Human Platelet Suspensions Research Articles

Novel Methods to Quantitate and Identity Dividing Platelets in Culture Conditions

Background: Unlike dividing cells, platelets are usually regarded as unproductive in that they are anuclear fragments of megakaryocyte and incapable of the canonical cell cycle. However, recent evidence showed that platelets could form progeny or at least undergo fission. The observation indicated the existence of a non-hematopoietic mechanism to determine platelet count in peripheral blood. The assessing the kinetic aspects of platelet division is not easy because automated cell counters often give equivocal results after culture not supporting those previous findings. Manual chamber counting is impractical because of the difficulty in differentiating singlets from any small sized clumps.Objective: We examined platelet division under in-vitro culture conditions to assess how many platelets and how fast they divide.Methods: We devised a method to track platelet division based on flow cytometry and fluorescent microscopic image. Briefly, buffered human platelet suspension was prepared and stained with cell tracking dyes followed by sorting and resuspension in cell culture medium and incubation. Platelet division was assessed quantitatively by observing a decrease in fluorescent intensity by flow cytometry after culture in a liquid medium or counting the number of single colored platelet doublets after culture in hydrogel medium.Results: After 6-hour culture of CFSE labelled platelet in M199 medium, we observed the appearance of a separate cluster of platelets with reduced CFSE intensity adjacent to the original platelet cluster, as is the typical finding of CFSE dilution assay for lymphocyte proliferation. We continued the culture for 20 hours. The division fraction of strongly labeled platelets was 48.7 % (range: 27.5 - 70.4) after 6 hours and 85.6 % (79.1 - 92.0) after 20 hours. For weakly labelled platelets, the division fraction after 6 hours was 35.0 % (24.6 - 43.4) and 75.5 % (34.5 - 97.0) after 20 hours. The division fractions at the two time points were much higher than expected and the effect of external stimuli such as shearing force exposure during the sorting procedure was to be considered. In another assay, we divided platelet suspension into two and stained with labelling dyes of different colors (CFSE and FarRed). The two fractions were mixed and cultured in hydrogel medium. At the beginning of the culture and after 6 hours, we counted platelet singlets and doublets in ten randomly selected fields of con focal microscopy. Single colored doublets can form both by division and contact. Those with two colors can form only by contact. The count of single- and two-colored doublets can be estimated by binary probability prediction if the platelets don't divide. Initially, the doublet counts follow the binary probability prediction (P: 0.65 - 0.88), but after 6 hours, single colored doublets were observed more frequently than two colored doublets (P<0.01, in the order of 10-6). The division was suppressed by treatment with taxol (P =0.26), nocodazole (P=0.38), and cytochalasin D (P=0.61). From the counted numbers of doublets and singlets of the two colors, we could derive a formula for the platelet division fraction. The division fraction of FarRed stained platelets were 8.3 % (95% CI: 2.2 - 14.4), and that of the CFSE stained platelets were 11.9 % (5.4 - 18.4).Conclusion: We introduce novel methods to assess platelet division credibly in culture conditions which can be easily combined with functional studies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Mechanism of free radical generation in platelets and primary hepatocytes: A novel electron spin resonance study.

Oxygen free radicals have been implicated in the pathogenesis of toxic liver injury and are thought to be involved in cardiac dysfunction in the cirrhotic heart. Therefore, direct evidence for the electron spin resonance (ESR) detection of how D‑galactosamine (GalN), an established experimental hepatotoxic substance, induced free radicals formation in platelets and primary hepatocytes is presented in the present study. ESR results demonstrated that GalN induced hydroxyl radicals (OH•) in a resting human platelet suspension; however, radicals were not produced in a cell free Fenton reaction system. The GalN‑induced OH• formation was significantly inhibited by the cyclooxygenase (COX) inhibitor indomethasin, though it was not affected by the lipoxygenase (LOX) or cytochrome P450 inhibitors, AA861 and 1‑aminobenzotriazole (ABT), in platelets. In addition, the present study demonstrated that baicalein induced semiquinone free radicals in platelets, which were significantly reduced by the COX inhibitor without affecting the formed OH•. In the mouse primary hepatocytes, the formation of arachidonic acid (AA) induced carbon‑centered radicals that were concentration dependently enhanced by GalN. These radicals were inhibited by AA861, though not affected by indomethasin or ABT. In addition, GalN did not induce platelet aggregation prior to or following collagen pretreatment in human platelets. The results of the present study indicated that GalN and baicalein may induce OH• by COX and LOX in human platelets. GalN also potentiated AA induced carbon‑centered radicals in hepatocytes via cytochrome P450. The present study presented the role of free radicals in the pathophysiological association between platelets and hepatocytes.

