Summary. Adenosine diphosphate (ADP) is an important platelet agonist and ADP released from platelet dense granules amplifies responses to other agonists. There are three known subtypes of ADP receptor on platelets: P2X 1 , P2Y 1 and P 2T receptors. Sustained ADP-induced aggregation requires co-activation of P2Y 1 and P 2T receptors. AR-C69931MX, a selective P 2T receptor antagonist and novel antithrombotic agent, was studied to characterize further the function of the P 2T receptor. The roles of the P2Y 1 receptor and thromboxane A 2 were assessed using the selective P2Y 1 antagonist A2P5P and aspirin respectively. Aggregation was measured by whole blood single-platelet counting and platelet-rich plasma turbidimetry, using hirudin anticoagulation. Dense granule release was estimated using [14 C]-5-hydroxytryptamine (HT)-labelled platelets. Ca 21 mobilization, P-selectin expression, Annexin V binding and microparticle formation were determined by flow cytometry. P 2T receptor activation amplified ADPinduced aggregation initiated by the P2Y 1 receptor, as well as amplifying aggregation, secretion and procoagulant responses induced by other agonists, including U46619, thrombin receptor-activating peptide (TRAP) and collagen, independent of thromboxane A 2 synthesis, which played a more peripheral role. P 2T receptor activation sustained elevated cytosolic Ca 21 induced by other pathways. These studies indicate that the P 2T receptor plays a central role in amplifying platelet responses and demonstrate the clinical potential of P 2T receptor antagonists.

The polyamines putrescine, spermidine and spermine are essential for cell renewal and, therefore, are needed to keep the body healthy. It was previously believed that polyamines are synthesized by every cell in the body when required. However, in the present paper evidence is provided to show that, as in the case of the essential amino acids, the diet can supply sufficient amounts of polyamines to support cell renewal and growth. Systematic analysis of different foods was carried out and from the data obtained, the average daily polyamine consumption of British adults was calculated to be in the range 350-500 pmol/person per d. The major sources of putrescine were fruit, cheese and non-green vegetables. All foods contributed similar amounts of spermidine to the diet, although levels were generally higher in green vegetables. Meat was the richest source of spermine. However, only a part of the polyamines supplied by the diet is available for use by the body. Based on experiments with rats it was established that polyamines were readily taken up from the gut lumen, probably by passive diffusion, and were partly metabolized during the process of absorption. More than 80% of the putrescine was converted to other polyamines and non-polyamine metabolites, mostly to amino acids. The enzyme responsible for controlling the bioavailability of putrescine was diamine oxidase (EC 1.4.3.6). For spermidine and spermine, however, about 7 W YO of the intragastrically intubated dose remained in the original form.Considering the limitations on bioavailability (metabolism and conversion), the amounts of polyamines supplied by the average daily diet in Britain should satisfy metabolic requirements.

Receptors for prostanoids on platelets include the EP3 receptor for which the natural agonist is the inflammatory mediator prostaglandin E(2) (PGE(2)) produced in atherosclerotic plaques. EP3 is implicated in atherothrombosis and an EP3 antagonist might provide atherosclerotic lesion-specific antithrombotic therapy. DG-041 (2,3-dichlorothiophene-5-sulfonic acid, 3-[1-(2,4-dichlorobenzyl)-5-fluoro-3-methyl-1H-indol-7-yl]acryloylamide) is a direct-acting EP3 antagonist currently being evaluated in Phase 2 clinical trials. We have examined the contributions of EP3 to platelet function using the selective EP3 agonist sulprostone and also PGE(2), and determined the effects of DG-041 on these. Studies were in human platelet-rich plasma or whole blood and included aggregometry and flow cytometry. Sulprostone enhanced aggregation induced by primary agonists including collagen, TRAP, platelet activating factor, U46619, serotonin and adenosine diphosphate, and enhanced P-selectin expression and platelet-leukocyte conjugate formation. It inhibited adenylate cyclase (measured by vasodilator-stimulated phosphoprotein phosphorylation) and enhanced Ca(2+) mobilization. It potentiated platelet function even in the presence of aspirin and/or AR-C69931 (a P2Y(12) antagonist). DG-041 antagonized the effects of sulprostone on platelet function. The effect of PGE(2) on platelet aggregation depended on the nature of the agonist and the concentration of PGE(2) used as a consequence of both pro-aggregatory effects via EP3 and anti-aggregatory effects via other receptors. DG-041 potentiated the protective effects of PGE(2) on platelet aggregation by inhibiting the pro-aggregatory effect via EP3 stimulation. DG-041 remained effective in the presence of a P2Y(12) antagonist and aspirin. DG-041 warrants continued investigation as a potential agent for the treatment of atherothrombosis without inducing unwanted bleeding risk.

