Platelets are central mediators of haemostasis at sites of vascular injury, but they also mediate pathologic thrombosis. Activated platelets stimulate thrombus formation in response to rupture of an atherosclerotic plaque or endothelial cell erosion, promoting atherothrombotic disease. They also interact with endothelial cells and leukocytes to promote inflammation, which contributes to atherosclerosis. Multiple pathways contribute to platelet activation, and current oral antiplatelet therapy with aspirin and a P2Y(12) adenosine diphosphate (ADP) receptor antagonist target the thromboxane A(2) and ADP pathways, respectively. Both can diminish activation by other factors, but the extent of their effects depends upon the agonist, agonist strength, and platelet reactivity status. Although these agents have demonstrated significant clinical benefit, residual morbidity and mortality remain high. Neither agent is effective in inhibiting thrombin, the most potent platelet activator. This lack of comprehensive inhibition of platelet function allows continued thrombus formation and exposes patients to risk for recurrent thrombotic events. Moreover, bleeding risk is a substantial limitation of antiplatelet therapy, because these agents target platelet activation pathways critical for both protective haemostasis and pathologic thrombosis. Novel antiplatelet therapies that provide more complete inhibition of platelet activation without increasing bleeding risk could considerably decrease residual risk for ischemic events. Inhibition of the protease-activated receptor (PAR)-1 platelet activation pathway stimulated by thrombin is a novel, emerging approach to achieve more comprehensive inhibition of platelet activation when used in combination with current oral antiplatelet agents. PAR-1 inhibition is not expected to increase bleeding risk, as this pathway does not interfere with haemostasis.

An association between platelets, angiogenesis, and cancer has long been recognized, but the mechanisms linking them remains unclear. Platelets regulate new blood vessel growth through numerous stimulators and inhibitors of angiogenesis by several pathways, including differential exocytosis of angiogenesis regulators. Herein, we investigated the differential release of angiogenesis stimulators and inhibitors from platelets. Activation of human platelets with adenosine diphosphate (ADP) stimulated the release of VEGF, but not endostatin whereas, thromboxane A(2) (TXA(2)) released endostatin but not VEGF. Platelet releasates generated by activation with ADP promoted migration and formation of capillary structures by human umbilical vein endothelial cells (HUV-EC-Cs) in in vitro angiogenesis models. Conversely, TXA(2)-stimulated platelet releasate inhibited migration and formation of capillary structures. Because tumor growth beyond 1-2 mm(3) is angiogenesis-dependent, we hypothesized that cancer cells preferentially stimulate platelets to secrete their pro-angiogenic payload. In support of this, the breast cancer cell line MCF-7 stimulated secretion of VEGF and a pro-angiogenic releasate from platelets. Furthermore, the antiplatelet agent aspirin inhibited platelet-mediated angiogenesis after exposure to ADP or MCF-7 cells providing a potential mechanism for how aspirin may impact malignancy. Manipulation of differentially mediated release of angiogenic factors from platelets may provide a new modality for cancer treatment.

Platelets are formed from mature megakaryocytes (MKs) and arise from the development of long and thin cytoplasmic extensions called proplatelets. After platelet release, the senescent MKs (nucleus surrounded by some cytoplasm) undergo cell death by apoptosis. To explore the precise role of apoptosis in proplatelet formation, we grew human MKs from CD34(+) cells and assessed the possible role of caspases. Proteolytic maturation of procaspase-3 and procaspase-9 was detected by immunoblots in maturing MKs as well as in proplatelet-bearing MKs and senescent MKs. Cleavage of caspase substrates such as gelsolin or poly adenosine diphosphate (ADP)-ribose polymerase (PARP) was also detected. Interestingly, activated forms of caspase-3 were detected in maturing MKs, before proplatelet formation, with a punctuate cytoplasmic distribution, whereas a diffuse staining pattern was seen in senescent and apoptotic MKs. This localized activation of caspase-3 was associated with a mitochondrial membrane permeabilization as assessed by the release of cytochrome c, suggesting an activation of the intrinsic pathway. Moreover, these MKs with localized activated caspase-3 had no detectable DNA fragmentation. In contrast, when apoptosis was induced by staurosporine, diffuse caspase activation was seen; these MKs had signs of DNA fragmentation, and no proplatelet formation occurred. The pan-caspase inhibitor z-VAD.fmk as well as more specific inhibitors of caspase-3 and caspase-9 blocked proplatelet formation, whereas an inhibitor of calpeptin had no effect. Overexpression of Bcl-2 also inhibited proplatelet formation in maturing MKs. Thus, localized caspase activation is causal to proplatelet formation. We conclude that proplatelet formation is regulated by a caspase activation limited to only some cellular compartments.