A Real-Time Monitoring System to Assess the Platelet Aggregatory Capacity of Components of a Tissue-Engineered Blood Vessel Wall.

Native blood vessels contain both an antiaggregatory intimal layer, which prevents platelet activation in the intact vessel, and a proaggregatory medial layer, which stimulates platelet aggregation upon vascular damage. Yet, current techniques for assessing the functional properties of tissue-engineered blood vessels may not be able to assess the relative effectiveness of both these pro- and antiaggregatory properties of the vessel construct. In this study, we present a novel technique for quantitatively assessing the pro- and antiaggregatory properties of different three-dimensional blood vessel constructs made using a layered fabrication method. This technique utilizes real-time measurements of cytosolic Ca2+ signaling to assess platelet activation in fluorescently labeled human platelet suspensions using fluorescence spectrofluorimetry, while also permitting examination of thrombus formation upon the surface of the construct using fluorescent imaging of DiOC6-labeled platelets. Experiments using this method demonstrated that type I collagen hydrogels, commonly used as scaffolds for vascular tissue engineering, were unable to support significant platelet activation, while type I and III neo-collagen secreted from human coronary artery smooth muscle cells cultured within these hydrogels as the medial layer were able to support thrombus formation. The incorporation of an intimal layer consisting of human umbilical vein endothelial cells on top of the medial layer inhibited platelet activation and aggregation. These data demonstrate that the methodology presented here is able to quantitatively compare the capacity of different constructs to trigger or prevent platelet activation. As such, this technique may provide a useful tool for standardizing the assessment of the functional properties of tissue-engineered blood vessel constructs developed using different culturing techniques.

The effect of electromagnetic radiation emitted by display screens on cell oxygen metabolism - in vitro studies.

IntroductionResearch studies carried out for decades have not solved the problem of the effect of electromagnetic radiation of various frequency and strength on the human organism. Due to this fact, we decided to investigate the changes taking place in human blood platelets under the effect of electromagnetic radiation (EMR) emitted by LCD monitors.Material and methodsThe changes of selected parameters of oxygen metabolism were measured, i.e. reactive oxygen species concentration, enzymatic activity of antioxidant defence proteins – superoxide dismutase (SOD-1) and catalase (CAT) – and malondialdehyde concentration (MDA). A suspension of human blood platelets was exposed to electromagnetic radiation of 1 kHz frequency and 150 V/m and 220 V/m intensity for 30 and 60 min. The level of changes of the selected parameters of oxidative stress was determined after the exposure and compared to the control samples (not exposed).ResultsThe measurements revealed an increase of the concentration of reactive oxygen species. The largest increase of ROS concentration vs. the control sample was observed after exposure to EMF of 220 V/m intensity for 60 min (from x = 54.64 to x = 72.92). The measurement of MDA concentration demonstrated a statistically significant increase after 30-min exposure to an EMF of 220 V/m intensity in relation to the initial values (from x = 3.18 to x = 4.41). The enzymatic activity of SOD-1 decreased after exposure (the most prominent change was observed after 60-min and 220 V/m intensity from x = 3556.41 to x = 1084.83). The most significant change in activity of catalase was observed after 60 min and 220 v/m exposure (from x = 6.28 to x = 4.15).ConclusionsThe findings indicate that exposure to electromagnetic radiation of 1 kHz frequency and 150 V/m and 220 V/m intensity may cause adverse effects within blood platelets’ oxygen metabolism and thus may lead to physiological dysfunction of the organism.

The impact of electromagnetic radiation of different parameters on platelet oxygen metabolism - in vitro studies.

