The miRNA Profile of Platelets Stored in a Blood Bank and Its Relation to Cellular Damage from Storage

Millions of blood products are transfused each year, and many lives are directly affected by transfusion. Platelet concentrate (PC) is one of the main products derived from blood. Even under good storage conditions, PC is likely to suffer cell damage. The shape of platelets changes after 5 to 7 days of storage at 22°C. Taking into consideration that some platelet proteins undergo changes in their shape and functionality during PC storage. Sixteen PC bags were collected and each PC bag tube was cut into six equal pieces to perform experiments with platelets from six different days of storage. Thus, on the first day of storage, 1/6 of the tube was used for miRNA extraction, and the remaining 5/6 was stored under the same conditions until extraction of miRNAs on each the following five days. Samples were sequenced on an Illumina Platform to demonstrate the most highly expressed miRNAs. Three miRNAs, mir127, mir191 and mir320a were validated by real-time quantitative PCR (RQ-PCR) in 100 PC bags tubes. Our method suggests, the use of the miRNAs mir127 and mir320a as biomarkers to assess the "validity period" of PC bags stored in blood banks for long periods. Thus, bags can be tested on the 5th day of storage for the relative expression levels of mir127 and mir320a. Thus, we highlight candidate miRNAs as biomarkers of storage damage that can be used as tools to evaluate the quality of stored PC. The use of miRNAs as biomarkers of damage is unprecedented and will contribute to improved quality of blood products for transfusions.     

MicroRNA 320a and Membrane Antigens as Tools to Evaluate the Pathophysiology of Platelets Stored in Blood Banks

Our research group, through the analysis of miRNomes in platelet concentrates (PCs) stored in blood banks, identified and validated the miR-127 and miR-320a miRNAs as biomarkers of platelet storage lesions (PSLs) in PCs. In order to validate the miRNAs 127 and 320a methodologically, as PSL biomarkers in a large number of PC bags, we also evaluated important immunological markers involved in the platelet activation/aggregation process—the CD62P receptor (P-selectin), the surface glycoproteins (GP) IIb/IIIa, and the purinergic P2Y12 receptor—via flow cytometry. The miRNAs miR-127 and miR-320a were quantified by real-time quantitative PCR (RT-qPCR). To carry out this study, 500 collection tubes were used at the upper edge of the PC bags containing platelets. Each tube was divided into seven equal parts (totaling 3500 samples) for platelet analysis from 7 different storage days, where the 1st day represents the high-quality control, and the 7th day corresponds to the low-quality control of the platelets. After analyzing all parameters during storage days, it was concluded that the relative quantification of miR-320a below 0.50 and the CD62P receptor below 27.92% are reliable indicators of the absence of storage lesions in blood banks. We believe that the values found in the expression of the CD62P receptor legitimize the use of the miR-320a and miR-127 miRNAs to build a kit capable of accurately measuring whether the stored platelets are suitable for transfusion.     

Storage of apheresis platelets in ethylene-vinyl acetate copolymer bags: relationship between the bag size and the number of platelets maintaining aerobic metabolism

The present study was designed to determine whether a bag made from ethylene-vinyl acetate copolymer (EV) with superior flexibility at subzero temperature is suitable for a storage container of single-donor apheresis platelets. Apheresis platelets were stored with 100 ml plasma in 1-liter bags made of EV or standard polyvinyl chloride (PVC) plastic at 22 degrees C with constant agitation. The oxygen permeability of the 1-liter EV bag averaged 1447 nmol/min/atm, which was about 1.5 times higher than that of PVC bags. The partial oxygen tension (PO2) of platelet concentrates (PC) has linearly decreased to 16 mm Hg with increasing platelet counts. The level of the partial carbon dioxide was always higher in EV bags than in PVC bags. Oxygen consumption rates of platelets stored in EV and PVC bags with a sufficient oxygen supply averaged 1.25 and 1.20 nmol/min/10(9) platelets, respectively. The rates of glucose consumption and lactate production were not changed in two bags. Ninety percent of the total ATP production of about 8 nmol/min/10(9) platelets were generated through the aerobic metabolism. The platelet counts in the 1-liter EV and PVC bags, at which PO2 is 16 mm Hg, were 2.2 and 1.5 x 10(11) platelets, respectively. The study indicates that apheresis platelets stored in EV bags at 22 degrees C have no different metabolic changes when compared with those of PVC bags. In addition, the number of platelets maintaining the aerobic metabolism is 1.5 times higher than that of PVC bags.     