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Summary. Adenosine diphosphate (ADP) released into blood induces platelet aggregation and contributes to hemostasis and thrombosis. Released ATP can also induce platelet aggregation and there is evidence that blood leukocytes and also erythrocytes play important roles in this. Rapid metabolism of ADP and ATP by endothelial cells is important in protecting platelets from their effects. Here we have performed a systematic investigation of adenine nucleotide metabolism in human blood and the involvement of blood cells. Conversion of ATP to ADP in blood was due almost exclusively to the presence of leukocytes; plasma, platelets and erythrocytes made little or no contribution. Mononuclear leukocytes (MNLs) and polymorphonuclear leukocytes (PMNLs) were equally effective. Conversion of ADP to AMP was also promoted by leukocytes, with no involvement of platelets or erythrocytes. Some ADP was also converted to ATP in blood, apparently via an enzyme present in plasma, but ATP was then rapidly removed by the leukocytes. Conversion of AMP to adenosine occurred via a plasma enzyme with little or no contribution from any cellular element. As expected, in blood the adenosine produced was removed very rapidly by erythrocytes and then converted to inosine and then hypoxanthine. In the absence of erythrocytes plasma supported only a slow conversion of adenosine to inosine and hypoxanthine, which was not influenced by platelets or leukocytes. This study has demonstrated that leukocytes and erythrocytes play a major role in adenine nucleotide metabolism in blood and that these cells, as well as endothelial cells, may be important determinants of the effects of ATP and ADP on platelets.

Objective-Effects on platelet aggregation of adenosine triphosphate (ATP) released from damaged cells and from platelets undergoing exocytosis have not been clearly established. In this study we report on the effects of ATP on platelet aggregation in whole blood. Methods and Results-Aggregation, measured using a platelet-counting technique, occurred in response to ATP and was maximal at 10 to 100 mol/L. It was abolished by MRS2179, AR-C69931, and creatine phosphate/creatine phosphokinase, implying that conversion to adenosine diphosphate (ADP) is required. ATP did not induce aggregation in platelet-rich plasma, but aggregation did occur when apyrase or hexokinase was added. Aggregation also occurred after addition of leukocytes to platelet-rich plasma (as a source of ecto-ATPase), and this was potentiated on removal of adenosine by adenosine deaminase, indicating that adenosine production modulates the response. Dipyridamole, which inhibits adenosine uptake into erythrocytes, inhibited aggregation induced by ATP in whole blood, and adenosine deaminase reversed this. DN9693 and forskolin synergized with dipyridamole to inhibit ATP-induced aggregation. 4 The released ADP can interact with P2Y 1 and P2Y 12 (formally known as P 2T ) receptors on platelets and induce platelet aggregation, 5-7 which contributes to normal hemostasis and to thrombus formation. However, the effect of the released ATP is unclear. It is known that ATP can interact with P2X 1 receptors on platelets, causing a transient Ca 2ϩ mobilization, 8,9 but this does not result in platelet aggregation, and the importance of P2X 1 receptors to overall platelet function is unknown. It is also known that ATP acts as an antagonist of the effects of ADP at P2Y 1 and P2Y 12 receptors 10,11 and that high concentrations can inhibit ADP-induced platelet aggregation. 12 When considering the possible effects of ATP on platelets in vitro and in vivo, the presence of enzymes present on blood cells and endothelial cells and in plasma that metabolize ATP must be taken into account. These include enzymes that convert ATP to ADP and ADP to AMP (NTPDase-1, also known as ATP diphosphohydrolase, CD 39 and EC 3.6.1.5), 13,14 ATP to AMP and ADP to AMP (5Ј-monophosphate phosphoanhydrolase/phosphodiesterase, NMPP), 15 and AMP to adenosine (5Ј-nucleotidase), 13 the latter being an inhibitor of platelet aggregation. 10,16 Also, adenosine can be taken up and neutralized by erythrocytes and other blood cells, thus limiting its potential inhibitory action. 17,18 Thus, the overall effect of ATP could depend on several competing influences. Conclusions-ATPIn this study, we have investigated the effects of ATP on platelet aggregation in whole blood and in platelet-rich plasma (PRP). We used hirudin as the anticoagulant to ensure that the conditions used were as near physiological as possible. We found that ATP induces platelet aggregation in whole blood but not in PRP, and we have investigated the mechanisms that are involved. Methods MaterialsHirudin (recombinant desulphato-hirudi...