Electromagnetic radiation emitted by a variety of devices, e.g. cell phones, computers and microwaves, interacts with the human body in many ways. Research studies carried out in the last few decades have not yet resolved the issue of the effect of this factor on the human body and many questions are left without an unequivocal answer. Various biological and health-related effects have not been fully recognized. Thus further studies in this area are justified. A comparison of changes within catalase enzymatic activity and malondialdehyde concentration arising under the influence of the electromagnetic radiation emitted by car electronics, equipment used in physiotherapy and LCD monitors. The suspension of human blood platelets at a concentration of 1 × 109/0.001 dm 3, obtained from whole blood by manual apheresis, was the study material. Blood platelets were exposed to an electromagnetic field for 30 min in a laboratory stand designed for the reconstruction of the electromagnetic radiation generated by car electronics, physiotherapy equipment and LCD monitors. The changes in catalase activity and malondialdehyde concentration were investigated after the exposure and compared to the control values (unexposed material). An increase in catalase activity and malondialdehyde concentration was observed after 30 min exposure of platelets to EMF regardless of the radiation source. The most significant changes determining the degree of oxidative stress were observed after exposure to the EMF generated by car electronics. The low frequency electromagnetic fields generated by car electronics, physiotherapy equipment and LCD monitors may be a cause of oxidative stress in the human body and may lead to free radical diseases.

In vitro platelet antiaggregatory properties of 4-methylcoumarins

Platelets play a crucial role in physiological haemostasis. However, in coronary arteries damaged by atherosclerosis, enhanced platelet aggregation, with subsequent thrombus formation, is a precipitating factor in acute myocardial infarction. Current therapeutic approaches are able to reduce approximately one quarter of cardiovascular events, but they are associated with an increased risk of bleeding and in some resistant patients are not efficient. Some coumarins possess antiplatelet activity and, due to their additional antioxidant effects, may be promising drugs for use in combination with the present therapeutic agents. The aim of this study was to analyse a series of simple 4-methylcoumarins for their antiplatelet activity. Human plasma platelet suspensions were treated with different aggregation inducers [arachidonic acid (AA), collagen and ADP] in the presence of the 4-methylcoumarins. Complementary experiments were performed to explain the mechanism of action. 5,7-Dihydroxy-4-methylcoumarins, in particular those containing a lipophilic side chain at C-3, reached the activity of acetylsalicylic acid on AA-induced aggregation. Other tested coumarins were less active. Some of the tested compounds mildly inhibited either collagen- or ADP-induced aggregation. 5,7-Dihydroxy-4-methylcoumarins did not interfere with the function of thromboxane synthase, but were competitive antagonists of thromboxane A(2) receptors and inhibited cyclooxygenase-1 as well. 5,7-Dihydroxy-4-methylcoumarins appear to be promising candidates for the extension of the current spectrum of antiplatelet drugs.

Inhibition of thrombin-induced Ca2+ influx in platelets by R59949, an inhibitor of diacylglycerol kinase

The aim of this study was to determine whether diacylglycerol kinase (DGK) is involved in transplasmalemmal Ca²⁺ influx of platelets. Effects of R59949, an inhibitor of diacylglycerol kinase, on intracellular Ca²⁺ concentration ([Ca²⁺](i) ) and mRNA expression of DGK isozymes were investigated using washed human platelet suspensions. Thrombin-induced increase in [Ca²⁺](i) was significantly inhibited by pretreatment of platelets with R59949, while thapsigargin-induced increase in [Ca²⁺](i) was comparable in platelets with and without R59949 pretreatment. Thapsigargin-induced increase in [Ca²⁺](i) was markedly attenuated in the presence of SKF-96365. In the presence of SKF-96365, thrombin-induced increase in [Ca²⁺](i) was significantly attenuated, and additional treatment with R59949 caused a further decrease in [Ca²⁺](i) . Pretreatment of platelets with 1-butanol significantly attenuated thrombin-induced increase in [Ca²⁺](i) , while thrombin-induced increase in [Ca²⁺](i) was augmented in the presence of propranolol. mRNA expression of DGK-α and DGK-γ, which are known to be inhibited by R59949, in platelets was confirmed by RT-PCR analysis. R59949 inhibited a store-depletion-insensitive component of transplasmalemmal Ca²⁺ entry induced by thrombin, while store-operated Ca²⁺ entry was not affected by R59949. The results of this study suggest that phosphatidic acid is involved in thrombin-induced Ca²⁺ influx of platelets.

