Adenine nucleotide metabolism of blood platelets. VIII. Transport of adenine into human platelets

Adenine uptake into human blood platelets is a carrier-mediated process with a K_m of 159±21 nM and a V of 100±10 pmoles/min per 10⁹ platelets (in citrated platelet-rich plasma). The Q₁₀ was 2.53±0.22. A pH optimum was found at 7.5. Washing of the platelets increased the K_m to 453±33 nM and V to 397±38 pmoles/min per 10⁹ platelets. The change in shape induced in platelets by ADP was accompanied by an increase in V (2 times) and K_m (1.5 times).Guanine (K_i 50 μM), hypoxanthine (K_i 390 μM), adenine-N′-oxide (K_i 40 μM), adenosine (K_i 100 μM), RA 233 (K_i 75 μM) and papaverine (K_i 15 μM) acted as competitive inhibitors. Adenosine at low concentrations, and prostaglandin E₁ gave inhibition at only high adenine levels. A similar inhibition was obtained with 2-deoxy-d-glucose. Sulfhydryl-group inhibitors, pyrimidines and ouabain had no effect.     

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.     

Receptors that activate platelets.

This review highlights the increasing knowledge of the biochemistry, pathology, and cell and molecular biology of platelet receptors. A receptor for ADP has been identified using the affinity label FSBA as aggregin, a 100-kDa membrane protein responsible for shape change, aggregation, and exposure of fibrinogen binding sites. A variety of putative receptors for collagen have been described, with GPIa/IIa and GPIV receiving the most attention recently. A thromboxane A2 receptor has been identified using receptor antagonists and photoaffinity labels. The alpha 2-adrenergic receptor has been cloned and expressed. The platelet thrombin receptor has been tentatively identified as GPIb. Following binding of thrombin to this receptor, activation of calpain occurs, with cleavage of aggregin leading to exposure of GPIIb/III alpha and platelet aggregation. Isolation, expression, or both of the ADP, collagen, and thrombin receptors as single gene products of the human platelet responsible for activation, and more complete understanding of stimulus-response coupling, should allow for greater specificity of drugs with selective therapeutic actions.     

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.     

Phospholipid-deacylating enzymes of rabbit platelets.

The inhibition of selenium-glutathione peroxidase by metal ions was studied by means of a direct spectrophotometric assay that monitors at 237 nm the decrease of GS^? concentration with time. Cadmium (II) and zinc (II) ions were the most potent inhibitors, while silver (I), mercury (II), cobalt (II), and lead (II) inhibited to a lesser extent. Inhibition by these metal ions was competitive with respect to the donor substrate, GSH. Competitive inhibition was verified for cadmium (II) ion by means of an assay employing Ellman's reagent, 5,5′-dithiobis-2-nitrobenzoic acid. Inhibition by cadmium (II) ion was noncompetitive with respect to the acceptor substrate, t-butyl hydroperoxide. Inhibitor constants obtained from Lineweaver-Burk plots and binding constants obtained from Scatchard plots were comparable. Correlation of inhibitor constants with chemical and physical properties showed a dependence on the softness of the metal ion as an acid and also a dependence on ionic size.     

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.     

Phosphoinositides of sheep platelets plasma membranes.

Phospholipid composition of sheep blood platelets and its various plasma membrane fractions have been analyzed. Based on their flotation rates in discontinuous sucrose density gradient centrifugation, three membrane fractions were isolated. 5'-Nucelotidase and alkaline phosphatase were distributed nearly equally in all the three membrane fractions. However these membrane fractions showed differences in the distribution of phosphatidyl ethanolamine, phosphatidyl choline and phosphoinositides. Phosphatidyl ethanolamine was predominant in fraction I (11.05 micrograms PLP/mg protein) while phosphatidyl choline was predominant in fractions II and III (110.10 and 68.30 micrograms PLP/mg protein respectively). Phosphatidyl inositol (Ptd-InsP) was equally distributed in all three membrane fractions. However, both Ptd-InsP and phosphatidyl inositol 4,5-bisphosphate were about 4-fold higher in fraction II (73.55 and 89.89 micrograms PLP/mg protein respectively).     

Signal transducing mechanisms in platelets.

Platelets respond through discrete receptors to a number of physiological stimuli and foreign surfaces with a sequence of measurable responses: shape change, aggregation, secretion and arachidonate liberation. Three secretory responses are distinguished: release of substances from 1) dense granules (ADP, serotonin), 2) alpha-granules (coagulation factors, platelet-specific proteins, adhesive proteins) and 3) lysosomes (acid hydrolases). The liberated arachidonate is converted to prostaglandins and thromboxanes which, together with secreted ADP and close cell contact, will cause further platelet activation through "positive feedback" (autocrine stimulation). Some agonists are "weak" (ADP, vasopressin, platelet-activating factor) and depend on positive feedback to promote the full sequence of responses, while other agonists are "strong" (thrombin, collagen) and stimulate the entire response sequence without positive feedback. Most agonists appear to stimulate platelet responses via G-protein-dependent activation of phospholipase C, resulting in diesteratic hydrolysis of phosphatidylinositol-4,5-bisphosphate yielding inositol-1,4,5-trisphosphate and diacylglycerol. These are signal molecules which mobilize cytoplasmic Ca2+ and stimulate protein kinase C, respectively. Cytoplasmic Ca2+ will in turn activate protein phosphorylations which eventually lead to execution of the various responses while activation of protein kinase C appears to be linked to regulation of intracellular pH through Na+/H+ exchanger and to termination of the Ca(2+)-mediated signal processing. Other agonists (prostaglandins I2 and D2) counteract platelet stimulation through classical activation of adenylate cyclase.     