Adenosine diphosphate (ADP)-ribosylation is a post-translational protein modification implicated in the regulation of a range of cellular processes. A family of proteins that catalyse ADP-ribosylation reactions are the poly(ADP-ribose) (PAR) polymerases (PARPs). PARPs covalently attach an ADP-ribose nucleotide to target proteins and some PARP family members can subsequently add additional ADP-ribose units to generate a PAR chain. The hydrolysis of PAR chains is catalysed by PAR glycohydrolase (PARG). PARG is unable to cleave the mono(ADP-ribose) unit directly linked to the protein and although the enzymatic activity that catalyses this reaction has been detected in mammalian cell extracts, the protein(s) responsible remain unknown. Here, we report the homozygous mutation of the c6orf130 gene in patients with severe neurodegeneration, and identify C6orf130 as a PARP-interacting protein that removes mono(ADP-ribosyl)ation on glutamate amino acid residues in PARP-modified proteins. X-ray structures and biochemical analysis of C6orf130 suggest a mechanism of catalytic reversal involving a transient C6orf130 lysyl-(ADP-ribose) intermediate. Furthermore, depletion of C6orf130 protein in cells leads to proliferation and DNA repair defects. Collectively, our data suggest that C6orf130 enzymatic activity has a role in the turnover and recycling of protein ADP-ribosylation, and we have implicated the importance of this protein in supporting normal cellular function in humans.

A nontoxic peptide (molecular weight, 26,000), which is active in catalyzing the adenosine diphosphate (ADP)-ribosylation of elongation factor 2, has been isolated from the culture supernatant of Pseudomonas aeruginosa strain 103 in stationary phase. Like fragment A from diphtheria toxin, the active peptide catalyzed the hydrolysis of nicotinamide adenine dinucleotide as well as the ADP-ribosylation of elongation factor 2 and showed similarities to fragment A in specific activity, kinetic constants, pH optimum, and ionic sensitivity. These results provide strong evidence for a high degree of homology in the structures of their active sites. That the peptide is not identical to fragment A is shown by the fact that it was not neutralized by fragment A-specific antiserum and was different in amino acid composition and pH and thermal labilities. Although definitive evidence is lacking, there are data suggesting that this peptide is a proteolytic fragment from the ADP-ribosylating toxin (exotoxin A; molecular weight, 66,000) produced by the same strain of P. aeruginosa.