Transcription and translation of human F11R gene are required for an initial step of atherogenesis induced by inflammatory cytokines

Background -The F11 Receptor (F11R; aka JAM-A, JAM-1) is a cell adhesion protein present constitutively on the membrane surface of circulating platelets and within tight junctions of endothelial cells (ECs). Previous reports demonstrated that exposure of ECs to pro-inflammatory cytokines causes insertion of F11R molecules into the luminal surface of ECs, ensuing with homologous interactions between F11R molecules of platelets and ECs, and a resultant adhesion of platelets to the inflamed ECs. The main new finding of the present report is that the first step in this chain of events is the de-novo transcription and translation of F11R molecules, induced in ECs by exposure to inflammatory cytokines.Methods -The experimental approach utilized isolated, washed human platelet suspensions and cultured human venous endothelial cells (HUVEC) and human arterial endothelial cells (HAEC) exposed to the proinflammatory cytokines TNF-alpha and/or IFN-gamma, for examination of the ability of human platelets to adhere to the inflamed ECs thru the F11R. Our strategy was based on testing the effects of the following inhibitors on this activity: general mRNA synthesis inhibitors, inhibitors of the NF-kappaB and JAK/STAT pathways, and small interfering F11R-mRNA (siRNAs) to specifically silence the F11R gene.Results -Treatment of inflamed ECs with the inhibitors actinomycin, parthenolide or with AG-480 resulted in complete blockade of F11R- mRNA expression, indicating the involvement of NF-kappaB and JAK/STAT pathways in this induction. Transfection of ECs with F11R siRNAs caused complete inhibition of the cytokine-induced upregulation of F11R mRNA and inhibition of detection of the newly- translated F11R molecules in cytokine-inflamed ECs. The functional consequence of the inhibition of F11R transcription and translation was the significant blockade of the adhesion of human platelets to inflamed ECs.Conclusion -These results prove that de novo synthesis of F11R in ECs is required for the adhesion of platelets to inflamed ECs. Because platelet adhesion to an inflamed endothelium is crucial for plaque formation in non-denuded blood vessels, we conclude that the de-novo translation of F11R is a crucial early step in the initiation of atherogenesis, leading to atherosclerosis, heart attacks and stroke.

Electrochemical Measurement of Endogenous Serotonin Release from Human Blood Platelets

Platelet aggregation in the bloodstream is tightly associated with the secretory function of platelets based on several types of cytoplasmic secretory granules, each sequestering distinct chemical messenger species. Dense-body granules are one prominent type of secretory granule responsible for storing small molecule chemical messengers. Upon platelet activation, the timely and rapid release of these small molecules is critical in facilitating platelet aggregation. Therefore, techniques capable of measuring real-time granule content release are needed to understand the fundamental properties of platelet secretion and aggregation. Existing techniques lack adequate time resolution or require potentially toxic exogenous reagents for real-time measurement of granule content release. Herein, we demonstrate a label-free electrochemical method based on the endogenous electroactive chemical messenger serotonin (5-hydroxytryptamine or 5-HT) for the real-time measurement of dense-body granule secretion from platelet suspensions; fast-scan cyclic voltammetry (FSCV) using carbon-fiber microelectrodes was chosen on the basis of its excellent temporal resolution, high sensitivity, and the ability to provide the electrochemical signature cyclic voltammograms for molecular identification. Real-time serotonin release from thrombin-stimulated human platelet suspensions was successfully measured, and the amount and time course of the bulk serotonin release were found to agree well with data obtained from single platelet measurements, thus confirming accurate characterization of granular secretion. Furthermore, this electrochemical method was applied to study the stimulation-secretion coupling in platelets, serotonin storage and release dynamics with applied pharmacological agents, and chemical messenger storage deficiency in Hermansky-Pudlak Syndrome (HPS) platelets, and the potential of this method to reveal secretion behavior in both normal and diseased platelets has clearly been demonstrated.

