Different metabolic behavior of long-chain n-3 polyunsaturated fatty acids in human platelets

Whereas numerous studies deal with the effects and metabolism of eicosapentaenoic acid (20:5(n - 3)) in platelets, very few concern docosahexaenoic acid (22:6(n - 3)), although both acids are consumed in equal amounts from most fish fat. The present paper reports the modulation of 22:6(n - 3) oxygenation as well as that of endogenous arachidonic acid (20:4(n - 6)) in 22:6(n - 3)-rich platelets. Like the oxygenation of 20:5(n - 3), the lipoxygenation of 22:6(n - 3) occurred at a low level when incubated alone, but was markedly increased in the presence of 20:4(n - 6), suggesting a similar peroxide tone dependency. 20:5(n - 3) could not replace 20:4(n - 6) in the increasing 22:6(n - 3) lipoxygenation, whereas 22:6(n - 3) shared the potentiating effect of 20:4(n - 6) on both the cyclooxygenation and the lipoxygenation of 20:5(n - 3). On the other hand, 20:5(n - 3), 22:6(n - 3) or 20:5(n - 3) + 22:6(n - 3) enrichment of platelet phospholipids inhibited the formation of cyclooxygenase but not lipoxygenase products from endogenous 20:4(n - 6) in thrombin-stimulated platelets. In doing so, 22:6(n - 3) appeared even more potent than 20:5(n - 3), although it was not liberated after acylation in phospholipids, the opposite of what was observed with 20:5(n - 3). Therefore, it seems that, in contrast to 20:5(n - 3), which may compete with endogenous 20:4(n - 6) at the cyclooxygenase level, 22:6(n - 3) would affect the latter enzyme activity in a different way. We conclude that 20:5(n - 3) and 22:6(n - 3) behave differently and might act synergistically on the inhibition of platelet functions after fish fat intake.     

The behavior of platelets at foreign surfaces

Many conditions affect the interaction of platelets with foreign surfaces, including the type of surface, modifications of the surface, conditions of blood flow, the adsorbed layer of plasma proteins, changes in this protein layer with time, and the animal species in which experiments are done. Platelets probably never adhere directly to a foreign surface in vivo, because upon exposure of the surface to blood, plasma proteins, principally fibrinogen, are adsorbed almost immediately. When platelets adhere to such a surface and spread on it, they are activated in much the same way as when they are exposed to a strong aggregating and release-inducing agent, but in contrast to aggregation caused by some agonists, adhesion is not dependent on the formation of TXA2 or the release of ADP. It does appear to depend on external Ca2+. Much less is known about the initial adhesion reaction than about platelet aggregation (thrombus formation) on the adherent platelets, although the morphological changes resulting from adhesion have been described. It is surmised that the metabolic and cytoskeletal changes upon adhesion are similar to those that are involved in the response of platelets to other activating agents. The consequences of adhesion include the formation of thrombi and thromboemboli, thrombocytopenia, reduced platelet survival, reduced platelet function in response to hemostatic stimuli, and the appearance in the circulation of products released or formed by activated platelets. Many efforts are being made to develop surfaces and to set up conditions that will minimize platelet adhesion, but it has not yet been possible to find a foreign surface that has and can maintain the nonthrombogenic characteristics of the normal endothelium.     

Phosphoproteome of resting human platelets

Beside their main physiological function in hemostasis, platelets are also highly involved in pathological processes, such as atherothrombosis and inflammation. During hemostasis, binding of adhesive substrates to tyrosine-kinase-linked adhesion receptors and/or soluble agonists to G-protein coupled receptors leads to a cascade of intracellular signaling processes based on substrate (de)phosphorylation. The same mechanisms are involved in platelet activation at sites of atherosclerotic plaque rupture, contributing to vessel occlusion and consequently to pathologic states, such as myocardial infarction, stroke, or peripheral artery disease. To gain a deeper insight into platelet function, we analyzed the phosphoproteome of resting platelets and identified 564 phosphorylation sites from more than 270 proteins, of which many have not been described in platelets before. Among those were several unknown potential protein kinase A (PKA) and protein kinase G (PKG) substrates. Because platelet inhibition is tightly regulated especially by PKA and PKG activity, these proteins may represent important new targets for cardiovascular research. Thus, our finding that GPIbalpha is phosphorylated at Ser603 in resting platelets may represent a novel mechanism for the regulation of one of the most important platelet receptor (GPIb-IX-V) mediated signaling pathways by PKA/PKG.     