Molecular link between membrane cholesterol and Na+/H+ exchange within human platelets.

Incubation of human platelets with cholesterol-poor, cholesterol-normal and cholesterol-rich liposomes revealed that: (i) acquisition or depletion of platelet membrane cholesterol was highly selective; (ii) variation in membrane cholesterol was highly selective. Variation in membrane cholesterol content (cholesterol-to-phospholipid molar ratio from 0.15-1.2) with respect to values found in unmodified normal platelets, was paralleled by the observed changes in amiloride-sensitive cytoplasmic pH, as well as phospholipase A2 activity. However, a decrease in cytoplasmic pH was accompanied by an increase in phospholipase A2 activity; (iii) membrane cholesterol-modulated changes in intra-platelet pH, as well as phospholipase A2 activity, was completely inhibited when platelets were pretreated with quinacrine (a specific phospholipase A2 inhibitor) before exposure to various types of liposomes. Although exposure of platelets (pretreated with amiloride) with various types of liposomes resulted in the inhibition of Na+/H+ exchange it had no noticeable effect upon the observed phospholipase A2 activity. Based upon these results we suggest that membrane cholesterol-modulated phospholipase A2 activity may be the basic mechanism responsible for the nature of Na+/H+ exchanger activity observed in cholesterol-enriched platelets, leading these platelets to a hypersensitized state.     

DNA damage in non-proliferating cells subjected to ionizing irradiation at high or low dose rates

Ionizing radiation damage to the genome of a non-cycling mammalian cell is analyzed using continuous time Markov chains. Immediate damage induced by the radiation is modeled as a batch Poisson arrival process of DNA double strand breaks (DSBs). Different kinds of radiation, for example gamma rays or alpha particles, have different batch probabilities. Enzymatic modulation of the immediate damage is modeled as a Markov process similar to the processes described by the master equation of stochastic chemical kinetics. An illustrative example is the restitution/complete exchange model, which postulates that radiation induced DSBs can subsequently either undergo enzymatically mediated repair (restitution) or can participate pairwise in chromosome exchanges, some of which make irremediable lesions such as dicentric chromosome aberrations. One may have rapid irradiation followed by enzymatic DSB processing or have prolonged irradiation with both DSB arrival and enzymatic DSB processing continuing throughout the irradiation period. A complete solution of the Markov chain is known for the case that the exchange rate constant is negligible so that no irremediable chromosome lesions are produced and DSBs are the only damage to the genome. Using PDEs for generating functions, a perturbation calculation is made assuming the exchange rate constant is small compared to the repair rate constant. Some non-perturbative results applicable to very prolonged irradiation are also obtained using matrix methods: Perron-Frobenius theory, variational methods and numerical approximations of eigenvalues. Applications to experimental results on expected values, variances and statistical distributions of DNA lesions are briefly outlined.Continuous time Markov chain models are the most systematic of those current radiation damage models which treat DSB-DSB interactions within the cell nucleus as homogeneous (e.g. ignore diffusion limitations). They contain most other homogeneous models as special cases, limiting cases or approximations. However, applying the continuous time Markov chain models to studying spatial dependence of DSB interactions, which is generally believed to be very important in some situations, presents difficulties.     

Freeze preservation of platelets using hydroxyethyl starch (HES); a preliminary report.