SUMMARY1. The actions of four volatile anaesthetics on the evoked synaptic potentials of in vitro preparations of the hippocampus were examined.2. All four anaesthetics (ether, halothane, methoxyflurane and trichloroethylene) depressed the synaptic transmission between the perforant path and the granule cells at concentrations lower than those required to maintain anaesthesia in intact animals.3. The population excitatory post-synaptic potential (e.p.s.p.) and massed discharge of the cortical cells (population spike) were depressed at concentrations of the anaesthetics lower than those required to depress the compound action potential of the perforant path nerve fibres. None of the anaesthetics studied increased the threshold depolarization required for granule cell discharge. Furthermore, frequency potentiation of the evoked cortical e.p.s.p.s was not impaired by any of the anaesthetics studied.4. It is concluded that all four anaesthetics depress synaptic transmission in the dentate gyrus either by reducing the amount of transmitter released from each nerve terminal in response to an afferent volley, or by decreasing the sensitivity of the post-synaptic membrane to released transmitter or by both effects together.

The effects on platelet function of temperatures attained during hypothermia used in cardiac surgery are controversial. Here we have performed studies on platelet aggregation in whole blood and platelet-rich plasma after stimulation with a range of concentrations of ADP, TRAP, U46619 and PAF at both 28 degrees C and 37 degrees C. Spontaneous aggregation was also measured after addition of saline alone. In citrated blood, spontaneous aggregation was markedly enhanced at 28 degrees C compared with 37 degrees C. Aggregation induced by ADP was also enhanced. Similar results were obtained in hirudinised blood. There was no spontaneous aggregation in PRP but ADP-induced aggregation was enhanced at 28 degrees C. The P2Y12 antagonist AR-C69931 inhibited all spontaneous aggregation at 28 degrees C and reduced all ADP-induced aggregation responses to small, reversible responses. Aspirin had no effect. Aggregation was also enhanced at 28 degrees C compared with 37 degrees C with low but not high concentrations of TRAP and U46619. PAF-induced aggregation was maximal at all concentrations when measured at 28 degrees C, but reversal of aggregation was seen at 37 degrees C. Baseline levels of platelet CD62P and CD63 were significantly enhanced at 28 degrees C compared with 37 degrees C. Expression was significantly increased at 28 degrees C after stimulation with ADP, PAF and TRAP but not after stimulation with U46619. Overall, our results demonstrate an enhancement of platelet function at 28 degrees C compared with 37 degrees C, particularly in the presence of ADP.