Kinetic and equilibrium properties are compared for a monomeric kinesin construct (K332) and a dimeric construct (K379). MtK379 has a low affinity (5 x 10(4) M(-1)) and a high affinity (5 x 10(6) M(-1)) binding site for mant ADP while MtK332 has a single low affinity site (5 x 10(4) M(-1)). Rate constants of dissociation of mant ADP are <1 s(-1) for the high affinity site and 75-100 s(-1) for the low affinity site for MtK379. For MtK332, the effective rate constant is 200-300 s(-1). It is proposed that the two heads of the dimer are different through the interaction with the microtubule, a strongly bound head with low affinity for 2'-(3')-O-(N-methylanthraniloyl) adenosine 5'-diphosphate (mant ADP), similar to the single strongly bound head of the monomer and a weakly bound or detached head with high affinity for mant ADP. Rate of binding of mant ADP gave an "S"-shaped dependence on concentration for MtK379 and a hyperbolic dependence for MtK332. Binding of K379 x mant ADP dimer to microtubules releases only one mant ADP at a rate of 50 s(-1). The second strongly bound mant ADP is released by binding of nucleotides to the other head. Rates are 100 s(-1) for ATP, 30 s(-1) for AMPPNP or ATPgammaS, and 2 s(-1) for ADP. The rate of binding of mant ATP to MtK379 showed an "S"-shaped concentration dependence and limiting rate at zero concentration is <1 s(-1) while MtK332 gave a hyperbolic dependence and limiting rate of 100 s(-1). The limiting rate is determined by the rate of dissociation of mant ADP in the hydrolysis cycle. The evidence is consistent with an interacting site model in which binding of ATP to one head is required for release of ADP from the other head in the hydrolysis cycle. This model, in which the cycles are maintained partly out of phase, is an extension of the alternating site model of Hackney (Hackney, D. D. (1994) Proc. Nat. Acad. Sci. U.S.A. 91, 6865-6869). It provides a basis for a processive mechanism.

Chemotaxis, the movement of cells along chemical gradients, is critical for the recruitment of immune cells to sites of inflammation; however, how cells navigate in chemotactic gradients is poorly understood. Here, we show that macrophages navigate in a gradient of the chemoattractant C5a through the release of adenosine triphosphate (ATP) and autocrine "purinergic feedback loops" that involve receptors for ATP (P2Y(2)), adenosine diphosphate (ADP) (P2Y(12)), and adenosine (A2a, A2b, and A3). Whereas macrophages from mice deficient in pannexin-1 (which is part of a putative ATP release pathway), P2Y(2), or P2Y(12) exhibited efficient chemotactic navigation, chemotaxis was blocked by apyrase, which degrades ATP and ADP, and by the inhibition of multiple purinergic receptors. Furthermore, apyrase impaired the recruitment of monocytes in a mouse model of C5a-induced peritonitis. In addition, we found that stimulation of P2Y(2), P2Y(12), or adenosine receptors induced the formation of lamellipodial membrane protrusions, causing cell spreading. We propose a model in which autocrine purinergic receptor signaling amplifies and translates chemotactic cues into directional motility.

BACKGROUND

After treatment with clopidogrel, patients with type 2 diabetes mellitus (T2DM) have reduced platelet inhibition compared with patients who are not diabetic. Whether platelet inhibition can be enhanced by increasing clopidogrel maintenance dosage in T2DM patients is unknown. The aim of this pilot study was to assess the functional impact of a high maintenance dose in T2DM patients with suboptimal clopidogrel-induced antiplatelet effects.

RESULTS

T2DM patients on chronic dual antiplatelet therapy were screened to identify suboptimal clopidogrel responders. The latter were randomized to 30-day treatment with a standard (75 mg; n=20) or high (150 mg; n=20) daily maintenance dose. Platelet function was assessed at 3 time points: baseline, 30 days after randomization, and 30 days after resuming standard dosing. Platelet function parameters included adenosine diphosphate-induced (20 and 5 micromol/L) maximal and late platelet aggregation, inhibition of platelet aggregation, platelet disaggregation, and P2Y12 reactivity index. A total of 64 T2DM patients were screened to identify 40 suboptimal responders. After randomization, maximal adenosine diphosphate-induced (20 micromol/L) platelet aggregation was significantly reduced in the 150-mg group compared with the 75-mg group (P=0.002; primary end point). However, suboptimal clopidogrel response was still present in 60% of patients on the 150-mg regimen. All other platelet function parameters showed enhanced clopidogrel-induced antiplatelet effects with 150 mg, which returned to baseline values after resumption of standard dosing.

CONCLUSIONS

A 150-mg maintenance dose of clopidogrel is associated with enhanced antiplatelet effects compared with 75 mg in high-risk T2DM patients. However, enhanced ex vivo platelet reactivity continues to persist, the clinical implications of which are unknown and need to be evaluated in large-scale clinical trials.

