteins, platelet response to ristocetin and thrombin, and levels of cyclic adenosine 3', 5' monophosphate (cAMP) and thromboxane B2 (TxB2). RESULTS: Control PCs stored at 4 degrees C underwent aggregation and displayed a significant decrease in the platelet number (40% on Day 5). By contrast, the ThromboSol-treated PCs maintained 80 percent of their initial platelet concentration after 9 days of storage at 4 degrees C. Compared to PCs stored at 22 degrees C, refrigerated PCs exhibited minor changes in metabolic values throughout storage, but the addition of ThromboSol induced a rise in metabolic rate during storage at 22 degrees C. Platelet responsiveness to both ristocetin and thrombin was maximally preserved in the ThromboSol-treated PCs stored at 4 degrees C. These units also maintained high levels of cAMP and low concentrations of TxB2 during storage. CONCLUSION: The pharmacologic supplementation of PCs with ThromboSol significantly favors the maintenance of in vitro integrity and responsiveness of platelets during extended storage at refrigerated temperature. This protective effect seems to be a consequence of the ability of ThromboSol's components to sustain high levels of cAMP and to inhibit TxB2 production during the entire extended-storage period.     

TEG技术在血小板保存过程中功能评价的应用研究

本研究旨在通过血栓弹力图（thmmbelastography，TEG）技术探讨血小板保存过程中功能的变化。随机选择各项指标均符合国家标准的单供者机采血小板12个单位并在（22±2）℃条件下振荡保存。分别在保存1、2、3、4、5天检测血栓弹力图参数，包括反应时间（R）、凝血时间（K）、α角（ANG）和最大振幅（MA），同时检测血小板计数、平均血小板体积、低渗休克反应（HSR）水平、CD62p阳性率及凝血酶激活CD62p再表达率的变化，综合评价血小板保存过程中体外功能的变化情况。结果显示：平均血小板体积随保存时间延长而轻度增大，但无统计学差异（p〉0．05）；血小板膜表面CD62p表达率随保存时间延长而显著升高（P〈0．01）；凝血酶激活后CD62p再表达率随保存时间的延长而显著下降（p〈0．01）；血小板低渗休克反应水平在1-5天无明显变化（P〉0.05）；R值随保存时间延长而明显延长（P〈0．01），K值无明显变化（P〉0.05），α角虽呈轻度下降趋势，但无显著差异（P〉0．05）；MA值在保存1-4天无显著变化，保存5天时仅有轻度下降（P〈0．05）。结论：虽然血小板随保存时间延长激活率明显升高，但反映血小板综合凝血功能的最大振幅（MA值）和HSR水平在整个保存期内并无显著变化，说明保存期末的血小板仍然具有良好的止血功能；血栓弹力图参数MA值可以作为一项重要指标用于血小板保存过程中的功能评价。     

L-carnitine decreases glycolysis in liquid-stored platelets

BACKGROUND: The platelet storage lesion is characterized metabolically by a pH decrease associated with lactic acid generation; a change in platelet morphology from discoid to spherical; a diminished response to in vitro challenge tests, such as the hypotonic shock response (HSR) and extent of shape change (ESC); increased surface P-selectin expression; and decreased in vivo recovery and survival. Altering storage conditions to improve these measures could allow for extension of the duration of in vitro storage. STUDY DESIGN AND METHODS: ABO-identical paired platelet concentrates were pooled and then equally divided into two plastic bags. Either L-carnitine (LC) or an equal volume of saline (control) was added to one container of each pair. Platelets were stored at 20 to 24 degrees C for 5 to 10 days or at 1 to 6 degrees C for 5 days at various concentrations of LC between 0.1 and 15 mM: At the end of storage, pH, glucose consumption, lactate generation, HSR, ESC, and surface P-selectin expression were measured. In different experiments, paired platelet concentrates were spiked with a Staphylococcus epidermidis suspension in the presence and absence of L-carnitine at a concentration of 5 mM: RESULTS: At 20 to 24 degrees C and concentrations of LC between 0.1 and 5 mM:, there was evidence of better pH preservation, less glucose consumption, and less lactate generation. Only with storage beyond 5 days was a difference present in either surface P-selectin expression or HSR. An L-carnitine concentration of 5 mM: appeared optimal. L-carnitine did not enhance the growth of bacteria after 7 to 8 days of storage. CONCLUSION: LC at 5 mM: may improve the quality of platelet concentrates that are stored beyond 5 days. There was no indication that LC at this concentration would promote bacterial growth. It may be a useful additive to platelet preservation.     

