The epidemiological characteristics of platelet aggregability were established in 958 participants in the Northwick Park Heart Study. The main analyses were based on the dose of adenosine diphosphate at which primary aggregation occurred at half its maximum velocity. Aggregability increased with age in both sexes, was greater in whites than blacks (particularly among men), and tended to decrease with the level of habitual alcohol consumption. Aggregability was, however, greater in women than men and in nonsmokers than smokers. There was no relation between aggregability on the one hand and obesity, current or past oral contraceptive use, menopausal state, or blood cholesterol and triglyceride concentrations on the other. Aggregability was somewhat, though not significantly, higher in men with a history of ischaemic heart disease and in those with electrocardiographic evidence of ischaemia than in those without. There was a strong association between the plasma fibrinogen concentration and aggregability. The widely held concept of platelet aggregability and its implications is probably an oversimplification. In the prevention of thrombosis it may be as useful to consider modifying external influences on platelet behaviour, such as plasma fibrinogen concentration or thrombin production, as it is to rely solely on platelet active agents.

BACKGROUND

Variations in platelet function among individuals may be related to differences in platelet-related genes. The major goal of our study was to estimate the contribution of inheritance to the variability in platelet function in unaffected individuals from white and African American families with premature coronary artery disease.

METHODS

Platelet reactivity, in the absence of antiplatelet agents, was assessed by in vitro aggregation and the platelet function analyzer closure time. Heritability was estimated using a variance components model.

RESULTS

Both white (n = 687) and African American (n = 321) subjects exhibited moderate to strong heritability (h(2)) for epinephrine- and adenosine diphosphate-induced aggregation (0.36-0.42 for white and >0.71 for African American subjects), but heritability for collagen-induced platelet aggregation in platelet-rich plasma was prominent only in African American subjects. Platelet lag phase after collagen stimulation was heritable in both groups (0.47-0.50). A limited genotype analysis demonstrated that the C825T polymorphism of GNB3 was associated with the platelet aggregation response to 2 muM epinephrine, but the effect differed by race.

CONCLUSIONS

Considering the few and modest genetic effects reported to affect platelet function, our findings suggest the likely existence of undiscovered important genes that modify platelet reactivity, some of which affect multiple aspects of platelet biology.

Triple negative breast cancer (TNBC), an aggressive variant of breast cancer, is characterized by lack of expression of the estrogen (ER) and progesterone receptors (PRs) and the human epidermal growth factor receptor (HER-2) that are commonly observed in other breast cancer subtypes. The TNBC subtype primarily occurs in younger women of African American or Hispanic descent and tumors tend to be high grade and initially responsive to chemotherapy. However, TNBC is characteristically aggressive with high recurrence, metastatic, and mortality rates. Treatment options are limited since the hormonal receptor and HER-2 antagonists typically used for other breast cancers are ineffective. As such, the mainstay of treatment of TNBC is traditional systemic cytotoxic chemotherapy. Potential future therapies for TNBC include targeted molecular strategies including poly (adenosine diphosphate ribose) polymerase (PARP) and epidermal growth factor receptor (EGFR) inhibitors and antiangiogenic agents. Further research aimed at identifying unique genetic characteristics of TNBC may allow development of other targeted molecular chemotherapy treatment options.

Reactive oxygen species and other oxidants are implicated in the mechanisms of biological ageing and exercise-induced tissue damage. The present study examined the effects of ageing and an acute bout of exercise on intracellular oxidant generation, lipid peroxidation, protein oxidation and glutathione (GSH) status in the heart and liver of young adult (8 month, N=24) and old (24 month, N=24) male Fischer 344 rats. Young rats ran on treadmill at 25 m min-1, 5% grade until exhaustion (55.4+/-2.7 min), whereas old rats ran at 15 m min-1, 5% until exhaustion (58.0+/-2.7 min). Rate of dichlorofluorescin (DCFH) oxidation, an indication of intracellular oxidant production, was significantly higher in the homogenates of aged heart and liver compared with their young counterparts. In the isolated heart and liver mitochondria, ageing increased oxidant production by 29 and 32% (P<0.05), respectively. Acute exercise increased oxidant production in the aged heart but not in the liver. When nicodinamide dinucleotide phosphate (reduced), adenosine diphosphate and Fe3+ were included in the assay, DCFH oxidation rate was 47 and 34% higher (P<0.05) in the aged heart and liver homogenates, respectively, than the young ones. The age differences in the induced state reached 83 and 140% (P<0.01) in isolated heart and liver mitochondria, respectively. Lipid peroxidation was increased in the aged liver and exercised aged heart, whereas protein carbonyl content was elevated only in the aged heart (P<0.05). Although our data using DCFH method probably underestimated cellular oxidant production because of time delay and antioxidant competition, it is clear that oxidative stress was enhanced in both heart and liver with old age. Furthermore, aged myocardium showed greater susceptibility to oxidative stress after heavy exercise.