An assay was developed to study the regulation of fruiting in Myxococcus xanthus. The nucleotides, adenosine 3':5'-cyclic monophosphate (cyclic AMP) and adenosine diphosphate (ADP), were found to greatly stimulate fruiting under the assay conditions. Very sharp concentration optima were observed. Even under conditions of starvation, these nucleotides greatly increased the number of aggregation sites. Nutrition was found to influence fruiting body morphology. The effect of amino acids on the nucleotide stimulation of fruiting was studied under our assay conditions. L-Methionine and L-isoleucine (1 mM) completely blocked either L-threonine or D,L-diaminopimelic acid synergistically enhanced the amount of fruiting in the presence of these nucleotides. The data presented suggest the existence of differentiation-related regulatory compounds in M. xanthus.

OBJECTIVE

We sought to compare the results obtained from six major platelet function tests in the assessment of the prevalence of aspirin resistance in patients with stable coronary artery disease.

RESULTS

201 patients with stable coronary artery disease receiving daily aspirin therapy >> or =80 mg) were recruited. Platelet aggregation was measured by: (i) light transmission aggregometry (LTA) after stimulation with 1.6 mM of arachidonic acid (AA), (ii) LTA after adenosine diphosphate (ADP) (5, 10, and 20 microM) stimulation, (iii) whole blood aggregometry, (iv) PFA-100, (v) VerifyNow Aspirin; urinary 11-dehydro-thromboxane B(2) concentrations were also measured. Eight patients (4%, 95% CI 0.01-0.07) were deemed resistant to aspirin by LTA and AA. The prevalence of aspirin resistance varied according to the assay used: 10.3-51.7% for LTA using ADP as the agonist, 18.0% for whole blood aggregometry, 59.5% for PFA-100, 6.7% for VerifyNow Aspirin, and finally, 22.9% by measuring urinary 11-dehydro-thromboxane B(2) concentrations. Results from these tests showed poor correlation and agreement between themselves.

CONCLUSIONS

Platelet function tests are not equally effective in measuring aspirin's antiplatelet effect and correlate poorly amongst themselves. The clinical usefulness of the different assays to classify correctly patients as aspirin resistant remains undetermined.

In the study of skeletal muscle bioenergetics, 31P magnetic resonance spectroscopy (MRS) allows frequent measurement of the cytosolic pH and the concentrations of phosphocreatine, inorganic phosphate, and adenosine diphosphate (ADP) during exercise and recovery, which can be supplemented by 1H MRS (or biopsy) measurements of muscle lactate content and 13C MRS (or biopsy) measurements of muscle glycogen. We review the many methods now described by which 31P MRS measurements can be made to yield quantitative estimates of adenosine triphosphate (ATP) turnover, oxidative capacity, and proton handling in skeletal muscle. In particular, we describe how to estimate the rates of glycogenolytic and aerobic ATP synthesis during exercise and oxidative ATP synthesis and proton efflux during recovery from exercise and how to assess oxidative capacity using data from steady-state exercise, work jumps, or recovery. We discuss the metabolic relationships that make these methods possible and the assumptions (e.g., about cytosolic buffer capacity and mitochondrial control mechanisms) on which they depend. We show how these methods, although sometimes based on apparently conflicting metabolic models, can be analysed in a common framework. Finally, we discuss some examples of the current and potential applications of these methods in clinical and experimental studies of skeletal muscle.

