The swirling phenomenon in stored platelets is influenced by their endogenous serotonin

The presence of the swirling phenomenon is useful to define platelet concentrates that are suitable for transfusion. If it is possible to identify donor-related factors which are related to persisting swirling during storage, it is possible to select platelet donors. Endogenous platelet serotonin content is stable and easily measured and related to agonist-induced serotonin secretion. During a 3-month period, the swirling in 825 single donor platelet concentrates was controlled before issue. Endogenous serotonin, % serotonin release and swirling were tested in 21 concentrates with poor or no swirling during storage. Sixty-three concentrates were randomly selected from the routinely prepared platelet concentrates and were routinely tested with the same analyses on days 1 and 7. To evaluate an obvious effect of endogenous serotonin on the swirling phenomenon, eight platelet concentrates prepared from buffy coat, each from four donors, were divided. One part was stored in the presence of 8.5 micromol serotonin L-1, and analysed as the control concentrates. The endogenous serotonin content in the 'low- swirling' concentrates was significantly lower than in the control group (P < 0.001). PCO2 and pH were significantly lower, and PO2 and MPV significantly higher than in the controls. In the control group, swirling after 7 days was significantly correlated with serotonin release. In the eight buffy-coat concentrates enriched in endogenous serotonin, both swirling and the percentage serotonin release were improved after storage for 10 days, compared with nonenriched concentrates. This study suggests that endogenous serotonin content and serotonin release are factors that may be of significance concerning preservation of the swirling phenomenon in platelet concentrates during storage.     

Thrombin-induced serotonin release as an in vitro indicator of the functional integrity of stored platelets

Measurements of endogenous serotonin and the thrombin-induced serotonin release reaction in platelet concentrates were compared with other methods of quality control: particle counting and pH and gas analyses. A discrepancy between the serotonin release reaction and the other data was observed after seven days of storage. The decline in the release reaction was not predictable from pH measurements or the number of platelets or leukocytes in the concentrate. There was a significant positive correlation between endogenous serotonin content and the thrombin-induced serotonin secretion after seven days of storage. We conclude that the current methods for routine control of platelet concentrates may not ensure the quality of the product after it has been stored for seven days. The thrombin-induced serotonin release may provide a valuable addition to the battery of tests available.     

Apoptotic activity in stored human platelets

BACKGROUND: Platelets possess some of the machinery required for apoptotic cell death. However, disruption of mitochondria function, implicated in several models of cell death, has not been extensively studied in platelets. Mitochondrial viability and several other measures of apoptotic death in stored and experimentally stressed platelets were evaluated. MATERIALS AND METHODS: Platelet mitochondrial transmembrane potentials (Deltapsim) were studied by staining platelets with JC-1, a dye that fluoresces at different wavelengths based on the state of mitochondrial polarization. Annexin V binding, a measure of phosphatidylserine (PS) exposure, and CD62P expression, an indicator of platelet activation, were determined by flow cytometry. Caspase-3 activity was measured with an enzyme assay and by Western blotting. Experimental platelet stressors included storage for 7 days, azide exposure, calcium ionophore stimulation, and plasma deprivation. RESULTS: As measured by flow cytometry, Deltapsim values were similar in freshly drawn platelets and in platelet concentrates stored for up to 7 days. However, compared to fresh platelets, stored platelet concentrates had significantly increased PS exposure (3.1 vs. 5.1%, p = 0.015), CD62P expression (6.5 vs. 13.5%, p = 0.0067), and caspase-3 activity. Azide exposure, which decreased ATP release 20 to 30 percent, did not affect the Deltapsim. Stressed platelets exhibited higher degrees of mitochondrial depolarization in response to calcium ionophore stimulation than platelets that were not stressed. Plasma deprivation also resulted in significant alterations in Deltapsim, PS exposure, and CD62P expression. CONCLUSIONS: Platelet mitochondria maintain Deltapsim when stored for up to 7 days under standard blood bank storage conditions. Therefore, changes in platelet mitochondria Deltapsim do not correlate with downstream markers of apoptotic death such as caspase activation and PS exposure.     