A comparative study has been made of platelets stored by freeze preservation following treatment with dimethyl sulfoxide (DMSO) or hydroxyethyl starch (HES) with fresh platelets and platelets stored at 4 °C for 48 hr. The indices studied were platelet recovery, pH, light microscope morphology, platelet Factor 3 (PF₃) availability and the hypotonic stress response. The DMSO preserved platelets gave a better response to hypotonic stress and incurred lesser degrees of membrane damage as demonstrated by PF₃ availability. There was however a significantly higher recovery of platelets treated with HES; with DMSO the osmotic damage inflicted during removal caused considerable lysis. Platelets frozen by DMSO or HES gave consistently better in vitro results than platelets stored at 4 °C for 48 hr. A preliminary clinical trial of HES preserved platelets has confirmed haemostatic effectiveness in vivo. HES being relatively nontoxic, platelets can be infused immediately after thawing and with minimal post thaw manipulation, thus maintaining a relatively closed system. It is concluded that cryopreservation with HES is a practical and effective means for long term platelet storage.     

Low density lipoproteins inhibit the Na+/H+ antiport in human platelets via activation of p38MAP kinase

Low density lipoproteins (LDL) inhibit the Na+/H+ antiport and thereby sensitize platelet towards agonist. However, mechanisms underlying the suppressing effect of LDL on Na+/H+ exchange are unclear. We here show that the lowering of intracellular pH and the suppression of the sodium propionate-induced Na+/H+ exchange in the presence of LDL are abolished by SKF86002, a selective inhibitor of p38MAP kinase (p38MAPK). The inhibitory effect of LDL on Na+/H+ exchange was mimicked by H2O2, which directly activates p38MAPK. Exposure of platelets to LDL or H2O2 led to phosphorylation of p38MAPK, its upstream regulator MAP kinase kinase 3/6 (MKK 3/6), and its downstream target heat shock protein 27 (HSP27), and this effect was abrogated in SKF86002-pretreated platelets. In addition, both LDL and H2O2 produced the SKF86002-sensitive phosphorylation of an oligopeptide encompassing p38MAPK phosphorylation sequence derived from NHE-1, a major Na+/H+ exchanger in platelets. We further show that the sensitizing effects of LDL on the thrombin-induced platelet activation, as reflected by aggregation and granule secretion, are abolished in cells pretreated with SKF86002. We conclude that activation of p38MAPK is required for the inhibitory effect of LDL on Na+/H+ antiport and thereby for LDL-dependent sensitization in human platelets.     

Decomposition of cellulose,soil organic matter and plant litter in a temperate grassland soil

The formation of mor humus in an experimental grassland plot, which has been acidified by long-term fertiliser treatment, has been studied by comparing the rates of cellulose, soil organic matter and plant litter decay with those in an adjacent plot with near-neutral pH and mull humus. The decomposition of cellulose filter paper in litter bags of 5 mm, 1-mm and 45-μm mesh size buried at 3 to 4 cm depth the plots was followed by measuring the weight loss and changes in glucose content over a 6 month period. Soil pH was either 5.3 or 4.3. Decomposition of native soil organic matter and plant litter in soil from the same plots were followed using CO₂ evolution in laboratory microcosms. Cellulose weight loss at pH 5.3 was greatest from the 5-mm mesh bags and least from the 45-um mesh bags. At pH 4.3 there was little weight loss from bags and no significant differences in weight loss between bags with different sized mesh. There was, however, a reduction in the glucose content of the hydrolysed and derivatised filter paper with time. The decomposition rate of native soil organic matter in the low pH soil was increased to that observed in the less acid soil when the pH of the former was increased from 4.3 to 5.3. The increase in decomposition rate of added plant litter in the more acid soil as a result of CA(OH)₂ addition was only 60% of that observed in the soil with pH 5.3. These data support the hypothesis that the absence of soil animals and the restricted microbial decomposition in the acidic soil was responsible for mor humus formation.     

