Platelet inhibition by nitrite is dependent on erythrocytes and deoxygenation

BACKGROUND: Nitrite is a nitric oxide (NO) metabolite in tissues and blood, which can be converted to NO under hypoxia to facilitate tissue perfusion. Although nitrite is known to cause vasodilation following its reduction to NO, the effect of nitrite on platelet activity remains unclear. In this study, the effect of nitrite and nitrite+erythrocytes, with and without deoxygenation, on platelet activity was investigated. METHODOLOGY/FINDING: Platelet aggregation was studied in platelet-rich plasma (PRP) and PRP+erythrocytes by turbidimetric and impedance aggregometry, respectively. In PRP, DEANONOate inhibited platelet aggregation induced by ADP while nitrite had no effect on platelets. In PRP+erythrocytes, the inhibitory effect of DEANONOate on platelets decreased whereas nitrite at physiologic concentration (0.1 μM) inhibited platelet aggregation and ATP release. The effect of nitrite+erythrocytes on platelets was abrogated by C-PTIO (a membrane-impermeable NO scavenger), suggesting an NO-mediated action. Furthermore, deoxygenation enhanced the effect of nitrite as observed from a decrease of P-selectin expression and increase of the cGMP levels in platelets. The ADP-induced platelet aggregation in whole blood showed inverse correlations with the nitrite levels in whole blood and erythrocytes. CONCLUSION: Nitrite alone at physiological levels has no effect on platelets in plasma. Nitrite in the presence of erythrocytes inhibits platelets through its reduction to NO, which is promoted by deoxygenation. Nitrite may have role in modulating platelet activity in the circulation, especially during hypoxia.     

Cholesterol exchange in platelets, erythrocytes and megakaryocytes

Cholesterol exchange between plasma and human platelets and erythrocytes and guinea pig platelets, erythrocytes and megakaryocytes was studied. The characteristics of exchange of cholesterol between [3H]cholesterol-labeled plasma and human platelets and erythrocytes were similar: exchange per cell was independent of cell concentration in whole plasma, decreased only 2-fold over a wide range of cell concentrations in low concentrations of plasma and approached a plateau at 1/3 normal plasma cholesterol concentration, and there was no net change in the cholesterol content of either cell. The activation energy for exchange for both cells was 47 kJ/mol. In all experiments, erythrocyte cholesterol was labeled to approximately twice the specific activity of platelet cholesterol. Guinea pig megakaryocyte cholesterol exchanged at 25-33% of the rate of guinea pig platelet cholesterol in vitro. Similarly, when guinea pigs were fed [3H]cholesterol, erythrocyte cholesterol specific activity after 24 h was 90%, platelet 50-65%, and megakaryocyte 20-26% that of plasma. Guinea pig platelets incubated with plasma radiolabeled in free and esterified cholesterol incorporated radioactivity from free but not esterified cholesterol. The similarity of free cholesterol exchange in platelets and erythrocytes in vitro and in vivo and the apparent inability of platelets to take up cholesterol esters from lipoproteins suggest that the interaction between normal platelets and normocholesterolemic plasma is limited to cholesterol exchange.     

Vitamin E exerts antiaggregatory effects without inhibiting the enzymes of the arachidonic acid cascade in platelets

Vitamin E (α-tocopherol) and tocopherol acetate produced a slightly increased amount of thromboxane in treated compared to untreated platelets. In tocopherol acetate-treated platelets significantly more lipoxygenase products were produced. α-tocopherol induced an increased, but not significant, production of thromboxane B₂ during blood clotting. α-tocopherol was not found to affect platelet phospholipase activity as determined by its effect on the release of labelled arachidonic acid from platelet phospholipids by challenging the platelets with calcium ionophore A23,187. α-tocopherol potentiated the incorporation of labelled arachidonate in the platelet phospholipids. Inspite of having no effect on the arachidonic acid cascade in platelets, α-tocopherol inhibited aggregation induced by several aggregating agents including A23,187. Inhibition of aggregation may be explained by the ability of α-tocopherol to inhibit intracellular mobilization of sequestered calcium from the dense tubular system to the cytoplasm.     

Erythrocytes infected by Plasmodium falciparum activate human platelets

Blood platelets are involved in Plasmodium falciparum malaria pathology as shown by thrombocytopenia and increased plasma level of two alpha granule proteins: beta thromboglobulin (beta TG) and platelet factor 4 (PF4). In this study we demonstrate that Plasmodium falciparum parasitized erythrocytes activate directly the secretion of beta TG and PF4 by human platelets. This secretion is related to parasitemia and occurs immediately after contact. Treatment of parasited erythrocytes by trypsin and diffusion chamber experiments suggest that platelet activation is triggered by parasitic substances shed on erythrocyte membrane and released in the culture medium.     

