The action of the adenyl compounds adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP) and adenosine was studied on the human blister base preparation. All 4 adenyl compounds produced pain which was slow in onset and not maintained. The threshold concentration for pain was of the order of 1-3 micron. The slopes of log concentration:pain intensity plots were relatively shallow and for moderate to severe pain a 100-fold increase of the threshold concentration was required. The adenyl compounds resembled 5-hydroxytryptamine and bradykinin with respect to onset and duration of action but were less potent. On the other hand, for threshold effects they were more potent than acetylcholine or potassium. Evidence was found for an interaction of adenyl compounds with 5-hydroxytryptamine but not with potassium, acetylcholine or bradykinin. Cyclic adenosine 3',5'-monophosphate or the chelation of extracellular calcium or magnesium were shown not to be involved in the algogenic action of adenyl compounds and the action of adenyl compounds on the rabbit isolated jejunum too was found to be unrelated to their algogenic action on the human blister base preparation.

The acute effects of reperfusion on myocardium reversibly damaged by 15 minutes of severe ischemia in vivo, were studied. Changes in the adenine nucleotide pool, cell volume regulation, myocardial calcium, and ultrastructure were studied at the end of 15 minutes of ischemia and after 0.5, 3.0, and 20 minutes of reflow. Before reperfusion, adenosine triphosphate and the adenylate pool decreased by 63% and 44% of control, respectively, and the adenylate charge was reduced to 0.65. After 3 minutes of reperfusion, the adenylate charge was restored to control by the rephosphorylation of adenosine mono- and diphosphate, but adenosine triphosphate was still reduced by 45%. Mild tissue edema was detected after 0.5 minute of reflow and persisted throughout 20 minutes of reperfusion. The increased tissue water was accompanied by a slight increase in sodium and a marked increase in tissue potassium. Although massive calcium accumulation develops when irreversibly injured tissue is reperfused, no calcium overload was detected during early reperfusion of reversibly injured myocytes. Reperfusion for 3 minutes exaggerated the mitochondrial swelling induced by 15 minutes of ischemia but after 20 minutes of reperfusion, myocardial ultrastructure was essentially normal except for rare swollen, or disrupted, mitochondria. Thus, the cellular abnormalities associated with brief periods of ischemia persist for variable periods of time after reperfusion of reversibly injured myocytes. First: although adenine nucleotide repletion occurs very slowly, the adenylate charge was restored after 3 minutes, indicating rapid resumption of mitochondrial adenosine triphosphate production. Second: calcium overload was not detected, but myocardial edema and increased potassium persisted throughout the 20 minutes of reperfusion. Third: the ultrastructural consequences of ischemia were nearly reversed after 20 minutes of reperfusion.

Inositol 1,4,5-trisphosphate (IP3) is thought to be a second messenger for intracellular calcium mobilization. However, in a cell-free system of islet microsomes, cyclic adenosine diphosphate-ribose (cADP-ribose), a nicotinamide adenine dinucleotide (NAD+) metabolite, but not IP3, induced calcium release. In digitonin-permeabilized islets, cADP-ribose and calcium, but not IP3, induced insulin secretion. Islet microsomes released calcium when combined with the extract from intact islets that had been incubated with high concentrations of glucose. Sequential additions of cADP-ribose inhibited the calcium release response to extracts from islets treated with high concentrations of glucose. Conversely, repeated additions of the islet extract inhibited the calcium release response to a subsequent addition of cADP-ribose. These results suggest that cADP-ribose is a mediator of calcium release from islet microsomes and may be generated in islets by glucose stimulation, serving as a second messenger for calcium mobilization in the endoplasmic reticulum.