Quantitative Analysis of Human Platelet Adhesions Under a Small‐Scale Flow Device

To realize real-time evaluation of human platelet adhesions onto material surfaces with small volumes of human platelet suspensions, we developed an apparatus consisting of a modified cone and plate-type viscometer, combined with an upright epi-fluorescence microscope. The apparatus allowed real-time evaluation of platelet-material interactions and the initial event of thrombus formation, using small platelet suspension volumes (7.5 microL) under shear flow conditions. To study the dynamic behavior of platelet-material interaction, we chose five representative opaque and transparent materials: acrylate resin (AC), polytetrafluoroethylene (PTFE), polyvynylchrolide (PVC), glass, and a monolayer of human normal umbilical cord vein endothelial cells (EC) on glass under shear flow conditions. The values of adhesiveness of human platelets to the test materials in descending order were as follows: AC > PTFE > PVC > glass > human EC. Under this new small-scale flow system, we could obtain highly reproducible data, which were comparable with results from a previously developed large-scale flow system. Therefore, the newly developed cone and plate-type rheometer is a useful instrument for testing and screening materials, and allows precise quantitative evaluation of human platelet adhesion.

LPS Mediates the Activation of Platelet by Toll-Like Receptor 4.

Abstract 3001Poster Board II-978Lipopolysaccsharide (LPS) is a principal outer membrane component of gram-negative bacteria. It initiates an inflammatory response to infection by activating Toll-like receptor-4 (TLR4) on various tissues or cells in host. Platelet contributes to the inflammation process through respond to invading pathogens, membrane adhesion molecule (CD62P, P-selectin) and CD40L on platelet are the indexes to determine platelet activation. The presentexperiment was designed to investigate the expression of Toll-like receptor 4 (TLR4) on platelet and to determine whether platelet TLR4 involves in platelet activation induced by Lipopolysaccsharide (LPS). Human platelet-rich plasma (PRP) and platelet suspension obtained from 15 healthy people were pretreated with a concentration of 0.2μg/ml of LPS in the presence or absence of thrombin (1 U/ml) for 1 hour. The expressions of TLR4,CD62P and CD40L on platelets were detected through flow cytometry, and platelet TLR4 was further determined by performing western blot analysis. The results show that the percentage of TLR4-positive platelet induced by thrombin was increased by 32.34% compared with the resting platelets (25.44%, P<0.01). TLR4 expression on platelets treated with LPS was remarkedly elevated in the presence or absent of thrombin. However, the expression level was much higher in presence of both than thrombin alone( 39.16%,P<0.01). Moreover, the similar results were found in Western blot analysis. Synchronously, the expressions of CD62P and CD40L on resting platelets were 6.39% and 2.45%, they were also markedly increased when treated with thrombin(42.68% and 14.80%) and LPS respectively, and the increase of CD62P was more significant when stimulated with both of LPS and thrombin(63.03%). Although anti-TLR4 antibody inhibited significantly the increases of TLR4, CD62P and CD40L on platelets induced by LPS, it didn't effect their increases induced by thrombin. The experiment results support the evidence that functional TLR4 can be expressed on human platelet. It may involve in platelet activation as an important mediator of LPS- induced CD62P and CD40L expressions on platelets. Disclosures:No relevant conflicts of interest to declare.

The effect of 50 hz magnetic field of different shape on oxygen metabolism in blood platelets: in vitro studies

The aim of the study was to assess the influence that the shape of low frequency magnetic field may have on catalase and superoxide dismutase activity, malondialdehyde concentration and free radicals generation in human blood platelets. The suspension of human blood platelets was exposed for 15 min to 50 Hz magnetic field of different shape, and flux density of 10 mT. The determinations of free radicals, malondialdehyde and catalase showed increased values compared with the initial level, regardless of the shape of the magnetic field applied. In contrast, superoxide dismutase activity was lower than at the onset of the experiment. The findings indicate that the oxidative stress resulting from exposure to 50 Hz magnetic field of 10 mT induction may produce a number of adverse effects within the cell and thus may lead to systemic disturbances in the human body.