Hydrolysis of guanosine and adenosine 3',5'-monophosphates by rat blood.

Cyclic nucleotide phosphodiesterase activity was measured in whole blood, plasma, and suspensions of platelets and erythrocytes from rats. In fresh whole blood, apparent phosphodiesterase activity was low, but it rose strikingly during the hour after blood withdrawal. The apparent phosphodiesterase activity in platelet-free plasma showed no such increase, but that in platelet-enriched plasma increased in parallel with that in whole blood. The apparent phosphodiesterase activity of blood or of platelet-enriched plasma also was increased markedly by sonication. The increase in rat blood phosphodiesterase activity with aging thus appeared to be due to damage of platelets. Most of the phosphodiesterase activity in rat erythrocytes and platelets was located in the soluble fraction of sonicated preparations, but the total enzyme activities from the two sources exhibited marked differences in substrate specificity. With erythrocyte preparations, the rate of hydrolysis of muM concentrations of cyclic AMP was approx. 50 times that of cyclic GMP, while with platelet preparations, cyclic GMP was hydrolyzed about 20 times faster than cyclic AMP at muM levels. The activity of phosphodiesterase in platelets was much greater than that in erythrocytes at all concentrations of both substrates.     

A high-performance liquid chromatographic method for the determination of tocopherol in plasma and cellular elements of the blood.

A rapid sensitive, and reproducible procedure is described for the analysis of alpha-tocopherol in blood cells and plasma using high-performance liquid chromatography and fluorometric detection. The cardinal feature for the increased sensitivity of this high-performance liquid chromatographic procedure is that the fluorometric analysis was carried out at a short excitation wavelength (205 nm) which increased the sensitivity of 20-fold over the usual excitation wavelength of 295 nm. Tocopherol levels can be measured in as little as 50 microliters of plasma and 200 microliters of erythrocytes. The tocopherol contentof plasma, red blood cells, platelets, polymorphonuclear leukocytes, and lymphocytes of normal subjects and subjects ingesting additional quantitites of vitamin E are reported. The values for the white cells are approximately 30 times higher than those of the red blood cells (polymorphonuclear leukocytes 4.47 +/- 0.62 micrograms/10(9), lymphocytes 3.89 +/- 0.85 micrograms/10(9), and erythrocytes 1.40 +/- 0.14 micrograms/10(10) cells). The tocopherol contents of the plasma and all the cellular elements of the blood were increased by oral feeding with vitamin E.     

Thrombin Stimulates Glucose Transport in Human Platelets via the Translocation of the Glucose Transporter GLUT-3 from α-Granules to the Cell Surface

Increased energy metabolism in the circulating blood platelet plays an essential role in platelet plug formation and clot retraction. This increased energy consumption is mainly due to enhanced anaerobic consumption of glucose via the glycolytic pathway. The aim of the present study was to determine the role of glucose transport as a potential rate-limiting step for human platelet glucose metabolism. We measured in isolated platelet preparations the effect of thrombin and ADP activation, on glucose transport (2-deoxyglucose uptake), and the cellular distribution of the platelet glucose transporter (GLUT), GLUT-3. Thrombin (0.5 U/ml) caused a pronounced shape change and secretion of most α-granules within 10 min. During that time glucose transport increased approximately threefold, concomitant with a similar increase in expression of GLUT-3 on the plasma membrane as observed by immunocytochemistry. A major shift in GLUT-3 labeling was observed from the α-granule membranes in resting platelets to the plasma membrane after thrombin treatment. ADP induced shape change but no significant α-granule secretion. Accordingly, ADP-treated platelets showed no increased glucose transport and no increased GLUT-3 labeling on the plasma membrane. These studies suggest that, in human blood platelets, increased energy metabolism may be precisely coupled to the platelet activation response by means of the translocation of GLUT-3 by regulated secretion of α-granules. Observations in megakaryocytes and platelets freshly fixed from blood confirmed the predominant GLUT-3 localization in α-granules in the isolated cells, except that even less GLUT-3 is present at the plasma membrane in the circulating cells (~15%), indicating that glucose uptake may be upregulated five to six times during in vivo activation of platelets.     