Amphotericin B-induced injury in stored human platelets

Administration of intravenous amphotericin B is a major factor in the reduction of the recovery of transfused platelet concentrates (PCs). For the study of possible drug-induced platelet lesions, fresh platelets and stored PCs were incubated with therapeutic concentrations of amphotericin B (10-50 micrograms/mL) and examined for membrane changes by scanning electron microscopy. Stored platelets demonstrated the formation of "pits" on the surface membrane, which were maximal immediately after preparation and gradually decreased with storage. The number of pits was significantly higher after exposure to amphotericin B. No increase was detected in fresh platelets following exposure to amphotericin B. The maximal effect of the drug was seen after 5 days of storage as PCs. Amphotericin B did not affect platelet shape or the number of pseudopodia. There was no correlation between pit formation and the pH, pO2, or pCO2 of the concentrates. Amphotericin B did not release 51Cr from prelabeled stored platelets after 2 hours' incubation at 37 degrees C. Thus, amphotericin B appears to exacerbate a membrane lesion induced by the preparation and storage of PCs.     

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.     

Influence of pH on stored human platelets

BACKGROUND: During storage at room temperature, platelets (PLTs) undergo several changes, a process known as PLT storage lesion. The pH is one of the variables changing and has been suggested to be a good surrogate marker for the quality of PLT concentrates. It is unknown whether the pH decrease as such induces the PLT storage lesion or that the deterioration of the PLTs results in the pH decrease. In this study, the responses of PLTs to applied pH values were investigated. STUDY DESIGN AND METHODS: PLTs were washed three times in PLT additive solution (PAS-IIIM) and were resuspended in PAS-IIIM buffers with or without 30 percent plasma with different pH values over a range from 6.0 to 7.5 (at 37 degrees C). The PLTs were stored in 50-mL culture flasks at 22 degrees C. RESULTS: During 3 days of storage in 100 percent additive solution (AS), the extracellular pH did not affect in vitro quality measures. Both at the lower and at the higher end of the pH range, we observed an increased glycolytic flux, accelerated at Day 6. Also in the presence of 30 percent plasma, the effect of extracellular pH was very limited, but all variables indicated better PLT quality with stable values up to Day 6 of storage. CONCLUSIONS: We conclude that PLTs stored in 100 percent AS are able to cope with high and low pH values without a strong deterioration within 3 days. PLTs stored in 30 percent plasma-70 percent AS are more capable in dealing with different pH values than PLTs stored in AS and remained stable for 6 days. We suggest that the pH decrease is a result of the PLT storage lesion and not the cause.     

The influence of irradiation on stored platelets

Platelet concentrates intended for transfusion to immunosuppressed patients are irradiated to minimize transfusion-induced graft-versus-host disease. Because few reports describe how irradiation influences stored platelets, the authors studied whether 5000 rad of gamma irradiation, the maximum dose currently used clinically, altered platelets in vitro. Platelet concentrates were stored for either 1 day or 5 days in plastic (PL 732) containers before gamma irradiation. One unit of a pair of identical platelet concentrates was irradiated; the second unit served as a control. Irradiation did not alter platelet morphology, mean platelet volume, expression of platelet-factor-3 activity, response to hypotonic stress, extent of discharge of lactate dehydrogenase, release of beta-thromboglobulin, formation of thromboxane B2, nor the ability to undergo synergistic aggregation. The lack of any substantial change was observed whether the platelet concentrates were stored initially for either 1 day or 5 days. These results suggest that stored platelets are not altered deleteriously by irradiation with 5000 rad.     

In vitro evaluation of platelets stored in CDP-adenine formulations

Little information is available about the effect of adenine and added glucose on stored platelets. Two new formulations, CPDA-2 and CPDA-3, contain 34 mg adenine per 63 ml preservative and extra glucose (1.75 and 2.0 times the glucose in standard CPD). We have studied the in vitro integrity of platelet concentrates stored in CPD, CPDA-1, CPDA-2, and CPDA-3 at 22 C for 72 hours. Morphology score, pH, platelet size, population distribution parameters, and electron microscopic ultrastructure did not show any adverse effects which could be ascribed to the presence of adenine or extra glucose or both. No differences in platelet adenosine triphosphate (ATP) concentration or plasma glucose utilization during storage were found between CPD and CPDA-1 platelets. The results suggest that adenine and added glucose in these preservatives are not detrimental to platelets in vitro by the measures employed.     