Although P2 receptors mediate a myriad of physiological effects of extracellular adenine nucleotides, study of this broad class of receptors has been compromised by a lack of P2 receptor-selective antagonist molecules. The adenine nucleotide-promoted inositol lipid hydrolysis response of turkey erythrocyte membranes, which has been used extensively as a model for P2Y receptors, has been applied to identify molecules that competitively block these receptors. Adenosine-3'-phosphate-5' -phosphosulfate (A3P5PS) promoted activation of phospholipase C that was only 10-25% of that observed with the full P2Y receptor agonists ATP, ADP, and 2-methylthio-ATP (2MeSATP). The small stimulatory effects of A3P5PS were saturable. Moreover, these effects were entirely the result of interaction with the P2Y receptor, because A3P5PS had no effect on activation of phospholipase C through the beta-adrenergic receptor and produced a concentration-dependent inhibition of 2MeSATP-promoted activity over the same range of A3P5PS concentrations that alone caused a small activation of phospholipase C. Increasing concentrations of A3P5PS produced a rightward shift of the concentration-effect curve for 2MeSATP, and Schild transformation of these data revealed that A3P5PS is a competitive P2Y receptor antagonist with a pKB of 6.46 +/- 0.17. The presence of a phosphate in the 2'- or 3'-position appears to be crucial for antagonist activity, because adenosine-3' -phosphate-5'- phosphate (A3P5P) and adenosine-2'- phosphate-5'-phosphate also exhibited competitive antagonist/partial agonist activities. Other 3'-substituted analogues, such as 3'-amino-ATP and 3'-benzoylbenzoyl-ATP, were full agonists with no antagonist activity. A3P5PS, A3P5P, and adenosine-2',5'-diphosphate also were competitive antagonists in studies with the cloned human P2Y1 receptor stably expressed in 1321N1 human astrocytoma cells. Moreover, both A3P5PS and A3P5P were devoid of agonist activity at the human P2Y1 receptor. The effects of these 2'- and 3'-phosphate analogues were specific for the phospholipase C-coupled P2Y1 receptor, because no agonistic or antagonistic effects on the adenylyl cyclase-coupled P2Y receptor of C6 glioma cells or on P2Y2, P2Y4, or P2Y6 receptors stably expressed in 1321N1 human astrocytoma cells were observed. These results describe specific competitive antagonism of the P2Y1 receptor by an adenine nucleotide derivative and provide a potential new avenue for P2 receptor drug development.

We have used cryo-electron microscopy of kinesin-decorated microtubules to resolve the structure of the motor protein kinesin's crucial nucleotide response elements, switch I and the switch II helix, in kinesin's poorly understood nucleotide-free state. Both of the switch elements undergo conformational change relative to the microtubule-free state. The changes in switch I suggest a role for it in "ejecting" adenosine diphosphate when kinesin initially binds to the microtubule. The switch II helix has an N-terminal extension, apparently stabilized by conserved microtubule contacts, implying a microtubule activation mechanism that could convey the state of the bound nucleotide to kinesin's putative force-delivering element (the "neck linker"). In deriving this structure, we have adapted an image-processing technique, single-particle reconstruction, for analyzing decorated microtubules. The resulting reconstruction visualizes the asymmetric seam present in native, 13-protofilament microtubules, and this method will provide an avenue to higher-resolution characterization of a variety of microtubule- binding proteins, as well as the microtubule itself.

The effects of chronic ketosis on cerebral metabolism were determined in adult rats maintained on a high-fat diet for approximately three weeks and compared to a control group of animals. The fat-fed rats had statistically significantly lower blood glucose concentrations and higher blood beta-hydroxybutyrate and acetoacetate concentrations; higher brain concentrations of bound glucose, glucose 6-phosphate, pyruvate, lactate, beta-hydroxybutyrate, citrate, alpha-ketoglutarate, alanine, and adenosine triphosphate (ATP); lower brain concentrations of fructose 1,6-diphosphate, aspartate, adenosine diphosphate (ADP), creatine, cyclic nucleotides, succinyl coenzyme A (CoA), acid-insoluble CoA, and total CoA; and similar brain concentrations of glucose, malate, calculated oxaloacetate, glutamate, glutamine, adenosine monophosphate, phosphocreatine, reduced CoA, acetyl CoA, sodium, potassium, chloride, and water content. The metabolite data in the chronically ketotic rats demonstrate an increase in the cerebral energy reserve and energy charge. These data also suggest negative modification of the enzymes phosphofructokinase, pyruvic dehydrogenase, and alpha-ketoglutaric dehydrogenase; positive modification of glycogen synthase; and possible augmentation of the hexose transport system. There was no demonstrable difference in brain pH, water content, or electrolytes in the two groups of animals. We speculate that the increased brain ATP/ADP ratio is central to most, if not all, the observed metabolic perturbations and may account for the increased neuronal stability that accompanies chronic ketosis.