Ca2+ transport across the platelet plasma membrane. A role for membrane glycoproteins IIB and IIIA

Human platelets maintain a low cytosolic free Ca2+ concentration in part by controlling plasma membrane Ca2+ transport. The present studies examine the role in this process of two well-characterized membrane proteins: glycoproteins IIb and IIIa. These glycoproteins form a Ca2+-dependent complex which serves as both the platelet fibrinogen receptor and the principle site for high affinity Ca2+ binding on the platelet surface. The kinetics of plasma membrane Ca2+ exchange were compared in normal platelets and in thrombasthenic platelets, which lack the IIb X IIIa complex. Under steady-state conditions, the maximum rate of plasma membrane Ca2+ exchange in the thrombasthenic platelets was half the rate observed in normal platelets. The size of the cytosolic exchangeable Ca2+ pool and the cytosolic free Ca2+ concentration, however, were normal. A quantitatively similar decrease in plasma membrane Ca2+ exchange was seen in normal platelets after incubation with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) at 37 degrees C, conditions that dissociate the IIb X IIIa complex. This decrease in the Ca2+ exchange rate in normal platelets could be prevented by preincubating platelets with a complex-specific anti-IIb X IIIa monoclonal antibody, but not by preincubating platelets with an anti-IIIa monoclonal antibody. In order to determine whether loss of the IIb X IIIa complex primarily affects Ca2+ influx or Ca2+ efflux, both processes were also examined under nonsteady-state conditions. An immediate decrease in the 45Ca2+ influx rate was seen when Ca2+ was added back to platelets preincubated with EGTA at 37 degrees C. The 45Ca2+ efflux rate, on the other hand, was not immediately affected. These data suggest, therefore, that an intact IIb X IIIa complex is necessary for normal Ca2+ homeostasis in platelets.     

Inhibition of Na+/H+ exchange reduces Ca2+ mobilization without affecting the initial cleavage of phosphatidylinositol 4,5-bisphosphate in thrombin-stimulated platelets

Stimulation of human platelets increases cytoplasmic pH (pHi) via activation of Na+/H+ exchange. We have determined the effect of inhibiting Na+/H+ exchange on (i) thrombin-induced Ca2+ mobilization and (ii) turnover of 32P-labelled phospholipids. Blocking Na+/H+ exchange by removal of extracellular Na+ or by ethylisopropylamiloride (EIPA) inhibited Ca2+ mobilization induced by 0.2 U/ml thrombin, whereas increasing pHi by NH4Cl enhanced the thrombin-induced increase in cytosolic free Ca2+. The effect of EIPA was bypassed after increasing pHi by moneasin. The thrombin-induced cleavage of phosphatidylinositol 4,5-bisphosphate (PIP2) was unaffected by treatments that blocked Na+/H+ exchange or increased pHi. It is concluded that activation of Na+/H+ exchange is a prerequisite for Ca2+ mobilization in human platelets but not for the stimulus-induced hydrolysis of PIP2.     

Serine base exchange enzyme in porcine lyophilised platelets: enzyme properties and modulation by AIF4-and different types of heparin

Phosphatidylserine is one of the PKC modulators and thus it may play an important role in signal transduction. Regulation of the synthesis of this phospholipid is not yet clarified. The contrasting reports are possibly related to the existence of different enzymes which, in mammalian tissues, catalyse the exchange between free serine and the nitrogen base of a membrane phospholipid. This study demonstrates that serine base exchange reactions of commercially available lyophilised porcine platelets exhibit similar pH optima, temperature and Ca²⁺ dependence as observed in fresh tissues. Analysis of fatty acids composition of the three phospholipid classes involved in base exchange reactions also demonstrated a similarity with fresh platelets. Serine and ethanolamine base exchange enzyme activities were assayed in parallel in platelet lysate subjected to preincubation at various temperatures (30-60°C). When dithioerithrol was omitted from the incubation medium, the two base exchange reactions were inhibited with a similar temperature-dependent pattern. Addition of the reducing agent enhanced the sensitivity to preincubation only for the serine base exchange reaction which was inhibited by 80% after preincubation at 45°C. With respect to its regulation, porcine platelet serine base exchange enzyme(s) was inhibited by fluoroalluminate, a widely used G-protein activator, and stimulated by unfractionated heparin. Low mol. wt. heparin did not influence enzyme activity. Unfractionated heparin greatly stimulated SBEE activity assayed at pH 7.4, a pH value far from the optimal pH.     

pH and urea dependence of amide hydrogen-deuterium exchange rates in the beta-trefoil protein hisactophilin.