Interaction of long-term stored platelets with vascular subendothelium

Platelets stored as platelet-rich plasma under mildly alkaline conditions in vitro maintain morphologic integrity and functional capability for 2 to 3 weeks. We used the Baumgartner method to assess the ability of long-term stored platelets to interact with exposed vascular subendothelium. After 3 days in storage the size and number of thrombi on damaged surfaces decreased. However, the percentage of vascular surface covered was not reduced significantly until the cells had been stored for 14 days. Treatment of vascular segments with chymotrypsin to increase thrombogenicity caused platelets stored for 14 days to adhere and form aggregates on denuded surfaces in a manner similar to that of fresh platelets, but the ability to develop thrombi remained depressed. Thus long-term storage that maintains physical and functional integrity also preserves a reasonable capacity of platelets to interact with damaged blood vessels.     

Energy substrate metabolism in fresh and stored human platelets

The latent capacity of human platelets for oxidizing several important energy-yielding substrates has been revealed by hypoosmolaric incubation conditions.The data show that the human platelet has a considerable capacity to oxidize both glucose and long-chain fatty acids. Long-chain fatty acids appear to rank favorably with glucose as a potential energy substrate. In a number of mammalian tissues, (-)-carnitine serves to regulate the rate at which long-chain fatty acids are oxidized. Evidence was obtained which suggests that (-)-carnitine functions in a similar role in the platelet.After storage of human platelets at 4 degrees C for 24 hr, the oxidative capacity for glucose was reduced by approximately 25% and for long-chain fatty acids by almost 50%. Investigation of the component parts of the metabolic pathways indicated that a marked decrease in the capacity of the Krebs cycle could be responsible for the decrement in energy substrate oxidation.     

贮存血小板形态变化与凋亡因子磷脂酰丝氨酸表达的研究

目的探讨储存期间机采血小板形态变化、凋亡因子磷脂酰丝氨酸(phosphatidyl serine,PS)表达以及两者之间的关系,为确立血小板体外保存时限提供实验依据。方法应用May-Grunwald-Giemsa染色方法观察机采血小板储存0-8d时的形态变化,同时应用流式细胞术检测血小板的膜PS表达率。结果储存至第4天,血小板形态出现损伤,表现为形态学计分与新鲜血小板相比显著下降(t=2.341,P<0.05);随着储存时间延长,血小板形态学计分持续下降,至第7天,形态学计分下降31%。血小板凋亡因子PS表达,储存1d组与新鲜血小板0d组相比有明显升高(t=3.088,P<0.05);储存1-3d,PS表达无明显变化,储存至第4天,PS表达显著增加(t=2.1612,P<0.05);储存5-8d,PS表达持续增加,各组间有统计学差异(P均<0.05)。血小板形态学变化分值与膜PS的表达随储存时间延长呈负相关性(r=-0.9923,P<0.01)。结论储存血小板形态学变化分值与膜PS表达率高度负相关。     

Decreased reuptake of serotonin in human platelets after surgery

We have documented earlier a decrease in platelet serotonin and a concurrent increase in plasma serotonin, 5‐hydroxytryptamin (5‐HT) after various forms of stress, suggesting a disturbed platelet 5‐HT reuptake mechanism following stress. In order to further elucidate these findings, we have studied platelet 5‐HT reuptake kinetics (V_max and K_m) in nine patients before and 4 days after major, uncomplicated abdominal surgery. We found a significant decrease in the maximal 5‐HT reuptake velocity (V_max) after surgery and changes in K_m, verifying alterations in the affinity of the platelet 5‐HT transport system. The present results thus confirm the hypothesis that 5‐HT reuptake kinetics are altered following adrenergic hyperactivity. A decrease in platelet 5‐HT reuptake may bear implications for our understanding of poststress adaptive changes in the cardiovascular system as well as in the central nervous system (CNS) serotonergic neurones following stressful stimulation.