Intraflagellar transport (IFT) machinery mediates the bidirectional movement of cargos that are required for the assembly and maintenance of cilia. However, little is known about how IFT is regulated in vivo. In this study, we show that the small guanosine triphosphatase (GTPase) adenosine diphosphate ribosylation factor-like protein 13 (ARL-13) encoded by the Caenorhabditis elegans homologue of the human Joubert syndrome causal gene ARL13B, localizes exclusively to the doublet segment of the cilium. arl-13 mutants have shortened cilia with various ultrastructural deformities and a disrupted association between IFT subcomplexes A and B. Intriguingly, depletion of ARL-3, another ciliary small GTPase, partially suppresses ciliogenesis defects in arl-13 mutants by indirectly restoring binding between IFT subcomplexes A and B. Rescue of arl-13 mutants by ARL-3 depletion is mediated by an HDAC6 deacetylase-dependent pathway. Thus, we propose that two conserved small GTPases, ARL-13 and ARL-3, coordinate to regulate IFT and that perturbing this balance results in cilia deformation.

Sirtuins (Sirt1-Sirt7) comprise a family of nicotinamide adenine dinucleotide (NAD(+))-dependent enzymes. While deacetylation reflects their main task, some of them have deacylase, adenosine diphosphate-ribosylase, demalonylase, glutarylase, and desuccinylase properties. Activated upon caloric restriction and exercise, they control critical cellular processes in the nucleus, cytoplasm, and mitochondria to maintain metabolic homeostasis, reduce cellular damage and dampen inflammation-all of which serve to protect against a variety of age-related diseases, including cardiovascular pathologies. This review focuses on the cardiovascular effects of Sirt1, Sirt3, Sirt6, and Sirt7. Most is known about Sirt1. This deacetylase protects from endothelial dysfunction, atherothrombosis, diet-induced obesity, type 2 diabetes, liver steatosis, and myocardial infarction. Sirt3 provides beneficial effects in the context of left ventricular hypertrophy, cardiomyopathy, oxidative stress, metabolic homeostasis, and dyslipidaemia. Sirt6 is implicated in ameliorating dyslipidaemia, cellular senescence, and left ventricular hypertrophy. Sirt7 plays a role in lipid metabolism and cardiomyopathies. Most of these data were derived from experimental findings in genetically modified mice, where NFκB, Pcsk9, low-density lipoprotein-receptor, PPARγ, superoxide dismutase 2, poly[adenosine diphosphate-ribose] polymerase 1, and endothelial nitric oxide synthase were identified among others as crucial molecular targets and/or partners of sirtuins. Of note, there is translational evidence for a role of sirtuins in patients with endothelial dysfunction, type 1 or type 2 diabetes and longevity. Given the availability of specific Sirt1 activators or pan-sirtuin activators that boost levels of the sirtuin cofactor NAD⁺, we anticipate that this field will move quickly from bench to bedside.

An in vitro culture system demonstrating the transitions from megakaryocyte progenitors to functional platelets is described. CD34-selected cells from normal human peripheral blood are cultured under conditions that promote megakaryocyte formation. After 8 to 11 days, enriched populations of mature megakaryocytes are replated under conditions that favor the development of proplatelets. Proplatelets express the platelet-specific proteins, glycoproteins Ib and IIb (GPIb and GPIIb), and fibrinogen and also contain microtubule coils equal in size to those found in plasma-derived platelets. In addition, proplatelets have ultrastructural features in common with plasma-derived platelets. Platelet-sized particles from the proplatelet culture supernatants are examined. Ultrastructurally, these particles are identical to plasma-derived platelets. Functionally, these culture-derived platelets aggregate in response to both thrombin and adenosine diphosphate (ADP) plus fibrinogen. This aggregation is specifically inhibited by the addition of a function-blocking anti-GPIIbIIIa antibody. Culture-derived platelets stimulated with agonists also express the activation-dependent antigens P-selectin and functional fibrinogen receptor. This is the first description of an in vitro culture system that sequentially demonstrates megakaryocyte growth, development, and platelet production.

