BACKGROUND

Thrombus propagation on disrupted plaque is a major cause of acute coronary events and serious complication after coronary intervention. 5-Hydroxytryptamine (5-HT) is a potent vasoactive and platelet-aggregating substance that is predominantly mediated by 5-HT2A receptor. However, the roles of 5-HT2A receptor in occlusive thrombus formation on disrupted plaque remain obscure.

OBJECTIVE

We investigated the role of 5-HT2A receptor in thrombus formation using a rabbit model of repeated balloon-injury.

METHODS

Three weeks after a first balloon-injury of the femoral arteries, luminal diameter, neointimal growth, and vasoconstriction by 5-HT in vitro were examined. Thrombus propagation and the role of 5-HT2A receptor after a second balloon-injury were evaluated using sarpogrelate, a selective 5-HT2A receptor antagonist.

RESULTS

Three weeks after the first balloon-injury, luminal stenosis was evident in the femoral arteries, where the neointima expressed tissue factor and 5-HT2A receptor. The hypercontractile response of the stenotic arteries to 5-HT was significantly reduced by sarpogrelate. Balloon-injury of the neointima with substantially reduced blood flow promoted the formation of occlusive thrombus that was immunoreactive against glycoprotein IIb-IIIa, 5-HT2A receptor and fibrin. Intravenous injection of sarpogrelate significantly inhibited ex vivo platelet aggregation induced by adenosine 5'-diphosphate, thrombin and collagen alone as well as with 5-HT, and significantly prevented occlusive thrombus formation in vivo.

CONCLUSIONS

The 5-HT2A receptor appears to play a crucial role in occlusive thrombus formation in diseased arteries via platelet aggregation and vasoconstriction. Inhibition of 5-HT2A receptor might help reduce the onset of acute coronary events and of acute coronary occlusion after the intervention.

Platelet activation is normally induced by primary agonists such as adenosine diphosphate (ADP), thrombin, and collagen, whereas other agonists, such as epinephrine, can play important accessory roles. It is now reported that the macrophage-derived chemokine (MDC), thymus activation-regulated chemokine (TARC), and stromal cell-derived factor one (SDF-1) are highly effective activators of platelet function under a variety of conditions, stimulating platelet shape change, aggregation, and adhesion to collagen or fibrinogen. Chemokine-mediated platelet activation was rapid and maximal (less than 5 seconds) under arterial flow conditions and depended strongly on the presence of low levels of primary agonists such as ADP or thrombin. Concentrations of ADP (0.05-0.25 microM) or thrombin (0.005-0.02 U/mL) that induced minimal aggregation caused major aggregation acting in combination with the chemokines. The ability of apyrase to block chemokine-dependent aggregation or adhesion was consistent with an important role for ADP. Chemokine-stimulated aggregation was also insensitive to indomethacin, suggesting that the activation of cyclo-oxygenase is not involved. TARC, MDC, and SDF-1 increased intracellular calcium concentrations [Ca(2+)](i) when combined with low levels of ADP. The MDC and TARC receptor CCR4 was expressed on platelets, and an anti-CCR4 antibody blocked aggregation induced by TARC or MDC. Treatment of platelets with SDF-1 and MDC rapidly exposed P-selectin (CD62P) on the cell surface but did not induce the secretion of serotonin. These findings suggest that the chemokines MDC, TARC, and SDF-1, which may be produced during inflammatory responses, coupled with low levels of ADP or thrombin, can serve as strong stimuli for activating platelet function.

BACKGROUND

Ligand activation of peroxisome proliferator-activated receptor gamma (PPARgamma) results in the inhibition of proliferation of various cancer cells. The aim of this study is to investigate the mechanisms of cell growth inhibition of hepatocellular carcinoma (HCC) cell lines by the PPARgamma ligand, troglitazone.

METHODS

Six HCC cell lines were used to study the effects of troglitazone on cell growth by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, on cell cycle by flow cytometry, and on the cell cycle-regulating factors of late G1 phase by Western blotting. Apoptosis assays were performed by flow cytometry using membrane, nuclear, cytoplasmic, and mitochondrial markers. Caspase inhibitors were used to analyze the mechanisms of apoptosis induced by troglitazone.