Carbonic anhydrase in human platelets.

The carbonic anhydrase activity of human platelets was investigated by measuring the kinetics of CO2 hydration in supernatants of platelet lysates by using a pH stopped-flow apparatus. An average carbonic anhydrase concentration of 2.1 microM was determined for pellets of human platelets. Analysis of the kinetic properties of this carbonic anhydrase yielded a Km value of 1.0 mM, a catalytic-centre activity kcat. of 130000 s-1 and an inhibition constant Ki towards ethoxzolamide of 0.3 nM. From these values, CO2 hydration inside platelets is estimated to be accelerated by a factor of 2500. When platelet lysates were subjected to affinity chromatography, only the high-activity carbonic anhydrase II could be eluted from the affinity column, whereas the carbonic anhydrase isoenzyme I, which is known to occur in high concentrations in human erythrocytes, appeared to be absent.     

Alpha-adrenergic receptors on human platelets.

[³H] dihydroergocyrptine, an α-adrenergic antagonist, binds specifically to sites on human platelet membranes. Prostaglandin E₁ (PGE₁) stimulates the production of cyclic AMP (cAMP) in human platelets. Alpha-adrenergic agonists, 1-epinephrine and 1-norepinephrine, and antagonists, phentolamine, phenoxybenzamine, and dihydroergocyrptine inhibit the binding of [³H] dihydroergocryptine. The α-adrenergic agonists inhibit PGE₁-stimulated cAMP production and the α-adrenergic antagonists phentolamine and dihydroergocryptine reverse this inhibition. The β-adrenergic agonist 1-isoproterenol and the β-adrenergic antagonists d1-propranolol and 1-alprenolol do not significantly alter binding or PGE₁-stimulated cAMP production. Clonidine, dopamine, and serotonin inhibit binding, but clonidine and dopamine are weak inhibitors of PGE₁-stimulated cAMP production, and serotonin is without effect. Tyramine, an amine without direct adrenergic activity fails to inhibit binding. Alpha-adrenergic agonists decrease the apparent affinity of a PGE₁-receptor activating cAMP production. The inhibition of PGE₁-stimulated cAMP production is a physiological measure of α-adrenergic agonist binding to the α-receptor.     

Effect of altitude exposure on platelets.

Since decompression from depth is known to produce a fall in platelet count, the effect of altitude decompression and high-altitude exposure on platelets was investigated. Sixteen subjects decompressed without hypoxia to 20,000 ft simulated altitude for two hours showed a significant (P less than 0.01) drop in circulating platelet count of approximately 10% for three days following decompression. Four of five subjects similarly exposed had a shortened autologous platelet survival compared to that prior to exposure. Subjects exposed to 9,800 ft and then 17,600 ft in a mountain environment showed a significant mean decrease in platelet count on day 2 of 7% and 25% respectively, which had returned to control by day 5. Nonhypoxic and hypoxic decompressed rabbits which received homologous chromium-51-labeled platelets had an increase in lung radioactivity compared with sea-level controls. It is postulated that altitude decompression produces platelet reductions similar to these seen after decompression from depth, and that platelets sequester in the pulmonary vascular bed.     

Identification of four major classes of sulfhydryl groups in human blood platelets. Ferricyanide titration of spin-labeled platelets.

Human blood platelets have been labeled with the sulfhydryl-specific spin labels, 4-iodoacetamido-2,2,6,6-tetramethylpiperidine-1-oxyl and 3-maleimido-2,2,5,5-tetramethylpyrrolidine-1-oxyl. First, the ESR spectra of platelets labeled with either reagent revealed two classes of sulfhydryl groups, a mobile class and an immobile class. Second, when spin-labeled platelets were titrated with high concentrations of potassium ferricyanide (greater than 10(-3) M), there was a decrease in the peak heights of the mobile class of sulfhydryl groups due to dipole-dipole exchange. Third, plots of peak heights of the mobile class versus ferricyanide concentration revealed three classes of mobile sulfhydryl groups compared to a single immobile class. This technique may be used to show the relative locations of spin-labeled groups on cell surfaces.     

Monoacylglycerol hydrolase in human platelets.

In the present paper we show for the first time monoacylglycerol hydrolase in human platelets. No monoacylglycerol hydrolase activity could be demonstrated in the other blood cells. The monoacylglycerol hydrolase of platelets could not be released from the cells by heparin, thus the enzyme is distinct from the postheparin plasma lipases. The enzyme could be solubilized by a non-ionic detergent, Triton X-100. The solubilized monoacylglycerol hydrolase from platelets was optimally active at pH between 7 and 8 and at ionic strength corresponding to [NaCl] between 0.1 and 0.3 M. The optimal assay temperature was 37 degrees C. The enzyme activity was sensitive to HgCl2 but not to NaF. Accordingly, it was stabilized by 2-mercaptoethanol.     

Immunological effect of co-trimoxazole on platelets.

Diminished survival of transfused platelets occurred in two patients given co-trimoxazole, and a third patient taking this drug developed thrombocytopenia. By means of an indirect immunofluorescence assay antibodies against donor platelets coated with co-trimoxazole were found in the sera in all cases. These antibodies were directed against the trimethoprim component of co-trimoxazole and not against sulphamethoxazole. Co-trimoxazole is a potent antimicrobial agent and is advocated for treatment and prophylaxis in leukaemia. Hence its adverse effect on platelets is of great importance.