Effect of recombinant human megakaryocyte growth and development factor coupled with polyethylene glycol on the platelet storage lesion

BACKGROUND: Platelet production is regulated by a thrombopoietic growth factor (Mpl ligand). The receptor for this platelet growth factor (Mpl) is expressed on the platelet surface membrane. A recombinant thrombopoietic cytokine, recombinant human megakaryocyte growth and development factor coupled with polyethylene glycol (PEG-rHuMGDF), was added to apheresis platelets in vitro to determine whether Mpl ligand-receptor binding produced any beneficial or adverse effect on the development of the platelet storage lesion during 5 days of storage. STUDY DESIGN AND METHODS: This study was designed as a dose-response protocol to determine the effects of adding increasing concentrations of PEG-rHuMGDF (0.0 [control], 2.5, 25, and 250 ng/mL) to apheresis platelets stored in two types of plastic storage containers. The increasing concentrations of PEG-rHuMGDF used simulated the theoretical peak plasma level attained in vivo, with an intravenous dose of 0, 0.1, 1.0 and 10 microg per kg of PEG-rHuMGDF. The platelets were stored with agitation at 20 to 24 degrees C for 5 days. A battery of in vitro assays was performed on storage Days 1 and 5, including pH, blood gases, platelet count, lactate dehydrogenase, mean platelet volume, glucose, lactate, osmotic recovery, morphology score, CD62P, and one-dimensional polyacrylamide gel electrophoresis analyses. RESULTS: Analysis of results on both Day 1 and Day 5 showed no significant differences among any of the three PEG-rHuMGDF doses and the control group, for any in vitro assay. One-dimensional polyacrylamide gel electrophoresis showed no changes among the platelet protein patterns for the three PEG-rHuMGDF doses studied or the control. Storage-induced changes, however, did occur equally in all four groups of platelets over the 5 days of storage. CONCLUSION: The addition to stored apheresis platelets of up to 10 microg per kg of PEG-rHuMGDF (250 ng/mL), followed by 5 days of storage at standard conditions, does not appear to promote or retard development of the platelet storage lesion.     

Translation of glycoprotein IIIa in stored blood platelets

BACKGROUND: Platelet (PLT) products have a short shelf life (5 days) owing in part to the deterioration of the quality of PLTs stored at 22 degrees C. This creates significant inventory challenges, and blood banks may suffer shortages and high wastage as a result. The precise biochemical pathways involved in the PLT storage lesion are unknown and must be understood before storage time can be extended. STUDY DESIGN AND METHODS: Informed by previous proteomics analysis, specific PLT glycoprotein (GP) concentration and surface expression were examined by Western blot and flow cytometry. mRNA concentration was determined by Northern blot and real-time polymerase chain reaction. Protein synthesis was confirmed by [(35)S]methionine labeling. RESULTS: Western blots of GPIIIa revealed a twofold increase in concentration on Day 7 of storage and a fourfold increase on Day 10. By flow cytometry, surface expression of the GPIIb/IIIa increased by 13.4 percent on Day 7 and 41.9 percent on Day 10. Full-length GPIIIa mRNA was present throughout this storage period and was shown to have a half-life of approximately 2.9 days. Translation of GPIIb and IIIa during storage was confirmed by [(35)S]methionine labeling. CONCLUSION: This article confirms that PLTs are capable of synthesizing biologically relevant proteins ex vivo throughout a 10-day storage period with particularly long-lived mRNA and provides a framework through which the biochemical mechanisms involved in the translational regulation of proteins thought to be involved in the initiation or exacerbation of the PLT storage lesion can be investigated.     