RESULTS

Troglitazone showed a potent dose-dependent effect on the growth inhibition of all six HCC cell lines, which were suppressed to under 50% of control at the concentration of 10 micromol/L. The growth inhibition was linked to the G1 phase cell cycle arrest through the up-expression of the cyclin-dependent kinase inhibitors, p21 and p27 proteins, and the hypophosphorylation of retinoblastoma protein. Troglitazone induced apoptosis by caspase-dependent (mitchondrial transmembrane potential decrease, cleavage of poly [adenosine diphosphate ribose] polymerase, 7A6 antigen exposure, Bcl-2 decrease, and activation of caspase 3) and caspase-independent (phosphatidylserine externalization) mechanisms.

CONCLUSIONS

Our data suggest that ligand activation of PPARgamma by troglitazone or modified analogs of the thiazolidinedione class of drugs is a novel target for effective therapy against HCC, because of the significant antiproliferative and programmed cell death induction capabilities demonstrated by troglitazone.

Mechanical activation of the mucosal lining of the colon by brush stroking elicits an intestinal neural reflex and an increase in short circuit current (Isc) indicative of electrogenic chloride ion transport. We tested whether endogenous nucleotides are physiologic regulators of mucosal reflexes that control ion transport. The brush stroking-evoked Isc response in mucosa and submucosa preparations (M-SMP) of rat colon was reduced by the P2Y1 receptor (R) antagonist 2'deoxy-N6-methyl adenosine 3',5'-diphosphate diammonium salt (MRS 2179) and further blocked by tetrodotoxin (TTX). M-SMP Isc responses to serosal application of the P2Y1 R agonist 2-methylthioadenosine-diphosphate (2MeSADP) or the P2Y2/P2Y4 R agonist 5'uridine-triphosphate (UTP) were reduced but not abolished by TTX. The potency profile of nucleotides for increasing Isc was 5'adenosine-triphosphate (ATP; effective concentration at half maximal response [EC50] 0.65 x 10(4) M) congruent with UTP (EC50 1.0 x 10(-4) M) congruent with 2MeSADP (EC50 = 1.60 x 10(-4) M). Mucosal touch and distention-induced Ca2+ transients in submucous neurons were reduced by apyrase and prevented by blocking the P2Y1 R with MRS 2179 and TTX; denervation of the mucosa. It did not occur by touching a ganglion directly. 2MeSADP Ca2+ responses occurred in subsets of neurons with or without substance P (SP) responses. The potency profile of nucleotides on the neural Ca2+ response was 2MeSADP (5 x 10(-7) M)>> UTP (6 x 10(-6) M)>> ATP (9 x 10(-5) M). The expression of P2Y R immunoreactivity (ir) in nerve cell bodies was in the order of P2Y1 R>> P2Y4 R>>) P2Y2 R. P2Y1R ir occurred in the cell somas of more than 90% of neuronal nitric oxide synthase, vasoactive intestinal peptide (VIP), calretinin, or neuropeptide Y (NPY)-ir neurons, 78% of somatostatin neurons, but not in calbindin or SP neurons. P2Y2 R ir was expressed in a minority of SP, VIP, NPY, vesicular acetylcholine transporter, and calcitonin gene-related peptide-ir varicose fibers (5-20%) and those surrounding calbindin (5-20%) neurons. P2Y4 ir occurred mainly in the cell somas of 93% of NPY neurons. Reverse transcriptase polymerase chain reaction of the submucosa demonstrated mRNA for P2Y1R, P2Y2, P2Y4, P2Y6, and P2Y12 Rs. Expression of P2Y1, P2Y2, and P2Y4 protein was confirmed by western blots. In conclusion, endogenous nucleotides acting at P2YRs transduce mechanically evoked reflex chloride ion transport in rat distal colon. Nucleotides evoke reflexes by acting primarily at postsynaptic P2Y1 Rs and P2Y4 R on VIP+/NPY+ secretomotor neurons, at P2Y2 Rs on no more than 2% of VIP+ secretomotor neurons, and 2Y2 Rs mainly of extrinsic varicose fibers surrounding putative intrinsic primary afferent and secretomotor neurons. During mucosal mechanical reflexes, it is postulated that P2Y1 R, P2Y2 R, and P2Y4 R are activated by endogenous ATP, UTP, and 5'uridine-diphosphate.