We and others have shown that an increased extracellular concentration of adenosine mediates the antiinflammatory effects of methotrexate and sulfasalazine both in vitro and in vivo, but the mechanism by which these drugs increase extracellular adenosine remains unclear. The results of the experiments reported here provide three distinct lines of evidence that adenosine results from the ecto-5'-nucleotidase- mediated conversion of adenine nucleotides to adenosine. First, pretreatment of a human microvascular endothelial cell line (HMEC-1) with methotrexate increases extracellular adenosine after exposure of the pretreated cells to activated neutrophils; the ecto-5'-nucleotidase inhibitor alpha, beta-methylene adenosine-5'-diphosphate (APCP) abrogates completely the increase in extracellular adenosine. Second, there is no methotrexate-mediated increase in extracellular adenosine concentration in the supernate of cells deficient in ecto-5'-nucleotidase, but there is a marked increase in extracellular adenosine concentration in the supernates of these cells after transfection and surface expression of the enzyme. Finally, as we have shown previously, adenosine mediates the antiinflammatory effects of methotrexate and sulfasalazine in the murine air pouch model of inflammation, and injection of APCP, the ecto-5'-nucleotidase inhibitor, abrogates completely the increase in adenosine and the decrement in inflammation in this in vivo model. These results not only show that ecto-5'-nucleotidase activity is a critical mediator of methotrexate- and sulfasalazine-induced antiinflammatory activity in vitro and in vivo but also indicate that adenine nucleotides, released from cells, are the source of extracellular adenosine.

We have assessed, whether six agents, that either inhibit free radical formation or scavenge free radicals once they are produced, can reduce the incidence of reperfusion-induced arrhythmias, whether a free radical-generating system (FeCl3 X adenosine diphosphate) can increase the incidence of reperfusion-induced arrhythmias, and whether "anti-free radical" interventions can reduce reperfusion rhythm disturbances caused by the addition of FeCl3 X adenosine diphosphate. With the isolated, perfused rat heart (n = 15 in each group), inclusion of L-methionine (1 and 10 mM), superoxide dismutase (2.5 X 10(4) and 1 X 10(5) U/liter), catalase (5 X 10(4), 5 X 10(5), and 1 X 10(6) U/liter), mannitol (50 mM), glutathione (10 microM), or desferrioxamine (150 microM) significantly reduced the incidence of reperfusion-induced ventricular fibrillation and, in many cases, the incidence of reperfusion-induced ventricular tachycardia. The mean duration of sinus rhythm during reperfusion was also increased significantly. Perfusion of hearts with boiled superoxide dismutase (1 X 10(5) U/liter) or boiled catalase (1 X 10(6) U/liter) did not decrease arrhythmias. Conversely, under conditions where, in the control group, the incidence of reperfusion arrhythmias was lowered by increasing perfusate potassium to 6.5 mM, the addition of the free radical-generating system FeCl3 X adenosine diphosphate (0.1 microM X 1 microM) to the perfusion fluid increased dramatically the incidence of reperfusion-induced ventricular fibrillation and tachycardia. Simultaneous perfusion with FeCl3 X adenosine diphosphate and superoxide dismutase (1 X 10(5) U/liter), catalase (1 X 10(6) U/liter), mannitol (50 mM), methionine (10 mM), or desferrioxamine (150 microM) again reduced the incidence of reperfusion-induced arrhythmias and increased the duration of normal sinus rhythm during the reperfusion phase. Thus, addition of six "anti-free radical" interventions reduced the incidence of reperfusion-induced arrhythmias, addition of a free radical-generating system increased the incidence of reperfusion-induced arrhythmias, and simultaneous perfusion of the hearts with FeCl3 X adenosine diphosphate and "anti-free radical" interventions again reduced reperfusion rhythm disturbances. These results are further evidence supporting the hypothesis that oxygen-derived free radicals play an important role in the genesis of reperfusion-induced arrhythmias.

Oxygen limitation is generally considered as impairment of mitochondrial respiration under hypoxia and ischemia. Low intracellular oxygen levels under normoxia, however, imply mild oxygen limitation, provide protection from oxidative stress, and result from economical strategies for oxygen transport through the respiratory cascade to cytochrome c oxidase. Both perspectives relate to the critical oxygen pressure, which inhibits mitochondrial respiration. Based on methodological considerations of oxygen kinetics and a presentation of high-resolution respirometry, mitochondrial oxygen affinities (1/P(50)) are reviewed with particular emphasis on the turnover effect under control of adenosine diphosphate ADP concentration, which increases the P(50) in active states. ADP/O(2) flux ratios are high even under severe oxygen limitation, as demonstrated by calorespirometry. Oxygen limitation reduces the uncoupled respiration observed under control by ADP, as shown by relationships derived between ADP/O(2) flux ratios, respiratory control ratios, and ADP kinetics. Bioenergetics at low oxygen versus oxidative stress must be considered in the context of limitation of maximum aerobic activity, ischemia-reperfusion injury, mitochondrial signalling to apoptosis, and mitochondrial theories of ageing.