Subventricular zone (SVZ)-derived adult neurospheres express two ectonucleotidases, nucleoside triphosphate diphosphohydrolase 2 (NTPDase2) and tissue non-specific alkaline phosphatase (TNAP). Agonists of the nucleotide receptors P2Y(1) and P2Y(2) as well as adenosine augment growth factor-mediated progenitor cell proliferation. NTPDase2 converts ATP and UTP to ADP and UDP, respectively, which are all P2Y receptor agonists. TNAP hydrolyzes nucleoside triphosphates and diphosphates and produces the P1 receptor agonist adenosine. In the SVZ, NTPDase2 is specifically expressed by type B cells. In order to further scrutinize the association of key molecules of the purinergic signaling pathway with neurogenic regions, we analyzed the expression of TNAP at the lateral ventricles of the adult and developing mouse brain. In the adult brain, TNAP was expressed by type B, type A and at least subsets of type C cells of the SVZ and throughout the rostral migratory stream. Almost 100% of the proliferating, Ki-67-positive cells of the adult SVZ stained for TNAP, supporting the notion of a ubiquitous association of TNAP with SVZ progenitors. In contrast, NTPDase2-positive progenitors of the dentate gyrus were TNAP-negative. Essentially all cells of the telencephalic vesicle at embryonic day (E) 14 revealed TNAP activity, including doublecortin-positive neuroblasts. During further embryonic development, enhanced TNAP activity became restricted to cells of the ventricular and SVZ. In contrast to TNAP, NTPDase2 was first expressed in the SVZ perinatally, in association with TNAP-positive SVZ border cells. During later development, NTPDase2-positive cells disappeared from the ventricular surface and began to form sheaths around clusters of subventricular doublecortin-positive cells, apparently transforming into type B cells. Our results identify TNAP and NTPDase2 as novel markers for subsets of progenitors in the adult and developing mouse brain. They further support the notion that signaling via extracellular nucleotides and nucleosides contributes to embryonic and adult neurogenesis.

OBJECTIVE

We sought to assess the impact of renal function on platelet reactivity in patients with diabetes mellitus (DM) and coronary artery disease on aspirin and clopidogrel therapy.

BACKGROUND

Diabetes mellitus is a key risk factor for chronic kidney disease (CKD). In aspirin-treated DM patients the presence of moderate/severe CKD is associated with reduced clinical efficacy of adjunctive clopidogrel therapy. Whether these findings may be attributed to differences in clopidogrel-induced effects is unknown.

METHODS

This was a cross-sectional observational study in which DM patients taking maintenance aspirin and clopidogrel therapy were studied. Patients were categorized into 2 groups according to the presence or absence of moderate/severe CKD. Platelet aggregation after adenosine diphosphate (ADP) and collagen stimuli were assessed with light transmittance aggregometry and defined patients with high post-treatment platelet reactivity (HPPR). Markers of platelet activation, including glycoprotein IIb/IIIa activation and P-selectin expression, were also determined using flow cytometry.

RESULTS

A total of 306 DM patients were analyzed. Patients with moderate/severe CKD (n = 84) had significantly higher ADP-induced (60 +/- 13% vs. 52 +/- 15%, p = 0.001) and collagen-induced (49 +/- 20% vs. 41 +/- 20%, p = 0.004) platelet aggregation compared with those without (n = 222). After adjustment for potential confounders, patients with moderate/severe CKD were more likely to have HPPR after ADP (adjusted odds ratio: 3.8, 95% confidence interval: 1.7 to 8.5, p = 0.001) and collagen (adjusted odds ratio: 2.4; 95% confidence interval: 1.1 to 5.4; p = 0.029) stimuli. Markers of platelet activation were significantly increased in patients with HPPR.