Rearrangement of aminophospholipids in bilayers from sheep platelet plasma membranes and platelet liposomes by increasing their cholesterol levels

Phospholipid orientation in platelet plasma membranes and other blood cells, such as erythrocytes, appears to be rather similar. The negatively charged phospholipids are almost exclusively located on the inner leaflet of the bilayer. No phosphatidylserine is present on the outer membrane bilayer. The results of the present study, using a specific reagent for amino groups, trinitrobenzenesulfanilic acid, showed that in sheep platelet plasma membranes enriched with free exogenous cholesterol, an alteration in the aminophospholipid topology occurs, with a portion of phosphatidylserine moving from the inner to the outer side. A progressive appearance of aminophospholipids in the outer membrane bilayer was also observed in artificial vesicles prepared with total lipids from sheep platelets supplemented with increased amounts of free cholesterol.     

Cytoplasmic free calcium concentration in porcine platelets: Regulation by an intracellular nonmitochondrial calcium pump and increase after thrombin stimulation

Mechanisms are assumed to exist in the resting platelet which maintain the concentration of cytoplasmic free calcium below that level required to activate cellular responses. To assess such processes the porcine platelet plasma membrane was selectively lysed with digitonin and the uptake (or flux) of free calcium monitored by an extracellular calcium electrode. Lysis resulted in an immediate lowering of the extracellular free calcium, due to the action of intracellular organelle(s) acting on the extracellular space through the permeabilized plasma membrane. In resting platelets, the rate of calcium uptake was first order with respect to the extracellular prelytic calcium concentration, and hence the cytoplasmic free concentration was found to be 1·10^?7 M by extrapolation to a point of zero flux (i.e., the null point). This approach could not be used with thrombin-stimulated platelets, as external calcium was required for both secretion of ATP + ADP and aggregation. Nevertheless, evidence for an increase in cytoplasmic free calcium after thromin stimulation was obtained. Metabolic inhibitors and agents known to inhibit calcium uptake by mitochondria had no effect on the calcium flux following lysis, indicating different mechanisms for calcium homeostasis in the platelet when compared with other cell types (e.g., liver). Levels of ionophore A23187, which caused platelet aggregation, gave a massive release of the nonmitochondrial pool of calcium into the cytoplasmic space. Thus, in porcine platelets an intracellular energy-requiring calcium pump, which sequesters calcium in a nonmitochondrial membranous compartment, is crucial for intracellular calcium homeostasis.     

Cytoplasmic free calcium concentration in porcine platelets. Regulation by an intracellular nonmitochondrial calcium pump and increase after thrombin stimulation

Mechanisms are assumed to exist in the resting platelet which maintain the concentration of cytoplasmic free calcium below that level required to activate cellular responses. To assess such processes the porcine platelet plasma membrane was selectively lysed with digitonin and the uptake (or flux) of free calcium monitored by an extracellular calcium electrode. Lysis resulted in an immediate lowering of the extracellular free calcium, due to the action of intracellular organelle(s) acting on the extracellular space through the permeabilized plasma membrane. In resting platelets, the rate of calcium uptake was first order with respect to the extracellular prelytic calcium concentration, and hence the cytoplasmic free concentration was found to be 1 X 10(-7) M by extrapolation to a point of zero flux (i.e., the null point). This approach could not be used with thrombin-stimulated platelets, as external calcium was required for both secretion of ATP + ADP and aggregation. Nevertheless, evidence for an increase in cytoplasmic free calcium after thrombin stimulation was obtained. Metabolic inhibitors and agents known to inhibit calcium uptake by mitochondria had no effect on the calcium flux following lysis, indicating different mechanisms for calcium homeostasis in the platelet when compared with other cell types (e.g., liver). Levels of ionophore A23187, which caused platelet aggregation, gave a massive release of the nonmitochondrial pool of calcium into the cytoplasmic space. Thus, in porcine platelets an intracellular energy-requiring calcium pump, which sequesters calcium in a nonmitochondrial membranous compartment, is crucial for intracellular calcium homeostasis.     

Spectrophotometric method for determination of tocopherol in red blood cells

A relatively rapid procedure is described for the spectrophotometric determination of total tocopherol in red blood cells (RBC) based on a modification of the original Emmerie-Engel reaction. The critical feature in this method is the presence of a large amount of an added antioxidant, pyrogallol or ascorbic acid, during the saponification and extraction stages and the use of thin-layer chromatography for tocopherol purification. The total tocopherol levels of plasma and erythrocytes were determined for a number of human subjects, for patients with abetalipoproteinemia, and for rats. It was found that these levels had a wide range in normal human subjects but that the ratio of RBC to plasma tocopherol was relatively constant and equal to 0.18, uncorrected, and 0.21 when both RBC and plasma values were corrected to 100% recovery. The RBC-to-plasma ratio for rats was 0.39. The accuracy of this ratio determined by the spectrophotometric procedure was verified by measuring the distribution of [(14)C]tocopherol in RBC and plasma when radioactive vitamin E was introduced into the blood by both in vitro and in vivo techniques. The addition of radioactive tocopherol to RBC or plasma at the initial stage of the analysis permits an accurate determination of the total tocopherol in RBC or plasma by calculations based on the recovery of the added isotope. This procedure for erythrocyte tocopherol analysis is compared with a gas-liquid chromatographic method in current use.     