Bisphosphonates effectively inhibit osteoclast-mediated bone resorption and are integral in the treatment of benign and malignant bone diseases. The evolution of bisphosphonates over the past 30 years has led to the development of nitrogen-containing bisphosphonates (N-BPs), which have a mechanism of action different from that of the nonnitrogen-containing bisphosphonates. Studies conducted over the past decade have elucidated the mechanism of action and pharmacologic properties of the N-BPs. N-BPs exert their effects on osteoclasts and tumor cells by inhibiting a key enzyme in the mevalonate pathway, farnesyl diphosphate synthase, thus preventing protein prenylation and activation of intracellular signaling proteins such as Ras. Recent evidence suggests that N-BPs also induce production of a unique adenosine triphosphate analogue (Apppi) that can directly induce apoptosis. Our increased understanding of the pharmacologic effects of bisphosphonates is shedding light on the mechanisms by which they exert antitumor effects. As a result of their biochemical effects on protein prenylation, N-BPs induce caspase-dependent apoptosis, inhibit matrix metalloproteinase activity, and downregulate alpha(v)beta(3) and alpha(v)beta(5) integrins. In addition, zoledronic acid (Zometa; Novartis Pharmaceuticals Corp.; East Hanover, NJ and Basel, Switzerland) exerts synergistic antitumor activity when combined with other anticancer agents. Zoledronic acid also inhibits tumor cell adhesion to the extracellular matrix and invasion through Matrigel trade mark and has antiangiogenic activity. A growing body of evidence from animal models demonstrates that zoledronic acid and other bisphosphonates can reduce skeletal tumor burden and prevent metastasis to bone. Further studies are needed to fully elucidate these biochemical mechanisms and to determine if the antitumor potential of bisphosphonates translates to the clinical setting.

The 5'-end of the flavivirus genome harbors a methylated (m7)GpppA(2'OMe) cap structure, which is generated by the virus-encoded RNA triphosphatase, RNA (guanine-N7) methyltransferase, nucleoside 2'-O-methyltransferase, and RNA guanylyltransferase. The presence of the flavivirus guanylyltransferase activity in NS5 has been suggested by several groups but has not been empirically proven. Here we provide evidence that the N-terminus of the flavivirus NS5 protein is a true RNA guanylyltransferase. We demonstrate that GTP can be used as a substrate by the enzyme to form a covalent GMP-enzyme intermediate via a phosphoamide bond. Mutational studies also confirm the importance of a specific lysine residue in the GTP binding site for the enzymatic activity. We show that the GMP moiety can be transferred to the diphosphate end of an RNA transcript harboring an adenosine as the initiating residue. We also demonstrate that the flavivirus RNA triphosphatase (NS3 protein) stimulates the RNA guanylyltransferase activity of the NS5 protein. Finally, we show that both enzymes are sufficient and necessary to catalyze the de novo formation of a methylated RNA cap structure in vitro using a triphosphorylated RNA transcript. Our study provides biochemical evidence that flaviviruses encode a complete RNA capping machinery.

This study aims to improve the health of patients suffering from chronic fatigue syndrome (CFS) by interventions based on the biochemistry of the illness, specifically the function of mitochondria in producing ATP (adenosine triphosphate), the energy currency for all body functions, and recycling ADP (adenosine diphosphate) to replenish the ATP supply as needed. Patients attending a private medical practice specializing in CFS were diagnosed using the Centers for Disease Control criteria. In consultation with each patient, an integer on the Bell Ability Scale was assigned, and a blood sample was taken for the "ATP profile" test, designed for CFS and other fatigue conditions. Each test produced 5 numerical factors which describe the availability of ATP in neutrophils, the fraction complexed with magnesium, the efficiency of oxidative phosphorylation, and the transfer efficiencies of ADP into the mitochondria and ATP into the cytosol where the energy is used. With the consent of each of 71 patients and 53 normal, healthy controls the 5 factors have been collated and compared with the Bell Ability Scale. The individual numerical factors show that patients have different combinations of biochemical lesions. When the factors are combined, a remarkable correlation is observed between the degree of mitochondrial dysfunction and the severity of illness (P<0.001). Only 1 of the 71 patients overlaps the normal region. The "ATP profile" test is a powerful diagnostic tool and can differentiate patients who have fatigue and other symptoms as a result of energy wastage by stress and psychological factors from those who have insufficient energy due to cellular respiration dysfunction. The individual factors indicate which remedial actions, in the form of dietary supplements, drugs and detoxification, are most likely to be of benefit, and what further tests should be carried out.

