Clopidogrel, like the homologous thienopyridine derivative tidopidine, selectively inhibits platelet aggregation induced by ADP. We have previously described two nucleotide-binding sites on platelets related to ADPmediated platelet responses. The first is a high-affinity binding she for 2-methylthio-ADP (2-MeSADP) that is linked to the inhibition of stimulated adenylate cyclase. The second is the 100-kd exofacial membrane protein aggregin, which is labeled by the reactive ADP analogue 5'-p-fluorosulfonyibenzoyl adenosine (FSBA) that is related to shape change and aggregation. We set out to determine if either of these sites is blocked in vivo by clopidogrel or its active metabolite. Six subjects were given clopidogrel (75 mg/day for 10 days) in a double-blind crossover experiment All of the subjects developed prolonged bleeding times while taking the drug. The rate of onset of the effect on bleeding time varied among subjects. Platelet aggregation induced by ADP or thrombin was significantly impaired by the drug treatment, but no effect was detected on shape change. The incorporation of [ 3 H]FSBA into aggregin was also unaffected. Inhibition of adenylate cyclase by ADP or by 2-MeSADP was greatly reduced in all subjects, and in the case of 2-MeSADP, there was evidence for a noncompetitive effect Inhibition of adenylate cyclase by epinephrine was unaffected. In the three subjects for whom binding measurements were made, the number of binding sites for [32 P]2-MeSADP was reduced from 534±44 molecules per platelet during control and placebo periods (11 determinations) to 199±78 molecules per platelet during drug treatment (three determinations). There was no consistent change in the binding affinity. The inhibition of platelet function by clopidogrel is associated with a selective reduction in the number of functional receptors mediating the inhibition of stimulated adenylate cyclase activity by ADP.
Glutathione is not effectively transported into human lymphoid cells, normal human skin fibroblasts, and fibroblasts from patients with genetic deficiencies of yglutamylcysteine synthetase or glutathione synthetase. On the other hand, the monoethyl ester of glutathione, in which the carboxyl group of the glycine residue is esterified, is readily transported into these cells and is hydrolyzed intracellularly. This leads to greatly increased cellular levels of glutathione, which often exceed those found normally. Glutathione ester was found to protect human lymphoid cells of the CEM line against the lethal effects of irradiation. Under the conditions employed, complete protection was found when the ester was added prior to irradiation. Addition of the ester after irradiation was partially effective, suggesting that GSH may also function in repair processes.There is currently much interest in procedures that increase cellular levels of glutathione (GSH) because of growing evidence that GSH plays an important role in the protection of cells against damage by radiation and by reactive oxygen compounds and other toxic substances (1, 2). Cellular levels of GSH may be raised to some extent by increasing the supply of the amino acids required for GSH synthesis, especially cysteine, which is a substrate for the first step in GSH biosynthesis catalyzed by y-glutamylcysteine synthetase. L-2-Oxothiazolidine-4-carboxylate, which is readily transported and converted to L-cysteine intracellularly by 5-oxoprolinase, promotes GSH synthesis (3, 4). Administration of yglutamylcysteine, a substrate of GSH synthetase, which catalyzes the second step, may increase cellular GSH levels to values higher than the feedback-regulated levels (5). Administered GSH does not seem to enter most cells to an appreciable extent; in contrast, there is considerable evidence that many cells export GSH (6).An ideal cellular delivery system for GSH might be based on a derivative of GSH that is (i) much more effectively transported into cells than is GSH itself and (ii) is readily converted to GSH after transport. Observations made previQusly in this laboratory showed that administration of the monoethyl (or methyl) ester of GSH to mice leads to substantial increases in the GSH levels of the liver and kidney, and the findings indicated that these compounds are transported and then hydrolyzed intracellularly (7). The use of esters of GSH is attractive because (i) many cells appear to have high levels of esterase capable of effectively converting such compounds to GSH; (ii) the formation of intracellular GSH from this type of derivative does not require intracellular energy as is required for de novo GSH synthesis; and (iii) since the delivery of GSH by means of its ester does not involve GSH synthesis, the levels of cellular GSH achieved would not be subject to feedback inhibition by GSH (8).In the present work, we have examined the potential usefulness of the monoethyl ester of GSH as a cellular GSH delivery system in studies on several human lym...