Congenital amegakaryocytic thrombocytopenia (CAMT) is a rare disease presenting with isolated thrombocytopenia in infancy and developing into a pancytopenia in later childhood. Thrombopoietin (TPO) is the main regulator of thrombocytopoiesis and has also been demonstrated to be an important factor in early hematopoiesis. We analyzed 9 patients with CAMT for defects in TPO production and reactivity. We found high levels of TPO in the sera of all patients. However, platelets and hematopoietic progenitor cells of patients with CAMT did not show any reactivity to TPO, as measured by testing TPO-synergism to adenosine diphosphate in platelet activation or by megakaryocyte colony assays. Flow cytometric analysis revealed absent surface expression of the TPO receptor c-Mpl in 3 of 3 patients. Sequence analysis of the c-mpl gene revealed point mutations in 8 of 8 patients: We found frameshift or nonsense mutations that are predicted to result in a complete loss of c-Mpl function in 5 patients. Heterozygous or homozygous missense mutations predicted to lead to amino acid exchanges in the extracellular domain of the receptor were found in 3 other patients. The type of mutations correlated with the clinical course of the disease. We propose a defective c-Mpl expression due to c-mpl mutations as the cause for thrombocytopenia and progression into pancytopenia seen in patients with CAMT.

We evaluated the response to clopidogrel among aspirin-resistant versus aspirin-sensitive patients undergoing elective coronary stenting. Patients (n = 150) treated with aspirin but not clopidogrel had blood samples drawn at baseline and 24 h after clopidogrel loading. Depending on the definition used, 9% to 15% were resistant to aspirin and 24% to clopidogrel. About half of the aspirin-resistant patients were also resistant to clopidogrel. As a group, aspirin-resistant patients had lower response to clopidogrel (assessed by platelet aggregation and activation markers) than aspirin-sensitive patients. Both aspirin- and clopidogrel-resistant patients had higher incidence of creatine kinase-MB elevation than the respective sensitive patients.

OBJECTIVE

We sought to evaluate the response to clopidogrel among aspirin-resistant versus aspirin-sensitive patients undergoing percutaneous coronary intervention (PCI).

BACKGROUND

Wide variability has been reported in response to aspirin and clopidogrel. There are limited data on the simultaneous responses to both drugs.

METHODS

Elective PCI patients (n = 150) who received aspirin for>> or = 1 week but not clopidogrel were included. All patients received bivalirudin during PCI. Blood samples were drawn at baseline and 20 to 24 h after a 300-mg clopidogrel dose. Aspirin resistance was defined by>> or = 2 of 3 criteria: rapid platelet function analyzer-ASA score>> or = 550, 5 micromol/l adenosine diphosphate (ADP)-induced aggregation>> or = 70%, and 0.5 mg/ml arachidonic acid-induced aggregation>> or = 20%. Clopidogrel resistance was defined as baseline minus post-treatment aggregation < or = 10% in response to 5 and 20 micromol/l ADP.

RESULTS

Nineteen (12.7%) patients were resistant to aspirin and 36 (24%) to clopidogrel. Nine (47.4%) of the aspirin-resistant patients were also clopidogrel resistant. Aspirin-resistant patients were more likely to be women and have diabetes than were aspirin-sensitive patients. They also had lower response to clopidogrel, assessed by platelet aggregation and activation markers (flow cytometry-determined PAC-1 binding and P-selectin expression). Elevation of creatine kinase-myocardial band after stenting occurred more frequently in aspirin-resistant versus aspirin-sensitive patients (38.9% vs. 18.3%; p = 0.04) and in clopidogrel-resistant versus clopidogrel-sensitive patients (32.4% vs. 17.3%; p = 0.06).

CONCLUSIONS

Aspirin-resistant patients as a group have reduced response to clopidogrel. Furthermore, we have identified a unique group of dual drug-resistant patients who may be at increased risk for thrombotic complications after PCI.

OBJECTIVE

We sought to determine whether patients receiving chronic clopidogrel therapy undergoing nonemergent stenting who display high on-treatment preprocedural platelet aggregation measured by standard light transmittance aggregometry and thrombelastography (TEG) will be at increased risk for poststenting ischemic events.

BACKGROUND

Patients exhibiting heightened platelet reactivity to adenosine diphosphate (ADP) might be at increased risk for recurrent ischemic events after coronary stenting.

METHODS

A total of 100 consecutive patients receiving chronic antiplatelet therapy consisting of aspirin (325 mg qd) and clopidogrel (75 mg qd) were studied before undergoing nonemergent stenting. Patients were followed for 1 year after coronary stenting for the occurrence of death, myocardial infarction, stent thrombosis, stroke, or ischemia requiring a hospital stay.