Sphingolipid composition of human platelets

Total lipid extracts from washed trypsinized human platelets were fractionated into neutral lipids, glycosphingolipids, and phospholipids by silicic acid chromatography. The concentrations and chemical structures of the neutral and acidic glycosphingolipids were then studied in detail. On the basis of sugar molar ratios, studies of permethylation products, and the action of stereospecific glycosidases on the lipids, identifications were made of four neutral glycosphingolipids. Lactosylceramide was the most abundant type and accounted for 64% of the total neutral glycolipid mixture. The major fatty acids of the lactosylceramide were 20:0, 22:0, 24:0, and 24:1; the major long-chain base was 4-sphingenine. The platelets were surprisingly rich in a ceramide fraction, which represented 1.3% of the total platelet lipids. It had a different fatty acid composition than the neutral glycosphingolipid and ganglioside fractions. Hematoside was also isolated from the total lipid fraction of platelets; the neuraminic acid component was N-acetylneuraminic acid. Treatment of platelets with trypsin, chymotrypsin, or thrombin increased the yield of hematoside as compared with a control, while the level of ceramides was not changed. It was concluded that the platelets are similar to leukocytes, liver, and spleen in that lactosylceramide and hematoside are the principal neutral and acidic glycosphingolipids. The presence of a relatively high proportion of ceramide in platelets may be a unique characteristic of this cellular fraction of blood.     

Diglyceride kinase in human platelets

Human platelets contain diglyceride kinase, an enzyme that catalyzes the phosphorylation of diacylglycerol by adenosine 5'-triphosphate to yield phosphatidic acid. The majority of the platelet enzyme is particulate-bound, and membrane fractions of platelet homogenates have a higher specific activity than granule fractions. Both deoxycholate and magnesium are necessary for optimal enzyme activity. The K(m) of the enzyme for adenosine 5'-triphosphate is 1.3 mm, and the apparent K(m) for diacylglycerol is 0.4 mm. The pH optimum is 6.6-6.8 in imidazole-HCl or maleate-NaOH buffer. The enzyme activity of platelets from normal subjects was similar to the activity from patients with renal and hepatic failure.     

Tocopherol in human platelets.

A spectrophotometric method was used to determine the total tocopherol levels in platelets, plasma, and erythrocytes from human subjects. The platelets contained about three times as much total tocopherol per cell as erythrocytes. This difference was not related to the content of polyunsaturated fatty acids in platelets and erythrocytes. In vitro incubation resulted in significant uptake of tocopherol by plasma and RBC, whereas no uptake was observed into platelets. A 3-month period of tocopherol treatment increased the level of tocopherol in plasma and erythrocytes, whereas the platelet level was unchanged. Tocopherol treatment did not interfere with platelet function or platelet lipid metabolism. The tocopherol fractions of platelets, red cells, and plasma were similar, and alpha-tocopherol was the main fraction.     

Lactoferrin binding to human platelets

• 1.1. Platelets bind specifically lactoferrin.
• 2.2. The lactoferrin binding to the platelets depends on the concentration of labelled lactoferrin, the number of platelets, the time of incubation and pH.
• 3.3. The binding was characterized by two types of binding site: one with high affinity and low capacity, and another with low affinity and high capacity (respectively k_{aff 1} = 13.6 × 10⁹1/mol and about 40 binding sites, and K_{aff 2} = 1.23 × 10⁹1/mol and about 135 binding sites per platelet).
• 4.4. Both human transferrin and bovine lactoferrin compete with human lactoferrin for the receptors.
• 5.5. The presence of lactoferrin receptors on the platelet membrane surface is connected most probably with the effect(s) on the cell function(s) of these cells.

     

Lysosomal enzymes in human platelets

In human freshly prepared platelets the following lysosomal enzymes were studied: alpha-mannosidase, alpha-fucosidase, beta-galactosidase, beta-glucosidase, beta-glucuronidase, beta-N-acetylglucosaminidase and acid phosphatase. For each of the examined enzymes the conditions providing maximal activity (pH, buffer), kinetic parameters (saturating substrate concentration and Km) as well as heat stability were established. On the basis of these parameters it is suggested that many of the serum glycohydrolases may be platelet derived.     

Interaction of terbium with human platelets

The effect of terbium on platelets has been studied by aggregation experiments and by fluorescence measurements. TbCl3 does not substitute for CaCl2 in the aggregation of platelets induced by ADP, but it may even inhibit, probably by a competition mechanism. It was impossible to observe a sensitized emission of Tb3+ in the presence of platelets. Instead the lanthanide, like Ca2+, significantly increases the aggregation of platelets induced by A23187. The fluorescence yield of this compound is greater in the presence of platelets than in buffer alone. Energy transfer appears to take place from the aromatic amino acids of the platelet membrane to the bound ionophore.     