CONCLUSIONS

In DM patients with coronary artery disease taking maintenance aspirin and clopidogrel therapy, impaired renal function is associated with reduced clopidogrel-induced antiplatelet effects and a greater prevalence of HPPR.

Activation of human platelets considerably enhanced their ability to accelerate factor Va inactivation by activated protein C (APC). The anticoagulant activity of platelet suspensions was markedly dependent on the kind of agonist used to activate platelets. APC-catalyzed factor Va inactivation in free solution was characterized by an apparent second-order rate constant of 2 x 10(5) (mol/L)-1 (seconds)-1. Nonstimulated platelets (2.4 x 10(8)/mL) and platelets stimulated with adenosine diphosphate or adrenalin accelerated factor Va inactivation fourfold. Rates of factor Va inactivation were increased 11-fold by thrombin-stimulated platelets, 29-fold after platelet stimulation with the Ca(2+)-ionophore A23187. At low platelet concentrations (3 x 10(7)/mL) only background levels of anticoagulant activity were observed in platelet suspensions that were nonstimulated or stimulated with thrombin or collagen. However, when such reaction mixtures were stirred during the activation procedure, platelet anticoagulant activity was increased more than 10-fold. Independent of platelet stimulation and stirring conditions, exogenously added purified plasma protein S increased platelet-dependent factor Va inactivation approximately twofold. Addition of a neutralizing antiprotein S antibody had little effect on the anticoagulant activity of platelets. This indicates that, under the reaction conditions tested, platelet-released protein S did not contribute to factor Va inactivation. Approximately 25% of the anticoagulant activity of stimulated platelet suspensions appeared to be associated with microparticles that were released on platelet activation. Such microparticles may provide an important source of anticoagulant activity. A similar distribution of procoagulant, ie, prothrombinase, activity between platelets and microparticles was observed for the same platelet suspensions. Because platelet stimulation and stirring also had the same overall effects on the ability of platelets and platelet microparticles to promote prothrombin activation and factor Va inactivation, it appears likely that the generation of potential platelet anticoagulant and procoagulant activities is coupled to the same platelet stimulation reactions.

The present study characterized platelet secretion and surface expression of proangiogenic stromal cell-derived factor-1α (SDF-1α) and vascular endothelial growth factor (VEGF) and antiangiogenic PF4 and endostatin on activation. The angiogenic factors presented in randomly distributed granules in resting platelets, which were peripherized on activation. Confocal and immunogold electron microscopy demonstrated that SDF-1α/CXCL12 and PF4/CXCL4 mostly present in different granules. Platelet activation induced marked SDF-1α and endostatin but mild PF4 or no VEGF surface expression. PAR1-activating peptide (PAR1-AP), adenosine diphosphate (via P2Y1/P2Y12), and glycoprotein VI-targeting collagen-related peptide induced massive SDF-1α and VEGF but modest PF4 or no endostatin release. In contrast, PAR4-AP triggered marked PF4 and sole endostatin release but limited SDF-1α or VEGF secretion. Distinct platelet release of SDF-1α and endostatin involved different engagements of intracellular signaling pathways. In conclusion, different platelet stimuli evoke distinct secretion and surface expression of proangiogenic and antiangiogenic factors. PAR1, adenosine diphosphate, and glycoprotein VI stimulation favors proangiogenic, whereas PAR4 promotes antiangiogenic, factor release.

OBJECTIVE

We sought to evaluate the response to antiplatelet therapy in patients with stent thrombosis (ST).

BACKGROUND

Stent thrombosis is associated with considerable morbidity and mortality. An impaired response to antiplatelet therapy might be related to an increased risk for ST.