In cells, stable microtubules (MTs) are covalently modified by a carboxypeptidase, which removes the C-terminal Tyr residue of alpha-tubulin. The significance of this selective detyrosination of MTs is not understood. In this study, we report that tubulin detyrosination in fibroblasts inhibits MT disassembly. This inhibition is relieved by overexpression of the depolymerizing motor mitotic centromere-associated kinesin (MCAK). Conversely, suppression of MCAK expression prevents disassembly of normal tyrosinated MTs in fibroblasts. Detyrosination of MTs suppresses the activity of MCAK in vitro, apparently as the result of a decreased affinity of the adenosine diphosphate (ADP)-inorganic phosphate- and ADP-bound forms of MCAK for the MT lattice. Detyrosination also impairs MT disassembly in neurons and inhibits the activity of the neuronal depolymerizing motor KIF2A in vitro. These results indicate that MT depolymerizing motors are directly inhibited by the detyrosination of tubulin, resulting in the stabilization of cellular MTs. Detyrosination of transiently stabilized MTs may give rise to persistent subpopulations of disassembly-resistant polymers to sustain subcellular cytoskeletal differentiation.

Platelet activation at sites of vascular injury is essential for primary hemostasis, but also underlies arterial thrombosis leading to myocardial infarction or stroke. Platelet activators such as adenosine diphosphate, thrombin or thromboxane A(2) (TXA(2)) activate receptors that are coupled to heterotrimeric G proteins. Activation of platelets through these receptors involves signaling through G(q), G(i) and G(z) (refs. 4-6). However, the role and relative importance of G(12) and G(13), which are activated by various platelet stimuli, are unclear. Here we show that lack of Galpha(13), but not Galpha(12), severely reduced the potency of thrombin, TXA(2) and collagen to induce platelet shape changes and aggregation in vitro. These defects were accompanied by reduced activation of RhoA and inability to form stable platelet thrombi under high shear stress ex vivo. Galpha(13) deficiency in platelets resulted in a severe defect in primary hemostasis and complete protection against arterial thrombosis in vivo. We conclude that G(13)-mediated signaling processes are required for normal hemostasis and thrombosis and may serve as a new target for antiplatelet drugs.

Circadian clocks are self-sustained biological oscillators that can be entrained by environmental cues. Although this phenomenon has been studied in many organisms, the molecular mechanisms of entrainment remain unclear. Three cyanobacterial proteins and adenosine triphosphate (ATP) are sufficient to generate oscillations in phosphorylation in vitro. We show that changes in illumination that induce a phase shift in cultured cyanobacteria also cause changes in the ratio of ATP to adenosine diphosphate (ADP). When these nucleotide changes are simulated in the in vitro oscillator, they cause phase shifts similar to those observed in vivo. Physiological concentrations of ADP inhibit kinase activity in the oscillator, and a mathematical model constrained by data shows that this effect is sufficient to quantitatively explain entrainment of the cyanobacterial circadian clock.

The control of flagellar activity in the biflagellate green alga, Chlamydomonas reinhardtii was investigated by the in vitro reactivation of the isolated flagellar apparatus (the 2 flagella attached to their respective basal bodies plus accessory structures). The waveform and beat frequency of the isolated apparatus in the presence of 1 mM adenosine triphophate (ATP) were comparable to those recorded for living cells. Equimolar concentrations of adenosine diphosphate (ADP) could be substituted for ATP with little change in beat frequency and no apparent change in waveform, suggesting that the latter is converted to ATP by axonemal adenylate kinase. No reactivation occurred in adenosine monophosphate (AMP). But frequencies in cytidine, guanosine and uridine triphosphates (CTP, GTP and UTP) were approximately 10% that obtained in ATP. Reactivation was optimal over a broad pH range between pH 6.4 and pH 8.9 in both APT and ADP. Isolated flagellar apparatus could be induced to change from forward to reverse motion in vitro by manipulation of exogenous calcium ions. The 2 types of motion were directly comparable to recorded responses of living cells. Forward swimming occurred at levels of calcium below 10(-6)M, the isolated apparatus changing to backward motion above this level. Motility was inhibited at concentrations above 10(-3)M. The threshold for reversal of motion by calcium was lowered to 10(-7)M when the flagellar membranes were solubilized with detergent, indicating that the flagellar membranes are involved in the regulaion of the level of calcium within the axoneme. The reversal of motion by calcium was itself freely reversible. The relationship of these observations to the known tactic responses of Chlamydomonas is discussed.