RESULTS

All patients were aspirin responsive. Patients with ischemic events (23 of 100, 23%) within 1 year had greater on-treatment prestent ADP-induced platelet aggregation than patients without ischemic events by aggregometry and TEG (p < 0.001 for both measurements). Of patients with an ischemic event, 70% and 87% displayed high on-treatment platelet reactivity at baseline by aggregometry and TEG, respectively. High on-treatment platelet reactivity as measured by aggregometry and TEG were the only variables significantly related to ischemic events (p < 0.001 for both assays). The administration of eptifibatide reduced periprocedural elevation in platelet reactivity, with no significant differences in bleeding events.

CONCLUSIONS

Patients receiving chronic clopidogrel therapy undergoing nonemergent percutaneous coronary intervention who exhibit high on-treatment ADP-induced platelet aggregation are at increased risk for postprocedural ischemic events. These findings might have implications for the alteration in clopidogrel maintenance dose and use of glycoprotein IIb/IIIa inhibitors in selected patients.

Opioids, although fundamental to the treatment of pain, are limited in efficacy by side effects including tolerance and hyperalgesia. Using an in vitro culture system, we report that morphine increased microglial migration via a novel interaction between mu-opioid and P2X(4) receptors, which is dependent upon PI3K/Akt pathway activation. Morphine at 100 nm enhanced migration of primary microglial cells toward adenosine diphosphate by 257, 247, 301, 394, and 345% following 2, 6, 12, 24, and 48 h of stimulation, respectively. This opioid-dependent migration effect was inhibited by naloxone and confirmed to be mu-opioid receptor-dependent through the use of selective agonists and antagonists. PPADS [pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid)], a P2X(1-3,5-7) antagonist, had no effect on microglial migration; however, TNP-ATP [2',3'-O-(2,4,6-trinitrophenyl)-ATP], a P2X(1-7) antagonist, inhibited morphine-induced migration, suggesting a P2X(4) receptor-mediated effect. The PI3K inhibitors wortmannin and LY294002 decreased morphine-induced microglial migration. Iba1 protein, a microglial marker, and P2X(4) receptor expression were significantly increased after 6, 12, 24, and 48 h of morphine stimulation. Together, these results provide evidence for two phases of morphine effects on microglia. The initial phase takes place in minutes, involves PI3K/Akt pathway activation and leads to acutely enhanced migration. The longer-term phase occurs on the order of hours and involves increased expression of Iba1 and P2X(4) receptor protein, which imparts a promigratory phenotype and is correlated with even greater migration. These data provide the first necessary step in supporting microglial migration as an attractive target for the prevention or attenuation of morphine-induced side effects including tolerance and hyperalgesia.

Platelet adhesion, activation and aggregation on the exposed subendothelial extracellular matrix (ECM) are essential for haemostasis, but may also lead to occlusion of diseased vessels. Binding of the glycoprotein (GP)Ib-V-IX complex to immobilised von Willebrand factor (VWF) initiates adhesion of flowing platelets to the ECM, and thereby enables the collagen receptor GPVI to interact with its ligand and to mediate platelet activation. This process is reinforced by locally produced thrombin and platelet-derived secondary mediators, such as adenosine diphosphate (ADP) and thromboxane A(2) (TxA(2)). Together, these events promote a shift of β1 and β3 integrins from a low to a high affinity state for their ligands through 'inside-out' signalling allowing firm platelet adhesion and aggregation. Formed platelet aggregates are stabilised by fibrin formation and signalling events between adjacent platelets involving multiple platelet receptors, such as the newly discovered C-type lectin-like receptor 2 (CLEC-2). While occlusive thrombus formation is the principal pathogenic event in myocardial infarction, the situation is more complex in ischaemic stroke where infarct development often progresses despite sustained early reperfusion of previously occluded major intracranial arteries, a process referred to as 'reperfusion injury'. Increasing experimental evidence now suggests that early platelet adhesion and activation events, orchestrate a 'thrombo-inflammatory' cascade in this setting, whereas platelet aggregation and thrombus formation are not required. This review summarises recent developments in understanding the principal platelet adhesion receptor systems with a focus on their involvement in arterial thrombosis and ischaemic stroke models.