METHODS

Eighty-two patients were included in the present study: 23 patients with previous ST, 50 matched controls (coronary stenting without ST), and 9 healthy volunteers. Platelet aggregation (PA) was studied (optical aggregometry) under monotherapy with acetylsalicylic acid (ASA) 100 mg daily for one month, followed by dual therapy with ASA 100 mg and clopidogrel 75 mg daily (loading dose 300 mg) for another month.

RESULTS

Maximal (5 and 20 micromol) adenosine diphosphate-induced PA was significantly higher in patients with ST compared with controls (5 micromol, p < 0.005; 20 micromol, p < 0.05) and volunteers (5 micromol, p < 0.005; 20 micromol, p < 0.05). Resistance to ASA (>20% aggregation with 0.5 mg/ml arachidonic acid) was more prevalent in patients with ST (48%) compared with control patients (32%, p = ns) and volunteers (0%, p = 0.01). Clopidogrel significantly reduced PA in all three groups, but intergroup differences persisted. Clopidogrel resistance (<10% relative change) was similar in patients with ST, control patients, and volunteers (4%, 6%, and 11%, respectively, all p = NS). However, combined ASA and clopidogrel resistance was more prevalent in patients with ST (52%) compared with controls (38%, p = NS) and volunteers (11%, p < 0.05).

CONCLUSIONS

Patients with previous ST show an impaired response to antiplatelet therapy with ASA compared with controls and volunteers. Additional treatment with clopidogrel is not able to overcome these differences in PA. Acetylsalicylic acid but not clopidogrel resistance appears to be associated with ST.

OBJECTIVE

The purpose of this study was to determine the impact of adjunctive cilostazol in patients with high post-treatment platelet reactivity (HPPR) undergoing coronary stenting.

BACKGROUND

Although addition of cilostazol to dual antiplatelet therapy enhances adenosine diphosphate (ADP)-induced platelet inhibition, it is unknown whether adjunctive cilostazol can reduce HPPR.

METHODS

Sixty patients with HPPR after a 300-mg loading dose of clopidogrel were enrolled. HPPR was defined as maximal platelet aggregation (Agg(max)) >50% with 5 micromol/l ADP. Patients were randomly assigned to receive either adjunctive cilostazol (triple group; n = 30) or high maintenance dose (MD) clopidogrel (high-MD group; n = 30). Platelet function was assessed at baseline and after 30 days with conventional aggregometry and the VerifyNow assay.

RESULTS

Baseline platelet function measurements were similar in both groups. After 30 days, significantly fewer patients in the triple versus high-MD group had HPPR (3.3% vs. 26.7%, p = 0.012). Percent inhibitions of 5 micromol/l ADP-induced Agg(max) and late platelet aggregation (Agg(late)) were significantly greater in the triple versus high-MD group (51.1 +/- 22.5% vs. 28.0 +/- 18.5%, p < 0.001, and 70.9 +/- 27.3% vs. 45.3 +/- 23.4%, p < 0.001, respectively). Percent inhibitions of 20 micromol/l ADP-induced Agg(max) and Agg(late) were consistently greater in the triple versus high-MD group. Percent change of P2Y12 reaction units demonstrated a higher antiplatelet effect in the triple versus high-MD group (39.6 +/- 24.1% vs. 23.1 +/- 29.9%, p = 0.022).

CONCLUSIONS

Adjunctive cilostazol reduces the rate of HPPR and intensifies platelet inhibition as compared with a high-MD clopidogrel of 150 mg/day.

1 The effects of adenosine triphosphate (ATP) and adenosine diphosphate (ADP) were investigated on evoked end-plate potentials (e.p.ps) and on miniature end-plate potentials (min. e.p.ps) recorded from muscle fibres of the rat diaphragm and the frog sartorius.2 ATP and ADP decreased the quantum content of the e.p.ps and the frequency of the min. e.p.ps. The maximum effects produced by the two substances were similar.3 The potency of ATP was found to be similar to that of adenosine. In the presence of adenosine, in a concentration producing its maximum effect, the addition of ATP had no further effect. This is compatible with the idea that ATP acts in the same way as adenosine.