The ultrastructure of defective human platelets

Summary Much of our current knowledge about the physiology of hemostasis has come from intensive study of platelets from patients with inherited and acquired bleeding disorders or an increased risk of thrombotic disease. Appreciation of the role of plasma proteins in platelet stickiness, of platelet surface membrane glycoproteins in aggregation, of the substances stored in platelet organelles in cell-cell interaction, vascular injury and atherosclerosis, and of endoperoxides and thromboxanes in platelet intercellular communication have resulted largely from investigations on various types of defective platelets. While the techniques of physiology and biochemistry have generated critical details about abnormal platelets, electron microscopy and ultrastructural cytochemistry have provided an improved morphological framework in which to integrate the new discoveries. The present review has attempted to correlate physiological, biochemical and ultrastructural concepts as they relate to the current understanding of inherited platelet disorders.     

Harman in human platelets.

The β-carbolines present in human platelets have been extracted with diethyl ether, isolated by liquid column chromatography and thin-layer chromatography (TLC), and identified by ultraviolet fluorimetry, gas liquid chromatography (GC) and mass spectrometry (MS). Harman (1-methyl-β-carboline) was the only β-carboline unequivocally identified in platelet samples with these techniques. Since harman is thought to be biosynthesized by the condensation of tryptamine and acetaldehyde, its formation may be of importance in the metabolism and the pharmacological-toxicological actions of alcohol.     

Putrescine transport in human platelets

Putrescine transport has been studied in human platelets. The uptake of putrescine is saturable and appears to be an energy-dependent process, since it is inhibited by the uncoupler 2,4-dinitrophenol and low temperature. The evidence presented suggests that the uptake process is complex and may be dependent upon pH gradient, membrane potential, and other unidentified factors. Putrescine transport is not inhibited by amino acids and is only slightly inhibited by spermidine and spermine. A membrane protein involved in putrescine transport has been identified and partially purified. Differential labeling with N-ethylmaleimide identified proteins with apparent molecular weights of 65000 and 23000 as determined by SDS-polyacrylamide gel electrophoresis. Column chromatographic purification on a putrescine affinity column revealed a Mr 55000 protein which copurified with the Mr 65000 protein. Additional evidence supporting the involvement of these proteins in putrescine transport was seen in putrescine protection against N-ethylmaleimide inhibition of putrescine uptake. Putrescine uptake may occur via the serotonin transport system, since imipramine inhibits transport and because of the similarities in the molecular weights of the proteins implicated in transport.     

Oxidative metabolism of human platelets.

The reducing capacity toward cytochrome c present in human resting platelets increases upon platelet stimulation, and is partially inhibited by superoxide dismutase. This activity therefore represents the generation of superoxide anion. In order to evaluate hydrogen peroxide formation a quantitative assay by mean of dichlorofluorescin (DCFH) has been set up. The DCFH, trapped inside the cell, is oxidized by hydrogen peroxide to the fluorescent compound DCF. Basal DCF increases during activation of platelets by agonists. Arachidonic acid, calcium ionophore A23187 and to a lesser extent PMA and thrombin are the most effective. N-ethylmaleimide induces a dose-dependent DCFH oxidation and potentiates the effect of agonists. NAD(P)H--cytochrome c reductase enzyme, which catalyzes superoxide anion production, is present in platelets at high specific activity, as well as those enzymes who protect the cells from oxygen reactive species.     

Movement of sodium into human platelets

Addition of ADP induces platelets in plasma to undergo shape change from a disc to a spiny sphere and to develop adhesiveness, i.e. to aggregate. The aggregation of human platelets by ADP is associated with a net uptake of Na⁺. The present experiments demonstrate that the induction of shape change by ADP in acidified or EGTA-treated plasma conditions which inhibit aggregation, is also associated with a movement of Na⁺ into platelets. When ADP-induced platelet shape change and aggregation is inhibited by prostaglandin E₁ Na⁺ uptake is also blocked. Platelets aggregated by epinephrine do not take up Na⁺. In a manner analogous to the effect of ADP, polylysine also induces Na⁺ uptake during aggregation. Vasopressin, in a manner analogous to epinephrine, induces aggregation without Na⁺ uptake. The increase in platelet Na⁺ resulting from ouabain inhibition of Na⁺ efflux induces an increase in the aggregation response to ADP and to epinephrine.