FcγRII mediates platelet aggregation caused by disintegrins and GPIIb/IIIa monoclonal antibody, AP2

Disintegrins, snake venom-derived Arg-Gly-Asp (RGD)-containing polypeptides, and GPIIb/IIIa antagonist (AP2) block fibrinogen binding to GPIIb/IIIa of activated platelets, however, the combination of these two agents caused platelet aggregation. We hypothesize that disintegrin initially binds to specific epitope of GPIIb/IIIa, causing conformational change, and the recruitment of FcgammaRII, which can be bound by AP2, and finally triggering platelet aggregation. We prepared human platelet suspensions and measured platelet aggregation, Ca2+ mobilization, thromboxane B2 formation, and signal transduction. Disintegrin (e.g., accutin) and AP2 (a monoclonal antibody [mAb]-raised against GPIIb/IIIa) individually inhibited human platelet aggregation caused by collagen. However, as both accutin and AP2 were sequentially added into platelet suspension, platelet aggregation occurred. Accutin/AP2 caused shape change, cytosolic Ca2+ mobilization, P-selectin expression, and thromboxane A2 formation. Tirofiban, FcgammaRII mAb, or indomethacin completely inhibited platelet aggregation caused by accutin/AP2. Accutin/AP2 also caused tyrosine phosphorylation of signal molecules. Disintegrins enhanced AP2 binding to platelets, and AP2 also promoted disintegrin binding to platelets. FcgammaRII mAb inhibited the enhanced fluorescein isothiocyanate-disintegrin binding to platelet caused by AP2. Immunoprecipitation of the lysates of disintegrin/AP2-treated platelets using FcgammaRII Ab showed complex formation of GPIIb/IIIa and FcgammaRII. FcgammaRII mediates platelet aggregation caused by disintegrin and AP2, triggering a phospholipase C, phospholipase A2, Src-, Syk kinases, and Ca2+-dependent activation process. AP2 triggers platelet aggregation via binding to accessible FcgammaRII and the conformation-altered GPIIb/IIIa caused by disintegrin.

Platelet counting-development of a reference method and a reference preparation.

A reference method has been defined for platelet counting by counting chamber haemocytometry, and a procedure has been developed, using platelet rich plasma (PRP) and a Coulter ZBI counter which provides a reliable, rapid and relatively simple method for platelet counting which is closely comparable to the reference method and can thus be used both as a secondary reference method and a routine method. The optimal method for obtaining PRP from EDTA blood was by augmented sedimentation with Boyum's methyl cellulose-metrizoate mixture at sp. gr. 1.08. This was shown to yield a platelet suspension which reflects closely the platelet content of the original blood. Calibration of electronic counters for platelet counting requires material which is stable and which parallels natural human platelets in size, distribution and other physical characteristics. A suspension of glutaraldehyde-fixed human platelets in glycerol appears to be suitable as a reference preparation. Its method of production is described.

Platelet Apoptosis in Fluid-Phase and Adherent Platelets and Thrombi-Like Platelet Aggregates.

Apoptosis, or programmed cell death, is the physiologic mechanism that serves for controlled deletion of unwanted cells. Apoptosis was initially attributed exclusively to nucleated cells but over the past decade it has been recognized that apoptosis also occurs in anucleated cytoplasts and platelets. In this study, using flow cytometry we analyzed in human platelets three critical manifestations of mitochondrial, cytoplasmic and plasma membrane apoptosis, mitochondrial inner transmembrane potential (Δψm) depolarization, caspase-3 activation and phosphatidylserine (PS) externalization, respectively. We found that these hallmarks of apoptosis can be induced in human platelet suspension by diverse stimuli, including human α-thrombin (1, 10, 100 nM), calcium ionophore A23187 (3, 5, 10 μM), high shear stresses generated by cone-and-plate viscometer (120, 200, 390 dyn/cm2) and prolonged storage of platelet concentrates in blood banking conditions at 22°C for 6 and 13 days. We also demonstrated that these apoptotic markers can be induced in mouse platelets in vivo in a murine model of immune thrombocytopenia caused by injection of anti-glycoprotein (GP) IIb (rat anti-mouse GPIIb, MWReg30) antibody. Other manifestations of apoptosis were detected in human platelets, including expression of proapoptotic members of Bcl-2 family proteins (Bax and Bak) induced by thrombin, and platelet shrinkage and shedding of microparticles induced by high shear stresses. In addition to apoptosis in fluid-phase platelets, apoptosis was also revealed by confocal fluorescent microscopy in adherent human platelets and thrombi-like platelet aggregates deposited on thrombogenic immobilized human vascular collagen types I and III, as detected by PS exposure and shedding of PS-exposed microparticles. Taken together, these data suggest that platelet apoptosis is a phenomenon that can be triggered by a wide diversity of chemical and physical stimuli using different mechanisms mediated by thrombin-, collagen- and integrin GPIIbIIIa-receptors, mechanoreceptors and Ca2+-overloading. These stimuli trigger platelet apoptosis by impacting on several intracellular apoptotic targets, including shifting the balance between Bcl-2 regulatory proteins in a proapoptotic direction, depolarizing the inner mitochondrial membrane, activating the executioner caspase-3, stimulating aberrant PS exposure on the platelet surface and, eventually, resulting in ‘terminal' stages of platelet apoptosis, such as platelet shrinkage and shedding of PS-exposed microparticles resembling apoptotic bodies. Platelet apoptosis can be induced both in fluid-phase and adherent platelets and thrombi-like platelet aggregates. These data also indicate that natural PL agonists thrombin and subendothelial vascular collagens and hemodynamic shear forces, can be involved not only in the processes of hemostasis, thrombosis and blood coagulation but also can trigger platelet death via apoptosis. Platelet apoptosis may contribute to the pathophysiology of thrombocytopenia in diseases associated with enhanced thrombin generation, such as sepsis and disseminated intravascular coagulation, as well as in autoimmune and alloimmune thrombocytopenias.