The isolated taenia coli of the guinea pig takes up tritiated adenosine, adenosine monophosphate, adenosine diphosphate, and adenosine triphosphate, in preference to tritiated inosine and adenine. After uptake, [(3)H]adenosine is converted and retained primarily as [(3)H]adenosine triphosphate. Tritium is released from taenia coli treated with [(3)H]adenosine upon activation of the nonadrenergic inhibitory nerves. These results are consistent with the previous evidence that adenosine triphosphate may be the transmitter from the nerves.

Release of prostacyclin was studied by superfusing small columns containing cells cultured on microcarrier beads. Transient dose-dependent stimulation of prostacyclin release by up to 500-fold was induced by adenosine 5'-triphosphate (ATP; 0.5-50 microM). Adenosine 5'-diphosphate (ADP) gave similar responses, whereas adenosine 5'-phosphate (AMP) and adenosine were essentially inactive. Of other natural nucleotides tested only uridine 5'-phosphate (UTP) was active. The L-enantiomers of ATP and ADP were inactive. 2-Cl-ATP was approximately 100 times more potent than ATP; 2-MeS-ATP was also more potent (threshold 0.05 microM) but its maximal effectiveness was less than 20% that of ATP; 2-EtS-ATP had a similar threshold to ATP but was even less effective than 2-MeS-ATP. Phosphorothioate nucleotide analogues of ATP or ADP were active, with no stereoselectivity between Rp and Sp diastereoisomers. No analogue tested showed antagonist activity. We conclude that ATP mediates endothelial prostacyclin release apparently via a P2Y receptor, although there are some striking differences from the previously described P2Y receptor mediating endothelium-dependent vasodilation in pig aorta.

OBJECTIVE

The aim of this study was to investigate the relationship between platelet reactivity (PR) after a loading dose (LD) of prasugrel and thrombotic events.

BACKGROUND

Post-treatment PR has been shown to be strongly associated with the occurrence of major adverse cardiac events (MACE) after percutaneous coronary intervention (PCI) in the clopidogrel era. Prasugrel is a new P2Y(12)-adenosine diphosphate receptor with a higher potency on PR.

METHODS

A prospective multicenter study included patients who underwent successful PCI for acute coronary syndromes and received prasugrel therapy. Vasodilator-stimulated phosphoprotein (VASP) index was measured after the prasugrel LD. High on-treatment PR was defined as a VASP index ≥50%. MACE included cardiovascular death, myocardial infarction, and definite stent thrombosis at 1 month.

RESULTS

Three hundred one patients were enrolled. The mean VASP index after 60 mg of prasugrel was 34.3 ± 23.1%. High on-treatment PR was observed in 76 patients (25.2%). Patients experiencing thrombotic events after PCI had significantly higher VASP indexes compared with those free of events (64.4 ± 14.4% vs. 33.4 ± 22.7%; range: 51% to 64% and 5% to 47.6%, respectively; p = 0.001). Kaplan-Meier analysis comparing good responders and patients with high on-treatment PR demonstrated a significantly higher rate of MACE in patients with suboptimal PR inhibition (log-rank p < 0.001). Receiver-operating characteristic curve analysis found a cutoff value of 53.5% of the VASP index to predict thrombotic events at 1 month (r = 0.86, p < 0.001). Patients with minor or major Thrombolysis In Myocardial Infarction unrelated to coronary artery bypass grafting bleeding and those without had similar VASP indexes (30 ± 17.8% vs. 34.3 ± 23%, p = 0.70).

CONCLUSIONS

Despite the use of prasugrel, a significant number of patients undergoing PCI in the setting of acute coronary syndromes do not achieve optimal PR inhibition. Such patients have a higher risk for MACE after PCI.