Electronic particle size measurements of platelet aggregates formed in vitro.

The aggregation of human platelets induced by adenosine diphosphate (ADP) was used to evaluate electronic particle size analyzer measurements of platelet aggregates in plasma. As platelets began to clump in plasma, the total volume and the diameter of individual aggregates increased; after a time dependent on experimental conditions, the diameter increased but the total volume remained unchanged. Similar but opposite changes in size distribution occurred during platelet deaggregation. The total volume of aggregates formed in plasma varied (linear correlation coefficient = 0.99) with the total volume of platelets which were available to clump and with simultaneous changes in optical density. The diameter of the aggregates varied with the concentration of, and time of exposure to, ADP and with the total volume of platelets and aggregates in plasma was not different from that of control platelets in untreated plasma, the individual platelets aggregated without an accompanying increase in size. This study demonstrates that platelet aggregation can be characterized by electronic measurements of the size distribution of platelet aggregates.     

Finite platelet size could be responsible for platelet margination effect

Blood flows through vessels as a segregated suspension. Erythrocytes distribute closer to the vessel axis, whereas platelets accumulate near vessel walls. Directed platelet migration to the vessel walls promotes their hemostatic function. The mechanisms underlying this migration remain poorly understood, although various hypotheses have been proposed to explain this phenomenon (e.g., the available volume model and the drift-flux model). To study this issue, we constructed a mathematical model that predicts the platelet distribution profile across the flow in the presence of erythrocytes. This model considers platelet and erythrocyte dimensions and assumes an even platelet distribution between erythrocytes. The model predictions agree with available experimental data for near-wall layer margination using platelets and platelet-modeling particles and the lateral migration rate for these particles. Our analysis shows that the strong expulsion of the platelets from the core to the periphery of the blood vessel may mainly arise from the finite size of the platelets, which impedes their positioning in between the densely packed erythrocytes in the core. This result provides what we believe is a new insight into the rheological control of platelet hemostasis by erythrocytes.     

An assay to measure the simultaneous uptake of [3H]dopamine and [14C]serotonin by the human platelet reveals an imipramine-insensitive [3H]dopamine uptake mechanism.

To determine whether a specific dopamine uptake mechanism is present on the human platelet the simultaneous uptake of [3H]dopamine and [14C]serotonin by platelets was measured. Utilising a dual radiolabel uptake technique, platelets have been shown to take up serotonin more rapidly and to a greater extent than they take up dopamine. Furthermore, at high concentrations serotonin was able to reduce dopamine uptake by platelets by 60% whereas dopamine had no effect on serotonin uptake. Similarly, imipramine and reserpine reduced (97% and 74% respectively) serotonin uptake by platelets in a dose-dependent manner, but did not affect the uptake of dopamine. Our data show that platelets take up dopamine by a mechanism independent of the imipramine-sensitive serotonin uptake mechanism. Furthermore, the increased capacity of platelets to store serotonin is because serotonin, unlike dopamine, is transported into the dense granules of the platelet.     

Synthetic byproducts of tocopherol oxidation as inhibitors of platelet function

Vitamin E and its fully oxidized form tocopherol quinone are known inhibitors of platelet aggregation. Previous results from our laboratory have shown that the quinone was approximately equal in effectiveness to vitamin E. A recent report of a far greater inhibitory activity of the quinone produced by nitric acid oxidation of vitamin E prompted this investigation. Our studies show that the unusually high inhibition of platelet aggregation, release and cyclooxygenase activity associated with nitric acid oxidized vitamin E is due to byproducts of the oxidation process and not due to tocopherol quinone. Treatment of vitamin E acetate, a substance of mild effect on aggregation and arachidonate metabolism of platelets, with nitric acid did not produce tocopherol quinone but exerted as potent an inhibition as oxidized vitamin E. We conclude that nitric acid oxidation is unsuitable for preparation of tocopherol quinone unless the latter is carefully isolated. Oxidation with permanganate proved to be an alternate method without these difficulties.     