Adenosine diphosphate ribosyl transferase (ADPRT) and X-ray repair cross-complementing 1 (XRCC1) are two major DNA base excision repair (BER) proteins and act interactively in stimulating and executing BER processes. Polymorphisms of ADPRT Val762Ala and XRCC1 Arg399Gln have been associated with altered protein function and BER activity. This case-control study examined the contribution of these two polymorphisms, alone and in combination, or in interaction with smoking, to the risk of developing lung cancer. We estimated the risk of lung cancer associated with these polymorphisms in 1,000 cases and 1,000 cancer-free controls using logistic regression models. Subjects having the ADPRT Ala/Ala genotype had an odds ratio (OR) of 1.68 [95% confidence interval (95% CI), 1.27-2.23] compared with those having the Val/Val genotype. A greater than multiplicative joint effect between the ADPRT polymorphism and smoking was observed. The ORs (95% CI) of the Ala/Ala genotype for nonsmokers and smokers who smoked < or =16, 16 to 28, or >28 pack-years were 1.13 (0.79-1.62), 1.35 (0.68-2.70), 2.46 (1.35-4.51) or 17.09 (8.15-35.83), respectively (P trend test < 0.001). Gene-gene interaction of ADPRT and XRCC1 polymorphisms increased risk of lung cancer in a supermultiplicative manner (OR for the presence of both ADPRT 762Ala/Ala and XRCC1 399Gln/Gln genotypes, 5.91; 95% CI, 2.09-16.72), although the XRCC1 polymorphism itself was not associated with the risk. In conclusion, the ADPRT Val762Ala polymorphism plays an important role in smoking-related lung cancer and the XRCC1 Arg399Gln polymorphism may serve as a risk modifier.

The patch-clamp method was applied to single atrioventricular (a.v.) node cells of the rabbit heart to study the characteristics of the K+ channel. When the electrode contained 5.4 mM-K+, depolarizations of the cell-attached patch membrane induced outward single channel currents characterized by burst-like openings; the open-state probability increased from 0.005-0.01 at -40 mV to 0.07-0.1 at +20 mV of membrane potential. The reversal potentials of the current at K+ concentrations of 5.4, 20 and 130 mM in the electrode agreed with those given by the Nernst equation, indicating that this channel is selective for K+ ions. The slope conductance of the channel decreased beyond 60-90 mV positive to the reversal potential (inward-going rectification). The conductance near the reversal potential increased with increasing K+ concentrations on either side of the membrane: from 31-32 pS at 5.4 mM-K+ to 41-42 pS at 20 mM-K+ on the outside, and from 19 pS at 90 mM-K+ to 29.3 pS at 130 mM-K+ on the inside. Superfusion of the cell with 5.4 mM-CN-, glucose-free Tyrode markedly increased the number of channel openings in the cell-attached patch. In the inside-out patch, application of 1 mM-adenosine-5'-triphosphate (ATP) at the inner surface of the patch membrane blocked reversibly the channel activity, while 1 mM-adenosine-5'-diphosphate (ADP) failed to block it. The conductance and kinetics of the channel were not modified by increasing the Ca2+ concentration from 10(-8) M to 5 X 10(-6) M on the inner side of the membrane, while a further increase in Ca2+ to 10(-4) M decreased the open-state probability. The probability density for the open time fitted well with an exponential distribution (time constant of 5.4 ms at 60 mV positive to the resting potential), while that for the closed time was separated into a fast and a slow component (time constants of 4.0 and 132.0 ms, respectively). The time constant of the slow component decreased significantly with depolarization in some preparations. However, neither the time constant of the fast component of the closed-time histogram nor that of the open-time histogram was voltage-dependent.

ATP-sensitive K+ (KATP) channels are present at high density in membranes of cardiac cells where they regulate cardiac function during cellular metabolic impairment. KATP channels have been implicated in the shortening of the action potential duration and the cellular loss of K+ that occurs during metabolic inhibition. KATP channels have been associated with the cardioprotective mechanism of ischemia-related preconditioning. Intracellular ATP (ATPi) is the main regulator of KATP channels. ATPi has two functions: 1) to close the channel (ligand function) and 2) in the presence of Mg2+, to maintain the activity of KATP channels (presumably through an enzymatic reaction). KATP channel activity is modulated by intracellular nucleoside diphosphates that antagonize the ATPi-induced inhibition of channel opening or induce KATP channels to open. How nucleotides will affect KATP channels depends on the state of the channel. K+ channel-opening drugs are pharmacological agents that enhance KATP channel activity through different mechanisms and have great potential in the management of cardiovascular conditions. KATP channel activity is also modulated by neurohormones. Adenosine, through the activation of a GTP-binding protein, antagonizes the ATPi-induced channel closure. Understanding the molecular mechanisms that underlie KATP channel regulation should prove essential to further define the function of KATP channels and to elucidate the pharmacological regulation of this channel protein. Since the molecular structure of the KATP channel has now become available, it is anticipated that major progress in the KATP channel field will be achieved.