The suppression of tension development by orthovanadate (Vi) was studied in mechanical experiments and by measuring the binding of radioactive Vi and nucleotides to glycerol-extracted rabbit muscle fibers. During active contractions, Vi bound to the cross-bridges and suppressed tension with an apparent second-order rate constant of 1.34 X 10(3) M-1s-1. The half-saturation concentration for tension suppression was 94 microM Vi. The incubation of fibers in Vi relaxing or rigor solutions prior to initiation of active contractions had little effect on the initial rise of active tension. The addition of adenosine diphosphate (ADP) and Vi to fibers in rigor did not cause relaxation. Suppression of tension only developed during cross-bridge cycling. After slow relaxation from rigor in 1 mM Vi and low (50 microM) MgATP concentration (0 Ca2+), radioactive Vi and ADP were trapped within the fiber. This finding indicated the formation of a stable myosin X ADP X Vi complex, as has been reported in biochemical experiments with isolated myosin. Vi and ADP trapped within the fibers were released only by subsequent cross-bridge attachment. Vi and ADP were preferentially trapped under conditions of cross-bridge cycling in the presence of ATP rather than in relaxed fibers or in rigor with ADP. These results indicate that in the normal cross-bridge cycle, inorganic phosphate (Pi) is released from actomyosin before ADP. The resulting actomyosin X ADP intermediate can bind Vi and Pi. This intermediate probably supports force. Vi behaves as a close analogue of Pi in muscle fibers, as it does with isolated actomyosin.

OBJECTIVE

This study was conducted to assess the thromboxane (TX) dependence of biochemical and functional indexes used to monitor the effect of low-dose aspirin.

BACKGROUND

Functional assays of the antiplatelet effects of low-dose aspirin variably reflect the TX-dependent component of platelet aggregation. Previous studies of aspirin resistance were typically based on a single determination of platelet aggregation.

METHODS

We assessed the TXB(2) dependence of biochemical and functional indexes, as well as their intersubject and intrasubject variability during administration of the drug and after its withdrawal in 48 healthy volunteers randomized to receive aspirin 100 mg daily for 1 to 8 weeks.

RESULTS

Serum TXB(2) was uniformly suppressed by 99% of baseline. Urinary 11-dehydro-TXB(2), arachidonic acid-induced aggregation, and VerifyNow Aspirin (Accumetrics Inc., San Diego, California) showed stable, incomplete inhibition (65%, 80%, and 35%, respectively). Adenosine diphosphate- and collagen-induced aggregation was highly variable and poorly affected by aspirin, with an apparent time-dependent reversal. Inhibition of platelet cyclooxygenase activity was nonlinearly related to inhibition of platelet aggregation. Platelet function largely recovered by day 3 post-aspirin, independently of treatment duration. With any functional assay, occasionally "resistant" subjects were found to be "responders" on previous or subsequent determinations.

CONCLUSIONS

Platelet cyclooxygenase activity, as reflected by serum TXB(2) levels, is uniformly and persistently suppressed by low-dose aspirin in healthy subjects. However, the effect of aspirin is variably detected by functional assays, potentially leading to misclassification of "responder" as "resistant" phenotypes owing to poor reproducibility of functional measurements. The nonlinearity of the relationship between inhibition of TX production and inhibition of platelet function has important clinical implications.

Cell surface ecto-nucleotidases are considered the major effector system for inactivation of extracellular adenine nucleotides, whereas the alternative possibility of ATP synthesis has received little attention. Using a TLC assay, we investigated the main exchange activities of 3H-labeled adenine nucleotides on the cultured human umbilical vein endothelial cells. Stepwise nucleotide degradation to adenosine occurred when a particular nucleotide was present alone, whereas combined cell treatment with ATP and either [3H]AMP or [3H]ADP caused unexpected phosphorylation of 3H-nucleotides via the backward reactions AMP ->> ADP ->> ATP. The following two groups of nucleotide-converting ecto-enzymes were identified based on inhibition and substrate specificity studies: 1) ecto-nucleotidases, ATP-diphosphohydrolase, and 5'-nucleotidase; 2) ecto-nucleotide kinases, adenylate kinase, and nucleoside diphosphate kinase. Ecto-nucleoside diphosphate kinase possessed the highest activity, as revealed by comparative kinetic analysis, and was capable of using both adenine and nonadenine nucleotides as phosphate donors and acceptors. The transphosphorylation mechanism was confirmed by direct transfer of the gamma-phosphate from [gamma-32P]ATP to AMP or nucleoside diphosphates and by measurement of extracellular ATP synthesis using luciferin-luciferase luminometry. The data demonstrate the coexistence of opposite, ATP-consuming and ATP-generating, pathways on the cell surface and provide a novel mechanism for regulating the duration and magnitude of purinergic signaling in the vasculature.