Low Concentrations of Resveratrol Potentiate the Antiplatelet Effect of Prostaglandins

Resveratrol, a polyphenolic compound found in grapes and other fruits, is thought to contribute to the cardioprotective effect of red wine. While resveratrol exhibits some antiplatelet effect in vitro, the concentrations needed are much higher than those in plasma after consumption of red wine. In the present study, we investigate if resveratrol is able to potentiate the effect of endogenous antiplatelet substances--prostaglandin (PG) I2 and PGE1. In human platelet suspension resveratrol at relatively low concentrations (2 or 5 microM), which did not affect platelet function, significantly enhanced the inhibitory activity of PGs on platelet aggregation caused by collagen. The mechanisms underlying this effect may be associated with the inhibition of protein kinase C activation and protein tyrosine phosphorylation, but not with cyclic nucleotide levels and intracellular calcium mobilization in platelets. Our results might provide a possible explanation for the in vivo antiplatelet effect of resveratrol despite the poor bioavailability and the weak in vitro activity.

Baicalein Induction of Hydroxyl Radical Formation via 12-Lipoxygenase in Human Platelets: An ESR Study

The pro-oxidant activities of baicalein, morin, myricetin, quercetin, and rutin were examined in various cell-containing systems including human platelets, rat vascular smooth muscle cells, human umbilical vein endothelial cells (HUVECs), human THP-1 cells, and fibroblast cells. Electron spin resonance (ESR) results showed that only baicalein generated hydroxyl radicals in a resting human platelet suspension, whereas the other flavonoids showed no effects on any of the resting cell systems. A low concentration of arachidonic acid (AA) increased the intensity of hydroxyl radicals, but a high concentration inhibited it. Collagen and thrombin, platelet aggregatory agents that can cause the release of AA by platelets, enhanced baicalein-induced hydroxyl radical formation, whereas ADP and U44619 showed no significant effects. Quinacrine and 5,8,11,14-eicosatetraenoic trifluoromethyl ketone, both PLA2 inhibitors, significantly attenuated baicalein-induced hydroxyl radical formation. These results suggest that baicalein-induced hydroxyl radical formation is associated with AA metabolite enzymes in human platelets. The formation of hydroxyl radicals was significantly inhibited by lipoxygenase inhibitors including nordihydroguaiaretic acid, (-)-epicatechin, (-)-epicatechin gallate, and hinokitiol, but was not affected by desferroxamine or the heme protein inhibitors KCN and NaN3. On the other hand, semiquinone free radicals were generated when baicalein was incubated with horseradish peroxidase/H2O2 or platelets/AA. The semiquinone radicals formed in the platelets/AA system could be extensively inhibited by desferroxamine, diethylenetriaminepentaacetic acid, KCN, and NaN3, indicating that prostaglandin H synthase (PGHS)-peroxidase may be involved. The results of this study led to the proposal that baicalein induces hydroxyl radical formation via 12-lipoxygenase and induces semiquinone radical formation via PGHS-peroxidase in human platelets.