Patients receiving dual antiplatelet treatment with aspirin and clopidogrel are commonly treated with proton pump inhibitors (PPIs). Attenuating effects on platelet response to clopidogrel have been reported solely for the PPI omeprazole. PPIs differ in their metabolisation properties as well as their potential for drug-drug interactions. The aim of this study was to investigate the impact of different PPIs (pantoprazole, omeprazole, esomeprazole) on platelet response to clopidogrel in patients with previous coronary stent placement under chronic clopidogrel treatment. In a cross-sectional observational study, consecutive patients under clopidogrel maintenance treatment (n = 1,000) scheduled for a control coronary angiography were enrolled. Adenosine diphosphate (ADP)-induced platelet aggregation (in AU*min) was measured with multiple electrode platelet aggregometry (MEA). From the entire study population, 268 (26.8%) patients were under PPI treatment at the time point of platelet function testing (pantoprazole, n = 162; omeprazole, n = 64; esomeprazole, n = 42). Platelet aggregation (median [interquartile range]) was significantly higher in patients with omeprazole treatment (295.5 [193.5-571.2] AU*min) compared to patients without PPI treatment (220.0 [143.8-388.8] AU*min; p = 0.001). Platelet aggregation was similar in patients with pantoprazole (226.0 [150.0-401.5] AU*min) or esomeprazole (209.0 [134.8-384.8] AU*min) treatment compared to patients without PPI treatment (p = 0.69 and p = 0.88, respectively). Attenuating effects of concomitant PPI treatment on platelet response to clopidogrel were restricted to the use of omeprazole. No attenuating effects on platelet response to clopidogrel were observed for pantoprazole or esomeprazole. Specifically designed and randomized clinical studies are needed to define the impact of concomitant PPI treatment on adverse events after percutaneous coronary intervention.

OBJECTIVE

The aim of this study was to evaluate the relationship between reticulated platelets (RPs), platelet size, and platelet function in patients with stable coronary artery disease (CAD) taking aspirin and clopidogrel.

BACKGROUND

Reticulated platelets are young platelets that are larger and possibly more active than non-RPs.

METHODS

Flow cytometry was used to measure RPs after staining with thiazole orange and to define the upper 20% and lower 20% of platelets by size. Platelet aggregation was measured with light transmission aggregometry (LTA); platelet activation was assessed by measuring activated platelet surface expression of P-selectin and glycoprotein (GP) IIb/IIIa.

RESULTS

Ninety patients were recruited and stratified into tertiles of %RPs. Patients in the upper tertile displayed greater platelet aggregation to 5-mumol/l adenosine diphosphate (ADP) (50.7 +/- 16.4% vs. 34.2 +/- 17.3%, p < 0.001), 1.5-mmol/l arachidonic acid (AA) (27.3 +/- 16.9% vs. 11.7 +/- 9.3%, p < 0.001), and 1-mug/ml collagen (18 +/- 11.6% vs. 12.1 +/- 8.7%, p < 0.05) and greater expression of GP IIb/IIIa (4.7 +/- 1.8% vs. 3.1 +/- 2.2%, p < 0.001). Frequency of low response to aspirin (AA LTA >20%) was higher in the upper tertile (53% vs. 17%, p < 0.001) compared with the lower tertile; low response to clopidogrel (ADP LTA >50%) was also elevated in the upper tertile (50% vs. 13%, p = 0.003). The larger platelet gate had a higher % of RPs compared with the smaller gate (15.4 +/- 16.7% vs. 1.7 +/- 2.3%, p < 0.001) and greater GP IIb/IIIa (5.7 +/- 3.1 vs. 2.1 +/- 1.2, p < 0.001) and P-selectin expression (7.8 +/- 4.9 vs. 4.6 +/- 2.7, p < 0.001).

CONCLUSIONS

The proportion of circulating RPs strongly correlates with response to antiplatelet therapy in patients with stable CAD. Large platelets exhibit increased reactivity despite dual antiplatelet therapy, compared with smaller platelets.