Cell survival is a critical issue in the onset and progression of neurodegenerative diseases and following pathological events including ischemia and traumatic brain injury. Oxidative stress is the main cause of cell damage in such pathological conditions. Here, we report that adenosine 5'-triphosphate (ATP) protects hippocampal astrocytes from hydrogen peroxide (H(2)O(2))-evoked oxidative injury in astrocyte monocultures. The effect of ATP was prevented by a selective antagonist of or siRNAs against P2Y(1)R. Interestingly, in astrocyte-neuron cocultures, ATP also produced neuroprotective effects against H(2)O(2)-evoked neuronal cell death, whereas ATP did not produce any neuroprotective effects in monocultures. The ATP-induced neuroprotection in cocultures was completely inhibited by silencing of astrocytic P2Y(1)R expression, indicating that ATP acts on astrocytes and enhances their neuroprotective functions by activating P2Y(1)R. Furthermore, this neuroprotective effect was mimicked by applying conditioned medium from astrocytes that had been stimulated by ATP, implying an involvement of diffusible factors from astrocytes. We found that, in both purified astrocyte cultures and astrocyte-neuronal cocultures, ATP and the P2Y(1)R agonist 2-methylthioadenosine 5' diphosphate (2MeSADP) induced the release of interleukin-6 (IL-6), but this did not occur in neuron monocultures. Moreover, exogenous IL-6 produced a neuroprotective effect, and the neuroprotection induced by P2Y(1)R-stimulated astrocytes was prevented in the presence of an anti-IL-6 antibody. Taken together, these results suggest that P2Y(1)R-stimulated astrocytes protect against neuronal damage induced by oxidative stress, and that IL-6 is a crucial signaling molecule released from astrocytes. Thus, activation of P2Y(1)R in astrocytes may rescue neurons from secondary cell death under pathological conditions.

Ethanol intake depletes the mitochondrial pool of reduced glutathione (GSH) by impairing the transport of GSH from cytosol into mitochondria. S-Adenosyl-L-methionine (SAM) supplementation of ethanol-fed rats restores the mitochondrial pool of GSH. The purpose of the current study was to determine the effect of ethanol feeding on the kinetic parameters of mitochondrial GSH transport, the fluidity of mitochondria, and the effect of SAM on these changes. Male Sprague-Dawley rats were fed ethanol-liquid diet for 4 weeks supplemented with either SAM or N-acetylcysteine (NAC). SAM-supplementation of ethanol-fed rats restored the mitochondrial GSH pool but NAC administration did not. Kinetic studies of GSH transport in isolated mitochondria revealed two saturable, adenosine triphosphate (ATP)-stimulated components that were affected significantly by chronic ethanol feeding: lowering Vmax (0.22 and 1.6 in ethanol case vs. 0.44 and 2.7 nmol/15 sec/mg protein in controls) for both low and high affinity components with the latter showing an increased Km (15.5 vs. 8.9, mmol/L in ethanol vs. control). Mitochondria from SAM-supplemented ethanol-fed rats showed kinetic features of GSH transport similar to control mitochondria. Determination of membrane fluidity revealed an increased order parameter in ethanol compared with control mitochondria, which was restricted to the polar head groups of the bilayer and was prevented by SAM but not NAC supplementation of ethanol-fed rats. The changes elicited in mitochondria by ethanol were confined to the inner membrane; mitoplasts from ethanol-fed rats showed features similar to those of intact mitochondria such as impaired transport of GSH and increased order parameter. A different mitochondrial transporter, adenosine diphosphate (ADP)/ATP translocator, was unaffected by ethanol feeding. Furthermore, fluidization of mitochondria or mitoplasts from ethanol-fed rats by treatment with a fatty acid derivative restored their ability to transport GSH to control levels. Thus, ethanol-induced impaired transport of GSH into mitochondria is selective, mediated by decreased fluidity of the mitochondrial inner membrane, and prevented by SAM treatment.