Inhibitory mechanisms of activated matrix metalloproteinase-9 on platelet activation

The intracellular mechanisms underlying the signaling pathways of activated matrix metalloproteinase-9 (MMP-9) in platelets are not yet completely understood. Therefore, the aim of this study was to further examine the effects of activated MMP-9 in preventing platelet aggregation. In this study, activated MMP-9 time-dependently (3–60 min) inhibited platelet aggregation in washed human platelet suspensions stimulated by agonists. However, activated MMP-9 had no significant effect on the binding of FITC-triflavin to the platelet glycoprotein IIb/IIIa complex. Triflavin is a specific antagonist of the glycoprotein IIb/IIIa complex purified from snake venom. Moreover, activated MMP-9 (21 and 90 ng/ml) markedly decreased the fluorescence intensity of platelet membranes tagged with diphenylhexatriene. The thrombin-evoked increase in pHi was inhibited in the presence of activated MMP-9 (21 and 90 ng/ml). In addition, activated MMP-9 (21 and 90 ng/ml) markedly reduced the electron spin resonance (ESR) signal intensity of hydroxyl radicals in collagen (1 μg/ml)-activated platelets. These results indicate that the antiplatelet activity of activated MMP-9 may involve the following pathways: (1) activated MMP-9 may initially induce conformational changes in platelet membranes and hydroxyl radical formation, leading to inhibition of platelet aggregation; and (2) activated MMP-9 also inhibits the Na +/H + exchanger, leading to reduced intracellular Ca 2+ mobilization, and ultimately to inhibition of platelet aggregation. This study further provides new insights concerning the effects of activated MMP-9 on platelet aggregation.

Pathogen Control in Complex Fluids with Water‐coupled Excimer Lamps at 282 and 308 nm

Water-coupled excimer lamp systems have been developed to inactivate microorganisms within complex, low-optical quality, fluids. Monochromatic lamps were selected to minimize UV-B and UV-C absorption within the carrier fluids while maximizing deposition within specific chemical targets. Fundamentals, system scaling and power supply design are discussed. This work used two large-surface area excimer lamps as intense sources of near monochromatic radiation at 308 and 282 nm. Data are presented for two distinct fluid systems: flow-through processing of large-volume metalworking fluids used in heavy industry and batch irradiation of human blood plasma and platelet suspensions used in transfusion medicine. In the first, a 200-600 L/min reactor is used to control bacterial concentrations within metalworking fluids used in large-scale metal machining processes. Control is defined as the maintenance of 10(3) to 10(4) CFU/mL in fluids that without treatment would have concentrations over 10(7) CFU/mL. The second is a batch process for viral inactivation in undiluted blood bank products. Samples of fresh frozen plasma and platelet suspensions were spiked with high titers of porcine parvovirus (PPV) and irradiated at 308 and 282 nm. Although both wavelengths were effective at reducing PPV levels, 308 nm light resulted in both higher rates of viral inactivation (greater than 6 log units) and lower rates of fluid degradation.

Mechanisms Involved in the Antiplatelet Activity of Ketamine in Human Platelets

The aim of this study was to systematically examine the inhibitory mechanisms of ketamine in platelet aggregation. In this study, ketamine concentration-dependently (100–350 µM) inhibited platelet aggregation both in washed human platelet suspensions and platelet-rich plasma stimulated by agonists. Ketamine inhibited phosphoinositide breakdown and intracellular Ca²⁺ mobilization in human platelets stimulated by collagen. Ketamine (200 and 350 µM) significantly inhibited thromboxane (Tx) A₂formation stimulated by collagen. Moreover, ketamine (200 and 350 µM) increased the fluorescence of platelet membranes tagged with diphenylhexatriene. Rapid phosphorylation of a platelet protein of Mr 47,000 (P47), a marker of protein kinase C activation, was triggered by phorbol-12,13-dibutyrate (100 nM). This phosphorylation was markedly inhibited by ketamine (350 µM). These results indicate that the antiplatelet activity of ketamine may be involved in the following pathways. Ketamine may change platelet membrane fluidity, with a resultant influence on activation of phospholipase C, and subsequent inhibition of phosphoinositide breakdown and phosphorylation of P47, thereby leading to inhibition of intracellular Ca²⁺ mobilization and TxA₂ formation, ultimately resulting in inhibition of platelet aggregation.