Platelet bacterial contamination and the use of a chemiluminescence- linked universal bacterial ribosomal RNA gene probe

BACKGROUND: Currently, the maximum outdate for platelets is 5 days, because of the increasing chance of bacterial growth over time. Various methods for rapid detection of bacterial contamination of blood components have been described, with mixed results and no general acceptance. A recently described, molecular biologic approach for the detection of bacterial contamination involves a chemiluminescence- linked universal DNA bacterial probe to a highly conserved bacterial region of ribosomal RNA (rRNA). STUDY DESIGN AND METHODS: A multicenter trial of a chemiluminescence-linked universal bacterial rRNA probe for the detection of bacterial contamination in platelet concentrates is described. At each of five sites, platelet concentrates (no older than 1 day from date of phlebotomy) were inoculated in triplicate with isolates of four bacterial species (Pseudomonas aeruginosa, Bacillus cereus, Staphylococcus epidermidis, and Staphylococcus aureus) to a final concentration of 10 to 50 colony-forming units (CFUs) per mL and in triplicate to a final concentration of 1000 CFUs per mL. At one site, an additional 6 platelet concentrates were inoculated with sterile saline to serve as controls. Inoculated units were then subjected periodically to quantitative cultures and probe analyses. A total of 126 platelet concentrates were studied over a period of 7 days (120 inoculated with bacteria and 6 with sterile saline). RESULTS: This assay was, in some cases, able to detect S. aureus bacterial contamination in the range of 100 to 1000 CFUs per mL; the majority of samples (B. cereus, P. aeruginosa, S. aureus, and S. epidermidis) with contamination exceeding 10(4) CFUs per mL; and all samples with contamination of 2.1 × 10(5) CFUs per mL or greater. Increasing the sample size from the recommended 0.4 mL to 1.0 mL resulted in an unacceptable loss of specificity (83.3%). CONCLUSION: The routine use of this assay would be expected to result in a decreased risk of septic platelet transfusion reactions and could lead to a lengthening of the current 5 day storage period for platelets. Further, the pooling of random-donor platelet concentrates before storage instead of immediately before transfusion may be possible if this rRNA probe is employed to detect bacteria in the pool.     

Thrombopoietin and the TPO receptorduring platelet storage

BACKGROUND: For most cells, the addition of a specific growth factor has improved cellular viability by preventing programmed cell death (apoptosis). To determine whether the platelet-specific hematopoietic growth factor thrombopoietin (TPO) might improve platelet viability, endogenous TPO and the platelet TPO receptor were analyzed during storage, and the effect of recombinant TPO on platelet viability was assessed. STUDY DESIGN AND METHODS: During platelet storage, TPO stability was assessed by SDS-PAGE, TPO receptor function was measured, and the platelet TPO receptor was characterized by a (125)I-rHuTPO competitive-binding assay. A recombinant TPO, pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF), was added to platelet concentrates during storage, and its effect on pH, LDH, and metabolic activity was determined. RESULTS: During storage, the molecular weight and concentration of endogenous TPO (125 +/- 19 pg/mL) and exogenous TPO (5720 +/- 140 pg/mL) were constant for 12 days; the number (33 +/- 4), binding affinity (149 +/- 33 pM), and function of the platelet TPO receptors were constant for 7 days. Metabolic activity measured with the MTT and MTS assays closely correlated with changes in the pH and LDH. The addition of PEG-rHuMGDF did not alter the pH, LDH, or metabolic activity of platelets during storage, but it did increase by 65 percent the uptake of (35)S-methionine into platelets. Finally, platelet concentrates obtained from donors treated with PEG-rHuMGDF retained normal metabolic activity for 12 days, as compared with 5 to 6 days for normal platelet concentrates. CONCLUSIONS: TPO and its platelet receptor are present in normal amounts and have normal function during platelet storage. The addition of recombinant TPO increased platelet methionine transport but did not alter platelet viability during storage. Other means to prevent apoptosis during platelet storage should be considered, and the measurement of platelet metabolic activity by MTT and MTS assays may assist this effort.     