Amide hydrogen/deuterium exchange rates were measured as a function of pH and urea for 37 slowly exchanging amides in the beta-trefoil protein hisactophilin. The rank order of exchange rates is generally maintained under different solution conditions, and trends in the pH and urea dependence of exchange rates are correlated with the rank order of exchange rates. The observed trends are consistent with the expected behavior for exchange of different amides via global and/or local unfolding. Analysis of the pH dependence of exchange in terms of rate constants for structural opening and closing reveals a wide range of rates in different parts of the hisactophilin structure. The slowest exchanging amides have the slowest opening and closing rates. Many of the slowest exchanging amides are located in trefoil 2, but there are also some slow exchanging amides in trefoils 1 and 3. Slow exchangers tend to be near the interface between the beta-barrel and the beta-hairpin triplet portions of this single-domain structure. The pattern of exchange behaviour in hisactophilin is similar to that observed previously in interleukin-1 beta, indicating that exchange properties may be conserved among beta-trefoil proteins. Comparisons of opening and closing rates in hisactophilin with rates obtained for other proteins reveal clear trends for opening rates; however, trends in closing rates are less apparent, perhaps due to inaccuracies in the values used for intrinsic exchange rates in the data fitting. On the basis of the pH and urea dependence of exchange rates and optical measurements of stability and folding, EX2 is the main exchange mechanism in hisactophilin, but there is also evidence for varying levels of EX1 exchange at low and high pH and high urea concentrations. Equilibrium intermediates in which subglobal portions of structure are cooperatively disrupted are not apparent from analysis of the urea dependence of exchange rates. There is, however, a strong correlation between the Gibbs free energy of opening and the denaturant dependence of opening for all amides, which suggests exchange from a continuum of states with different levels of structure. Intermediates are not very prominent either in equilibrium exchange experiments or in quenched-flow kinetic studies; hence, hisactophilin may not form partially folded states as readily as IL-1 beta and other beta-trefoil proteins.     

Sodium fluoride prevents receptor- and protein kinase C-mediated activation of the human platelet Na+/H+ exchanger without inhibiting its basic pHi-regulating activity

When human platelets are stimulated with thrombin or activators of protein kinase C, cytosolic pH (pHi) increases due to activation of Na+/H+ exchange. In order to further elucidate the molecular mechanisms that regulate the exchanger, we used sodium fluoride, which is a known activator of guanine nucleotide-binding proteins (G proteins) in platelets. Although NaF induced the mobilization of Ca2+ from intracellular storage sites in fura2-loaded platelets, it failed to raise pHi as determined from the fluorescence of 2,7-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein-loaded platelets. Furthermore, when thrombin (0.1 unit/ml) or the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) had raised pHi from 7.13 +/- 0.05 to 7.35 +/- 0.07 (n = 30), addition of NaF (2.5-10 mM) rapidly restored pHi to values found before stimulation. Conversely, preincubation of platelets with low concentrations of NaF (2.5 mM) completely prevented alkalinization in response to thrombin or TPA. Unlike ethylisopropylamiloride, which completely blocked Na+/H+ exchange, NaF did not prevent the recovery of pHi from an artificial acid load. Hence, the inhibitory action of NaF is restricted to receptor-mediated activation of the antiport. In order to investigate whether the NaF effect was attributable to a G protein, platelets were preincubated with N-ethylmaleimide (50 microM), which is known to inhibit the adenylyl cyclase-inhibitory G protein. N-Ethylmaleimide treatment not only prevented inhibition of adenylyl cyclase by epinephrine but also completely reversed the inhibitory effect of NaF on the Na+/H+ exchanger. Our data suggest the existence of a novel G protein which is activated by fluoride and functions as a negative regulator of the Na+/H+ exchanger in platelets.     