The release of nucleotides in extracellular fluids can result from cell necrosis, exocytosis of secretory granules (such as platelet dense granules), or efflux through membrane channels. In addition, recent evidence suggests that vesicular trafficking is an important pathway of nucleotide release. Once in the extracellular fluids, they are rapidly degraded by ectonucleotidases, such as CD39, that play a key role in neutralizing the platelet aggregatory action of adenosine diphosphate (ADP), and act on two families of receptors: the ionotropic P2X receptors and the G-protein-coupled P2Y receptors. The family of P2X receptors encompasses seven genes. Currently, there are eight genuine P2Y receptors that can be subdivided into two structurally distinct subfamilies. Whereas P2X receptors are receptors of ATP, the different P2Y receptors are activated by distinct nucleotides, diphosphates or triphosphates, or purines or pyrimidines, some of them being conjugated to sugars. The study of knockout mice has demonstrated that P2X receptors play important roles in the neurogenic control of smooth muscle contraction, in pain and visceral perception, and in macrophage functions. The phenotype of P2Y null mice so far is more restricted: inhibition of platelet aggregation to ADP and increased bleeding time in P2Y (1)(-/-) and P2Y (12)(-/-) mice and lack of epithelial responsiveness to nucleotides in airways (P2Y (2)(-/-)) and intestine (P2Y (4)(0/-)).

CS-747 (prasugrel, LY640315) is a member of the thienopyridine class of oral platelet aggregation inhibitors that includes ticlopidine and clopidogrel. A single oral administration of CS-747 produced a dose-related inhibition of platelet aggregation in rats that was approximately 10- and 100-fold more potent than that of clopidogrel and ticlopidine, respectively. The antiaggregatory effect of CS-747 was evident at 30 minutes and lasted until 72 hours after dosing, indicating fast onset and long duration of action. CS-747 showed more potent antithrombotic activity compared with clopidogrel and ticlopidine with the same rank order as the antiaggregatory potencies. Combined administration of CS-747 with aspirin to rats produced substantially greater inhibition of both platelet aggregation and thrombus formation compared with each agent alone. The antiplatelet action of CS-747 is due to irreversible and selective blockade of platelet P2Y (12) adenosine diphosphate (ADP) receptors by its active metabolite R-138727. In phase I studies, a single oral dose of CS-747 (30 and 75 mg) produced>> 50% inhibition of ADP-induced platelet aggregation, with rapid onset (1 hour) and long duration >> 48 hours) of action. In healthy volunteers, once-daily administration of 10 mg CS-747 for 10 days showed significant cumulative inhibition of platelet aggregation from 2 days after the first dose until at least 2 days after the final dose. Studies conducted to date indicate that CS-747 is a highly effective antiplatelet and antithrombotic agent and is anticipated to be effective in the treatment of atherothrombotic and other ischemic vascular diseases.

The platelet von Willebrand factor (vWF) receptor, glycoprotein Ib-IX (GPIb-IX), mediates platelet adhesion and induces signaling leading to integrin activation. Phosphoinositol 3-kinase (PI3K) is important in GPIb-IX-mediated signaling. PI3K-dependent signaling mechanisms, however, are unclear. We show that GPIb-IX-induced platelet aggregation and stable adhesion under flow were impaired in mouse platelets deficient in PI3K effectors, Akt1 and Akt2, and in human platelets treated with an Akt inhibitor, SH-6. Akt1 and Akt2 play important roles in early GPIb-IX signaling independent of Syk, adenosine diphosphate (ADP), or thromboxane A2 (TXA2), in addition to their recognized roles in ADP- and TXA2-dependent secondary amplification pathways. Knockout of Akt1 or Akt2 diminished platelet spreading on vWF but not on immobilized fibrinogen. Thus, Akt1 and Akt2 are both required only in the GPIb-IX-mediated integrin activation (inside-out signaling). In contrast, PI3K inhibitors abolished platelet spreading on both vWF and fibrinogen, indicating a role for PI3K in integrin outside-in signaling distinct from that in GPIb-IX-mediated inside-out signaling. Furthermore, Akt1- or Akt2-deficiency diminished vWF-induced cGMP elevation, and their inhibitory effects on GPIb-IX-dependent platelet adhesion were reversed by exogenous cGMP. Thus, Akt1 and Akt2 mediate GPIb-IX signaling via the cGMP-dependent signaling pathway.