In an attempt to reveal certain aspects of the pathogenesis of the bleeding disorder in dengue hemorrhagic fever, hemostatic and platelet kinetic studies were carried out in 61 children with this disease. As has been shown by others, thrombocytopenia and hypofibrinogenemia were the two most prominent hemostatic defects constantly discovered. Increased intravascular clotting seemed to be one responsible factor, though not an outstanding one. This was evidenced by mildly and variably low factors II, V, VII, VIII, IX, X, and XII, and by mild to moderate increase of fibrin degradation products as well as low platelet counts and fibrinogen. In 11 cases platelet kinetic study revealed increased destruction as a main cause for the thrombocytopenia, most probably due to the underlying immunologic mechanism, i.e., via the immune complexes formed. Another factor was platelet dysfunction--the release of adenosine diphosphate.

The interactions of simian virus 40 (SV40) large T antigen with DNA carrying the viral origin of DNA replication, as well as its interactions with cellular replication proteins, have been investigated by using fluorescent ATP analogues as specific probes. The enhanced fluorescence of 3'(2')-O-(2,4, 6-trinitrophenyl)adenosine diphosphate (TNP-ADP) induced by T antigen binding to the nucleotide was decreased upon binding of T antigen to origin DNA. Similarly, the enhanced fluorescence induced by T antigen binding to TNP-ADP or TNP-ATP was decreased upon binding to human DNA polymerase alpha-primase (pol alpha), but not to replication protein A (RPA). Fluorescence titrations revealed noncompetitive inhibition of TNP-ADP binding by origin DNA, and noncompetitive inhibition of TNP-ADP and TNP-ATP binding by pol alpha, suggesting that T antigen complexed with either origin DNA or pol alpha was not able to bind the TNP nucleotide. From these titrations, we have measured a binding stoichiometry of 11.5 +/- 0.8 T antigen monomers per viral origin DNA, in agreement with the double hexamer assembly of T antigen on the origin as reported earlier. The stoichiometry of pol alpha binding to T antigen was measured to be 5.5 +/- 0.6 mol of T antigen per mole of pol alpha. While monomeric T antigen-nucleotide complex was a preferred ligand over free T antigen in the double hexamer assembly reaction, preformed T antigen hexamers were incapable of forming double hexamers on the DNA. The results support a model in which double hexamer assembly on the viral origin occurs by successive binding of 12 free T antigen or monomeric T-nucleotide complexes to the DNA. In contrast with this stepwise assembly of T antigen monomers on DNA, hexameric T antigen was able to bind directly to pol alpha with concomitant release of the bound TNP nucleotide. The possible implications of these results for the mechanism of initiation of SV40 DNA replication are discussed.

Mutational analysis has established that the cytoplasmic tail of the integrin beta 3 subunit binds c-Src (termed as Src in this study) and is critical for bidirectional integrin signaling. Here we show in washed human platelets that a cell-permeable, myristoylated RGT peptide (myr-RGT) corresponding to the integrin beta 3 C-terminal sequence dose-dependently inhibited stable platelet adhesion and spreading on immobilized fibrinogen, and fibrin clot retraction as well. Myr-RGT also inhibited the aggregation-dependent platelet secretion and secretion-dependent second wave of platelet aggregation induced by adenosine diphosphate, ristocetin, or thrombin. Thus, myr-RGT inhibited integrin outside-in signaling. In contrast, myr-RGT had no inhibitory effect on adenosine diphosphate-induced soluble fibrinogen binding to platelets that is dependent on integrin inside-out signaling. Furthermore, the RGT peptide induced dissociation of Src from integrin beta 3 and dose-dependently inhibited the purified recombinant beta 3 cytoplasmic domain binding to Src-SH3. In addition, phosphorylation of the beta 3 cytoplasmic tyrosines, Y(747) and Y(759), was inhibited by myr-RGT. These data indicate an important role for beta 3-Src interaction in outside-in signaling. Thus, in intact human platelets, disruption of the association of Src with beta 3 and selective blockade of integrin alpha IIb beta 3 outside-in signaling by myr-RGT suggest a potential new antithrombotic strategy.