Simultaneous flow cytometric measurements of thrombin-induced cytosolic pH and Ca2+ fluxes in human platelets

Human platelets exhibit an extremely rapid increase in cytoplasmic Ca2+ concentrations ((Ca2+]in) and a dose-dependent cytoplasmic pH change ((pH]in) upon thrombin stimulation. A cytoplasmic alkalinization, maximal by 60 s, is preceded by a very rapid acidification, which is masked by the alkalinization when saturating thrombin doses are used. Using the pH probe 2',7'-bis-(carboxyethyl)-5(6)-carboxyfluorescein we report here the kinetics of simultaneous cytoplasmic pH and Ca2+ changes in thrombin-stimulated platelets, measured in single cells by flow cytometry. This permits analysis of the responding subpopulation. Maximal thrombin stimulation (greater than or equal to 4.5 nM) induces a dose-dependent increase in pHin from approximately 7.0 to 7.30 and a maximal [Ca2+]in transient of up to 800 nM. The Ca2+ transient coincides temporally with the rapid initial acidification, while the alkalinization is maximal considerably later. The Ca2+ transients occur maximally in each responding cell, but occur only in a subpopulation of the platelets at subsaturating (less than 4.5 nM) thrombin doses; in contrast, the dose-dependent cytoplasmic acidification appears to occur uniformly in all platelets. The rapid increase in [Ca2+]in is not dependent on the alkalinization, and the former occurs maximally in amiloride treated, Na+/H+ exchange inhibited human platelets. These results indicate that the acidification and the rise in [Ca2+]in may be interrelated, whereas the cytoplasmic alkalinization (maximal considerably later than either the acidification or the [Ca2+]in rise) may be independent of these earlier, temporally correlated increases in H+ and Ca2+ concentrations.     

Thyroid hormone stimulates expression of 6-phosphofructo-2-kinase in rat liver

Gel-filtrated human platelets were stimulated with thrombin in the absence and presence of adrenaline. Adrenaline markedly enhanced the thrombin-induced increase in cytoplasmic pH (pH_i) in BCECF-loaded platelets. This rise in pH_i was strongly inhibited by the Na⁺/H⁺ exchange blocker EIPA. The potentiation by adrenaline of thrombin-induced PLC activation measured as [³²P]PA formation and final platelet responses was, however, not blocked by EIPA, even at low concentrations of thrombin. These results indicate that the enhancement by adrenaline of thrombin-induced cytoplasmic alkalinization may be a secondary effect which is not essential for the potentiation by adrenaline of platelet activation by thrombin.     

Microbiology of pozol,a Mexican fermented maize dough

Mexican fermented maize dough, pozol, including traditional banana leaf-wrapped samples and material in plastic bags, was purchased. All samples were pH 4.7 to 5.7 approx. 12 h after preparation, pH declining to 3.6 to 3.9 after 6 to 9 days storage at ambient temperature. These latter samples had dry matter contents of 31% to 48% (w/w), 0.35% to 0.75% titratable acidity as lactic acid and lactic acid bacteria as predominant microbial flora at about 10⁸ c.f.u./ml. The lactic acid bacteria included strains of Leuconostoc mesenteroides, Lactobacillus plantarum, Lactobacillus confusus, Lactococcus lactis and Lactococcus raffinolactis. Fungi were not found in the samples stored in plastic bags. The samples wrapped in banana leaf, however, developed a large surface mycoflora within 2 days. This included Geotrichum candidum, yeasts and moulds. The majority of the lactic acid bacteria and approx. 50% of yeasts hydrolysed starch to some extent. No Geotrichum isolate hydrolysed starch. Lactate was assimilated by all the Geotrichum isolates and by 17 of 39 yeast strains.     

Na+/H+ metabolic activity in the thrombocytes of spontaneously hypertensive rats

The sodium-proton exchange was determined in platelets of spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). The platelets were suspended in sodium propionate; the cytoplasmic acidification activated the exchanger and intracellular pH (the increasing) and volume of the platelets (the swelling) were registered. The activity of Na+/H+ exchange was inhibited by isopropyl amiloride. The platelets' volume and the exchange rate constant of SHR were increased on 30-40% as compared with those of WKY.     

Leaching of plasticizers from and surface characterization of PVC blood platelet bags.