BACKGROUND

Interindividual variability of platelet inhibition after aspirin or clopidogrel administration has been described. Additionally, aspirin resistance and clopidogrel resistance occur in some individuals. Because the prodrug clopidogrel is activated by hepatic cytochrome P450 (CYP) 3A4, we hypothesized that interindividual variability in clopidogrel efficacy might be related to interindividual differences in CYP3A4 metabolic activity.

RESULTS

Platelet aggregation was measured before and after clopidogrel treatment in 32 patients undergoing coronary artery stent implantation and in 35 healthy volunteers. The erythromycin breath test was used to measure CYP3A4 activity in vivo in 25 of the healthy volunteers. Individual platelet aggregation was studied in 10 healthy volunteers after the coadministration of clopidogrel and rifampin (a CYP3A4 inducer). Clopidogrel nonresponders, low responders, and responders were defined by a relative inhibition of adenosine diphosphate (20 micromol/L)-induced platelet aggregation of <10%, 10% to 29%, and>> or =30%, respectively. Among patients, 22% were clopidogrel nonresponders, 32% were low responders, and 47% were responders. Among volunteers, 16% were nonresponders, 12% were low responders, and 72% were responders. Percent platelet aggregation after clopidogrel inversely correlated with CYP3A4 activity (r=-0.6, P=0.003). Improved platelet inhibition in volunteers resistant to clopidogrel was observed with the coadministration of clopidogrel and rifampin.

CONCLUSIONS

Clopidogrel administration results in interindividual variability in platelet inhibition, which correlates with CYP3A4 metabolic activity. Measurement of antiplatelet drug efficacy with a point-of-care device and alternative antithrombotic strategies for aspirin or clopidogrel nonresponders and low responders could reduce the incidence of thrombotic events that continue to occur despite oral antiplatelet therapy.

The 70-kDa heat shock protein (HSP70) family of molecular chaperones represents one of the most ubiquitous classes of chaperones and is highly conserved in all organisms. Members of the HSP70 family control all aspects of cellular proteostasis such as nascent protein chain folding, protein import into organelles, recovering of proteins from aggregation, and assembly of multi-protein complexes. These chaperones augment organismal survival and longevity in the face of proteotoxic stress by enhancing cell viability and facilitating protein damage repair. Extracellular HSP70s have a number of cytoprotective and immunomodulatory functions, the latter either in the context of facilitating the cross-presentation of immunogenic peptides via major histocompatibility complex (MHC) antigens or in the context of acting as "chaperokines" or stimulators of innate immune responses. Studies have linked the expression of HSP70s to several types of carcinoma, with Hsp70 expression being associated with therapeutic resistance, metastasis, and poor clinical outcome. In malignantly transformed cells, HSP70s protect cells from the proteotoxic stress associated with abnormally rapid proliferation, suppress cellular senescence, and confer resistance to stress-induced apoptosis including protection against cytostatic drugs and radiation therapy. All of the cellular activities of HSP70s depend on their adenosine-5'-triphosphate (ATP)-regulated ability to interact with exposed hydrophobic surfaces of proteins. ATP hydrolysis and adenosine diphosphate (ADP)/ATP exchange are key events for substrate binding and Hsp70 release during folding of nascent polypeptides. Several proteins that bind to distinct subdomains of Hsp70 and consequently modulate the activity of the chaperone have been identified as HSP70 co-chaperones. This review focuses on the regulation, function, and relevance of the molecular Hsp70 chaperone machinery to disease and its potential as a therapeutic target.