Lipid composition of subcellular particles from sheep platelets. Location of phosphatidylethanolamine and phosphatidylserine in plasma membranes and platelet liposomes

The lipid composition of whole sheep platelets and their subcellular fractions was determined. The basic lipids show similar distributions in granules, microsomes, plasma membranes and whole platelets. Phospholipid (about 70% of total lipids) and cholesterol (25% of total lipids) are the principal lipid components. Free cholesterol represents about 98% of the total, whereas cholesteryl ester is a minor component. The phospholipid composition found in intact platelets and their subcellular particles is about: 35% phosphatidylethanolamine (PE), 30% phosphatidylcholine (PC), 20% sphingomyelin and 15% phosphatidylserine (PS). We also investigated aminophospholipid topology in intact platelet plasma membranes and platelet liposomes by using the nonpenetrating chemical probe trinitrobenzenesulfonic acid (TNBS), because they are the major components of total lipids. In intact platelets, PS is not accessible to TNBS during the initial 15 min of incubation, whereas 18% PE is labelled after 15 min. In contrast, in phospholipid extracted from platelets 80% PE and 67% PS react with TNBS within 5 min, while 27 and 25% PE and 15 and 19% PS from liposomes and isolated plasma membranes, respectively, were modified after 15 min of incubation. In view of this chemical modification, it is concluded that 22% of PE and less than 1% of PS are located on the external surface of intact platelet plasma membranes. The asymmetric orientation of aminophospholipids is similar between liposomes and isolated plasma membrane. PS (23 and 28%) and PE (34 and 31%) are scarcely represented outside the bilayer. The data found are consistent with the nonrandom phospholipid distribution of blood cell surface membranes.     

In vitro oxidation of alpha-tocopherol (vitamin E) in human platelets upon incubation with unsaturated fatty acids, diamide and superoxide

Incubation of human blood platelets in vitro in Tyrode solution with unsaturated fatty acids, diamide or superoxide (generated in situ) resulted in the oxidation of tocopherol in the platelets. Arachidonate concentrations of (3-5).10(-4) M caused a 50% decrease in platelet alpha-tocopherol. The addition of saturated fatty acids or platelet-active substances such as ADP, dibutyryl cyclic AMP, and some prostaglandins, or peroxidizing agents such as hydrogen peroxide and tert-butylhydroperoxide to the incubation medium did not cause any change in platelet tocopherol content. During incubations of platelets with arachidonate, malonaldehyde as well as alpha-tocopherolquinone were produced. The latter was also produced during incubations with diamide or superoxide. The oxidation of tocopherol induced by unsaturated fatty acids may be one factor responsible for the well-known increase in dietary vitamin E requirements induced by polyunsaturated fatty acids. The oxidative consumption of tocopherol in the membranes could be expected to take place during localized release of oxidants such as superoxide and polyunsaturated fatty acids during normal biological function (e.g., phagocytosis) or pathological processes (e.g., ischemia). Tocopherol utilization is kept low probably by the regeneration of the compound by vitamin C and/or the preferential utilization of the other biological antioxidants.     

Platelet serotonin content and uptake in spontaneously hypertensive rats

Platelet serotonin (5-HT) content and uptake were studied in male SHR and WKY at various ages. Blood was withdrawn from the carotid artery under anesthesia and 5-HT levels determined from platelet rich plasma (PRP) using a HPLC technique coupled with an electrochemical detection method. Platelet 5-HT uptake was studied by incubating PRP at 37 degrees C for 10 sec with increasing concentrations of 3H-5HT. Lineweaver- Burk plots of 3H-5HT uptake were linear suggesting simple Michaelis- Menten uptake kinetics. The SHR had more platelets than age-matched controls and consequently a higher blood circulating pool of 5-HT. Nevertheless, the 5-HT platelet levels were similar to those of their age-matched rats. The 5 week-old SHR and WKY had greater numbers of platelets and higher 5-HT platelet levels than the older rats of both strains. The affinity constants (Km) and the maximal velocities (Vmax) of platelet 5-HT uptake did not differ significantly between the 12 week- and the 6 month-old SHR and WKY. These data suggest that the SHR do not show the same impairment in platelet 5-HT metabolism as observed in essential hypertension in man.     

Rouleau formation of erythrocytes is influenced by thrombocytes

Thrombocytes are activated by shearing and influence possibly the flow properties of blood. Erythrocytes resuspended in platelet poor plasma show a significantly increased deformability as well as reduced aggregability after addition of ADP as well as adrenaline in comparison with control. In platelet rich plasma the aggregation of erythrocytes becomes enhanced. This is discussed in respect of a release of macromolecules of platelets.