The leaching of phthalate plasticizers from four types of blood platelet bags was investigated. The anticoagulant solutions used in the blood collection bags had pH values of 5.64 +/- .04 and contained no detectable amounts of phthalates. Platelet bag materials from each bag were soaked in normal salines for up to 5 days. The salines were tested for the leached phthalates from the bags but none could be found. However, di-(2-ethylhexyl) phthalate (DEHP) leached out of the PL-146 and Terumo bags into bovine calf serum used for soaking the bag materials. There was an increase in the amount of DEHP leached from about 1.1 mg at the end of one day to about 3.3 mg per gm of bag material at the end of a five day extraction with the serum. In PL-732 sets, a platelet bag made of a specialty polyolefin, the amount of DEHP leached out was less than 0.02 mg per g of bag material. CLX bags, which contained tri-(2-ethylhexyl) trimelliate (TETM) as a plasticizer, showed a negligible amount of it leaching into the calf serum. Infra-red spectra showed that PL-146 bags had been coated with a layer of a fatty acid amide while the Terumo bags contained a layer of a silicone fluid on their inner surfaces. CLX bags showed a coating of stearates, which were probably soaps of calcium or zinc. Scanning electron microscopy (SEM) showed that the inner surfaces of each brand of the bag were distinctly different morphologically. The two PVC bags were very similar whereas the surfaces morphology of PL-732 was rougher. Terumo bag had a different surface morphology than those of the other bags whereas the CLX bags had a very regular surface pattern. The exact significance of the surface morphology is not certain but excessively rough surfaces may not be desirable for the bags.     

Cytosolic alkalinization alone is not sufficient for Ca2+ mobilization, phosphatidic acid formation, and protein phosphorylation in human platelets

One of the earliest events following stimulation of human platelets with thrombin is a rise in the cytosolic pH, pHi, mediated by Na+/H+ exchange, and an increase in the cytosolic free Ca2+ concentration, [Ca2+]i. In the present study we investigated whether an increase in pHi alone, induced by the Na+/H+ ionophore monensin, is sufficient for platelet activation. Although monensin (20 microM) raised pHi from 7.10 +/- 0.05 (n = 21) to 7.72 +/- 0.17 (n = 13), neither Ca2+ influx nor mobilization were detectable upon this treatment in fura2-loaded platelets. In contrast, thrombin (0.05 U/ml) raised pHi to 7.31 +/- 0.10 (n = 10) and increased [Ca2+]i by more than 250 nM both in the presence and absence of extracellular Ca2+. Thrombin also caused the formation of phosphatidic acid and phosphorylation of the 20 kDa and 47 kDa proteins in platelets labeled with 32P. Monensin, however, induced none of these responses. It is concluded that an increase in pHi alone is not a sufficient trigger for platelet activation but enhances intracellular signal transduction in platelets stimulated by natural agonists.     

Molecular pathways used by platelets to initiate and accelerate atherogenesis

PURPOSE OF REVIEW: The response to injury model in the development of atherosclerosis is broadly accepted by the scientific audience. Platelets are generally not believed to be involved in the initiation of atherosclerosis. New data imply, however, that the response to injury model is too simple for a complete understanding of the inflammatory disease atherosclerosis. The involvement of platelets in the initiation of atherosclerotic lesion formation is critical in directing the atherosclerotic process into regeneration or ongoing vascular injury. RECENT FINDINGS: Platelets internalize oxidized phospholipids and promote foam cell formation. Platelets also recruit circulating blood cells including progenitor cells to the vessel, that are able to differentiate into foam cells or endothelial cells depending on conditions. Platelets express various scavenger receptors that are able to regulate LDL-uptake. LDL-laden platelets are internalized by adherent progenitor cells that in turn differentiate into macrophages and foam cells. SUMMARY: An expanding body of evidence continues to build on the role of platelets as initial actors in the development of atherosclerotic lesions. Platelets bind to leukocytes, endothelial cells, and circulating progenitor cells and initiate monocyte transformation into macrophages. Therefore platelets regulate the initiation, development and total extent of atherosclerotic lesions.