Administration of y-glutamylcysteinylglycyl monomethyl (or monoethyl) ester to mice leads to substantial increases in the levels of glutathione in the liver and kidney. Mice depleted of glutathione by treatment with buthionine sulfoximine, a potent inhibitor of y-glutamylcysteine synthetase, exhibited about a 4-fold increase in liver and kidney glutathione levels after administration of glutathione monomethyl ester. This ester also prevented the marked decline in liver glutathione level found after giving mice acetaminophen, and it protected mice from toxicity due to this compound. The findings indicate that the monomethyl and monoethyl esters of glutathione are transported into 'cells and hydrolyzed to glutathione. Such esters may be useful in experimental work on glutathione metabolism and function and may provide a relatively safe method for protecting cells against damage by toxic compounds, oxygen, and radiation.Several studies have shown that intracellular glutathione (GSH) is of major importance in protecting' cells against damage by toxic compounds, reactive oxygen compounds, and radiation (1-3). For this reason there has been interest in the factors that influence intracellular GSH synthesis and especially in ways of increasing cellular levels of GSH. Although the availability of cysteine may be a limiting factor in GSH synthesis, administration of this amino acid does not always lead to substantially increased GSH levels; cysteine is rapidly metabolized and it is also toxic, possibly in part because of its extracellular effects. Administration of compounds that are transported and converted intracellularly into cysteine, such as L-2-oxothiazolidine-4-carboxylate (4,5), are useful in increasing cellular GSH. Another promising approach for increasing kidney GSH levels is administration of y-glutamylcysteine (and related compounds), which are transported and stimulate GSH synthesis by providing a substrate for GSH synthetase (6). The possibility that administration of GSH itself might-lead to increased cellular GSH levels has also been considered. Recent studies on the metabolism of GSH indicate that GSH-is transported out of many cells, and that such export is a discrete step in GSH metabolism (2, 7,8). One might think that GSH export would'be reversible, but this does not seem to be the case. Although it is possible that some cells may take up intact GSH, there is as yet no conclusive evidence for transport of GSH into cells. Thus, the repletion of intracellular GSH in human lymphoid cells by exogenously supplied GSH was found to involve extracellular degradation of GSH, uptake of the products, and intracellular synthesis (9). The apparent uptake of GSH in the basolateral circulation of the kidney is evidently explained by cleavage of GSH to its constituent amino acids (10). No evidence for GSH uptake by the liver was obtained in studies on rats (11), and the moderate increase in kidney GSH levels observed after administration of GSH to mice may be explained by formation and transport of y-glut...
Platelet activation is central to the pathogenesis of hemostasis and arterial thrombosis. Platelet aggregation plays a major role in acute coronary artery diseases, myocardial infarction, unstable angina, and stroke. ADP is the first known and an important agonist for platelet aggregation. ADP not only causes primary aggregation of platelets but is also responsible for the secondary aggregation induced by ADP and other agonists. ADP also induces platelet shape change, secretion from storage granules, influx and intracellular mobilization of Ca2+, and inhibition of stimulated adenylyl cyclase activity. The ADP-receptor protein mediating ADP-induced platelet responses has neither been purified nor cloned. Therefore, signal transduction mechanisms underlying ADP-induced platelet responses either remain uncertain or less well understood. Recent contributions from chemists, biochemists, cell biologists, pharmacologists, molecular biologists, and clinical investigators have added considerably to and enhanced our knowledge of ADP-induced platelet responses. Although considerable efforts have been directed toward identifying and cloning the ADP-receptor, these have not been completely successful or without controversy. Considerable progress has been made toward understanding the mechanisms of ADP-induced platelet responses but disagreements persist. New drugs that do not mimic ADP have been found to inhibit fairly selectively ADP-induced platelet activation ex vivo. Drugs that mimic ADP and selectively act at the platelet ADP-receptor have been designed, synthesized, and evaluated for their therapeutic efficacy to block selectively ADP-induced platelet responses. This review examines in detail the developments that have taken place to identify the ADP-receptor protein and to better understand mechanisms underlying ADP-induced platelet responses to develop strategies for designing innovative drugs that block ADP-induced platelet responses by acting selectively at the ADP-receptor and/or by selectively interfering with components of ADP-induced platelet activation mechanisms.
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