Thrombin receptor levels in platelet concentrates during storage and their impact on platelet functionality

BACKGROUND: Quality control of platelet (PLT) concentrates is challenging, due to PLT lesions, which are difficult to detect with routine methods. The search for reliable PLT lesion biomarkers is focused on the role of PLTs in primary hemostasis. PLT transfusions also have a significant impact on secondary hemostasis. In this phase, responsiveness of PLTs to small amounts of thrombin is crucial. PAR1 and PAR4 are protease‐activated receptors and are responsible for thrombin reactivity of human PLTs. This study should elucidate if levels of those two receptors are changing in PLT concentrates during storage and if those changes have an impact on PLT aggregation and support of thrombin generation. STUDY DESIGN AND METHODS: PLT concentrates from buffy coat preparations were stored in SSP+ solution for 9 days at 22 ± 2°C on a horizontal flatbed agitator, and samples were taken daily for analysis. PAR1 and PAR4 levels were evaluated using Western blot analysis. PLT aggregation was measured using Born aggregometry and specific PAR1 or PAR4 agonists. Thrombin generation was measured using calibrated automated thrombography. RESULTS: Levels of both receptors (PAR1 and PAR4) started to decrease after 5 days of storage. PAR1‐mediated PLT aggregation remained constant, whereas PAR4‐mediated PLT aggregation decreased with storage time. Rate of thrombin generation was accelerated after 5 days of storage. CONCLUSION: Decreasing levels of PARs in PLT concentrates after 5 days of storage influenced PAR4‐mediated, but not PAR1‐mediated, aggregation. Thrombin generation with senescent PLTs was increased, which may be attributed to other mechanisms promoting increased phosphatidylserine exposure.     

Recovery of in vitro functional activity of platelet concentrates stored at 4 degrees C and treated with second-messenger effectors

BACKGROUND: The potential for bacterial contamination limits the storage of platelets at 22 degrees C to 5 days. Refrigerated storage at 4 degrees C would abrogate this problem but would also result in a rapid loss of in vitro viability and functional activity and in vivo viability. The inhibition of platelets during storage by a combination of specific, reversible, second-messenger effectors has been investigated to allow prolonged storage at 4 degrees C with significant retention of in vitro viability and functional activity. STUDY DESIGN AND METHODS: The combination of effectors was added directly to platelet concentrates, and this step was followed by storage at 4 degrees C. Control units were incubated at 4 degrees C without the effectors and at 22 degrees C according to standard blood-banking techniques. At 1, 5, and 9 days, the units were tested for recovery of cell number, recovery of in vitro functional activity and viability, and expression of platelet surface markers. RESULTS: Treated platelets stored at 4 degrees C for 9 days, while spherical in shape, displayed no loss of cell number and had a recovery of viability and functional activity, as compared with control platelets stored at 22 degrees C for 5 days, as follows: ADP and collagen aggregation responses of 250 and 100 percent, respectively; a 70-percent recovery of hypotonic shock response; and a 60-percent recovery of extent of shape change. The treated platelets also expressed an equivalent amount of the surface marker glycoprotein lb and a lower amount of the activation marker alpha-granule membrane protein-140 on the membrane surface. CONCLUSION: Second-messenger effectors added to platelets significantly maintained in vitro functional activity with storage at 4 degrees C. In vitro analysis demonstrates the potential for extended 4 degrees C storage of platelets with numerical and functional recovery comparable to that achieved with current methods. Refrigerated storage of platelet concentrates has the potential to reduce the risk of bacterial contamination.     

Stored platelets alter glycerophospholipid and sphingolipid species,which are differentially transferred to newly released extracellular vesicles