Mitogens evoke many alterations in gene expression in eukaryotic cells. Genes which are activated rapidly and transiently, that are evolutionarily conserved and whose induction is shared by diverse cell types when exposed to different growth stimuli are likely to be of critical importance in transducing mitogenic signals and regulating cellular proliferation. c-myc and c-fos are the only known genes fulfilling these criteria. We report on the molecular cloning of a novel early growth response (egr) gene which also satisfies these conditions. In response to serum, its 3.7 kb mRNA is induced dramatically in mouse fibroblasts reaching a peak level at about 30 minutes that is ten times higher than the maximal value attained by c-fos mRNA. This transcript is induced by the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate and is "superinduced" by serum and cycloheximide together. Importantly, the gene is highly induced by different mitogens in a wide array of cell types: insulin stimulated rat hepatoma cells, adenosine diphosphate treated monkey kidney epithelial cells, and phytohemagglutinin stimulated human peripheral blood lymphocytes. Given the many properties that this gene shares with c-myc and c-fos, it may play a key role in the control of cell growth and perhaps in oncogenesis.

Transcriptional feedback loops are a feature of circadian clocks in both animals and plants. We show that the plant circadian clock also incorporates the cytosolic signaling molecule cyclic adenosine diphosphate ribose (cADPR). cADPR modulates the circadian oscillator's transcriptional feedback loops and drives circadian oscillations of Ca2+ release. The effects of antagonists of cADPR signaling, manipulation of cADPR synthesis, and mathematical simulation of the interaction of cADPR with the circadian clock indicate that cADPR forms a feedback loop within the plant circadian clock.

Current available data show that about 4% to 30% of patients treated with conventional doses of clopidogrel do not display adequate antiplatelet response. Clopidogrel resistance is a widely used term that remains to be clearly defined. So far, it has been used to reflect failure of clopidogrel to achieve its antiaggregatory effect. The interpatient variability in clopidogrel response is multifactorial. It can be due to extrinsic or intrinsic mechanisms. Among extrinsic mechanisms are the possibility of clopidogrel underdosing in patients undergoing stenting or with acute coronary syndrome, and drug-drug interactions involving CYP3A4. Intrinsic mechanisms include genetic polymorphisms of the P2Y(12) receptor and of the CYP3As, accrued release of adenosine diphosphate, or up-regulation of other platelet activation pathways. Presently, there is no definite demonstration of an association between low responsiveness to clopidogrel and thrombotic events. The optimal level of clopidogrel-induced platelet inhibition, which will correlate quantitatively with clopidogrel's ability to prevent atherothrombotic events is still lacking. Furthermore, because there is no single and validated platelet function assay to measure clopidogrel's antiplatelet effect, it is not justified to routinely look for clopidogrel resistance in the clinical setting. This review discusses currently available evidence surrounding the variability in the antiplatelet response to clopidogrel.

The transient potential receptor melastatin-2 (TRPM2) channel has emerged as an important Ca(2+) signalling mechanism in a variety of cells, contributing to cellular functions that include cytokine production, insulin release, cell motility and cell death. Its ability to respond to reactive oxygen species has made TRPM2 a potential therapeutic target for chronic inflammation, neurodegenerative diseases, and oxidative stress-related pathologies. TRPM2 is a non-selective, calcium (Ca(2+))-permeable cation channel of the melastatin-related transient receptor potential (TRPM) ion channel subfamily. It is activated by intracellular adenosine diphosphate ribose (ADPR) through a diphosphoribose hydrolase domain in its C-terminus and regulated through a variety of factors, including synergistic facilitation by [Ca(2+)](i), cyclic ADPR, H(2)O(2), NAADP, and negative feedback regulation by AMP and permeating protons (pH). In addition to its role mediating Ca(2+) influx into the cells, TRPM2 can also function as a lysosomal Ca(2+) release channel, contributing to cell death. The physiological and pathophysiological context of ROS-mediated events makes TRPM2 a promising target for the development of therapeutic tools of inflammatory and degenerative diseases.