BACKGROUND: Stored platelet concentrates (PLCs) for transfusion develop a platelet storage lesion (PSL), resulting in decreased platelet (PLT) viability and function. The processes leading to PSL have not been described in detail and no data describe molecular changes occurring in all three components of stored PLCs: PLTs, PLC extracellular vesicles (PLC‐EVs), and plasma. STUDY DESIGN AND METHODS: Fifty PLCs from healthy individuals were stored under standard blood banking conditions for 5 days. Changes in cholesterol, glycerophospholipid, and sphingolipid species were analyzed in PLTs, PLC‐EVs, and plasma by mass spectrometry and metabolic labeling. Immunoblots were performed to compare PLT and PLC‐EV protein expression. RESULTS: During 5 days, PLTs transferred glycerophospholipids, cholesterol, and sphingolipids to newly formed PLC‐EVs, which increased corresponding lipids by 30%. Stored PLTs significantly increased ceramide (Cer; +53%) and decreased sphingosine‐1‐phosphate (?53%), shifting sphingolipid metabolism toward Cer. In contrast, plasma accumulated minor sphingolipids. Compared to PLTs, fresh PLC‐EVs were enriched in lysophosphatidic acid (60‐fold) and during storage showed significant increases in cholesterol, sphingomyelin, dihydrosphingomyelin, plasmalogen, and lysophosphatidylcholine species, as well as accumulation of apolipoproteins A‐I, E, and J/clusterin. CONCLUSION: This is the first detailed analysis of lipid species in all PLC components during PLC storage, which might reflect mechanisms active during in vivo PLT senescence. Stored PLTs reduce minor sphingolipids and shift sphingolipid metabolism toward Cer, whereas in the plasma fraction minor sphingolipids increase. The composition of PLC‐EVs resembles that of lipid rafts and confirms their role as carriers of bioactive molecules and master regulators in vascular disease.     

The use of a chemiluminescence-linked universal bacterial ribosomal RNA gene probe and blood gas analysis for the rapid detection of bacterial contamination in white cell-reduced and nonreduced platelets

Because of the rising incidence of bacterial growth and septic platelet transfusions in aging units, platelet storage is currently limited in the United States to 5 days. This approved shelf life of platelets might be altered if methods were devised to rapidly detect infected units and/or to decrease the incidence of bacterially contaminated platelets. An investigation was conducted on the effect of a prototype blood collection system with an in-line filter for the production of white cell-reduced platelet-rich plasma on the growth of bacteria in platelets prepared from whole blood that had been inoculated with Staphylococcus epidermidis. Additional studies were conducted with a chemiluminescence-linked ribosomal RNA (rRNA) gene probe and with blood gas analysis to identify possible methods for the rapid detection of bacterial contamination. All units were followed for 9 days of storage. The filtration of the platelet-rich plasma resulted in an approximate 2 log10 reduction in white cells with an average loss of 6.7 percent of platelets. Filtration did not appear to alter bacterial growth. In all platelet units that supported growth, pO2 dropped to negligible values and pCO2 rose relative to culture-negative units. The changes were most sensitive and specific beyond 5 days of storage. The universal bacterial rRNA probe assay was able to detect S. epidermidis in concentrations as low as 1 × 10(3) colony-forming units per mL in some cases and reliably detected all units contaminated at a concentration of 1 × 10(4) colony-forming units per mL.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alterations in cytoskeletal organization and tyrosine phosphorylation in platelet concentrates prepared by the buffy coat method

BACKGROUND: Numerous morphologic and biochemical changes occurring during platelet storage may result in the impairment of platelet function. STUDY DESIGN AND METHODS: The effect of preparation and storage conditions on platelet function was analyzed through evaluation of cytoskeletal organization and signaling mechanisms involved in the activation of platelets by thrombin. Samples of platelets prepared by the buffy coat method were obtained before and after the platelet concentrates were prepared during storage for 1, 3, and 5 days. Thrombin-induced aggregation was monitored, and changes in the organization of proteins in the cytoskeleton were analyzed by gel electrophoresis. For the analysis of tyrosine phosphorylation, proteins were transferred to nitrocellulose membranes and probed with a specific antibody. RESULTS: The aggregation and the cytoskeletal organization induced by thrombin activation were markedly impaired immediately after preparation of platelet concentrates, although they normalized after the first 24 hours of storage and decreased progressively after 3 days of storage. Results in tyrosine phosphorylation paralleled those obtained with cytoskeletal organization, except for samples obtained immediately after processing to obtain platelet concentrates. CONCLUSION: These data indirectly suggest that the stress induced by the preparation method has an activating effect on platelet function that may imply a delayed platelet response to further stimuli. This effect may result in a deficient redistribution of signaling molecules within platelets.