The sirtuin proteins are nicotinamide adenine dinucleotide dependent deacetylases and adenosine diphosphate (ADP)-ribosyl transferases associated with metabolic balance and lifespan extension. Sirtuin 1 (SIRT1) and sirtuin 4 (SIRT4) have been reported to regulate insulin secretion, but their association with the development of insulin resistance and nonalcoholic fatty liver disease remain undefined. The aim of this study was to determine the expression of SIRT1 and SIRT4 in the liver and pancreas of rats fed with different diets and analyze the association of these proteins with insulin resistance and nonalcoholic fatty liver disease. Male Sprague-Dawley rats were randomly divided into the following 4 diet treatment groups: normal control (NC), calorie restriction (CR), high-fat (HFa), and high-fructose (HFr), and these groups were maintained for 12 weeks. Blood biochemical analysis and histopathology indicated that HFa and HFr groups were insulin resistant and developed nonalcoholic fatty livers. SIRT1 was present in the nucleus and cytoplasm of the pancreatic beta-cells, while SIRT4 was located in the cytoplasm. Treatment with the CR diet increased the expression of SIRT1 in both the pancreas and liver, while treatment with the HFa and HFr diets caused a decrease. SIRT4 was upregulated in the liver of rats treated with the HFa diet, but did not change with the CR diet treatment. These data suggest that SIRT1 and SIRT4 were both involved in the development of insulin resistance and nonalcoholic fatty liver disease.

Members of the cytohesin protein family, a group of guanine nucleotide exchange factors for adenosine diphosphate ribosylation factor (ARF) guanosine triphosphatases, have recently emerged as important regulators of signal transduction in vertebrate and invertebrate biology. These proteins share a modular domain structure, comprising carboxy-terminal membrane recruitment elements, a Sec7 homology effector domain, and an amino-terminal coiled-coil domain that serve as a platform for their integration into larger signaling complexes. Although these proteins have a highly similar overall build, their individual biological functions appear to be at least partly specific. Cytohesin-1 had been identified as a regulator of beta2 integrin inside-out regulation in immune cells and was subsequently shown to be involved in mitogen-associated protein kinase signaling in tumor cell proliferation as well as in T-helper cell activation and differentiation. Cytohesin-3, which had been discovered to be strongly associated with T-cell anergy, was very recently described as an essential component of insulin signal transduction in Drosophila and in human and murine liver cells. Future work will aim to dissect the mechanistic details of the modes of action of the cytohesins as well as to define the precise roles of these versatile proteins in vertebrates at the genetic level.

The pathophysiology of thrombocytopenia in the syndrome of thrombocytopenia with absent radii (TAR) is not yet understood. We examined thrombopoietin (TPO) serum levels and the in vitro reactivity of platelets to TPO in five patients affected with TAR syndrome. We found elevated TPO serum levels in all patients tested, excluding a TPO production defect as cause for thrombocytopenia in TAR syndrome. In addition, we found similar expression of the TPO receptor c-Mpl on the surface of platelets from TAR patients (5 of 5) and a similar molecular weight of the receptor as compared with healthy controls (4 of 4). Platelet response to adenosine diphosphate or thrombin receptor agonist peptide SFLLRN (TRAP) was normal in TAR patients. However, in contrast to results with healthy controls we could show absence of in vitro reactivity of platelets from TAR patients to recombinant TPO as measured by testing TPO synergism to adenine diphosphate and TRAP in platelet activation. TPO induced tyrosine phosphorylation of platelet proteins was completely absent (3 of 4) or markedly decreased (1 of 4). Our results indicate that defective megakaryocytopoiesis/thrombocytopoiesis in TAR syndrome is not caused by a defect in TPO production but a lack of response to TPO in the signal transduction pathway of c-Mpl.

The purinergic system is highly involved in the regulation of microglial physiological processes. In addition to the accepted roles for the P2 X4,7 and P2 Y12 receptors activated by adenosine triphosphate (ATP) and adenosine diphosphate, respectively, recent evidence suggests a role for the adenosine A2A receptor in microglial cytoskeletal rearrangements. However, the expression and function of adenosine A1 receptor (A1AR) in microglia is still unclear. Several reports have demonstrated possible expression of A1AR in microglia, but a new study has refuted such evidence. In this study, we investigated the presence and function of A1AR in microglia using biomolecular techniques, live microscopy, live calcium imaging, and in vivo electrophysiological approaches. The aim of this study was to clarify the expression of A1AR in microglia and to highlight its possible roles. We found that microglia express A1AR and that it is highly upregulated upon ATP treatment. Moreover, we observed that selective stimulation of A1AR inhibits the morphological activation of microglia, possibly by suppressing the Ca(2+) influx induced by ATP treatment. Finally, we recorded the spontaneous and evoked activity of spinal nociceptive-specific neuron before and after application of resting or ATP-treated microglia, with or without preincubation with a selective A1AR agonist. We found that the microglial cells, pretreated with the A1AR agonist, exhibit lower capability to facilitate the nociceptive neurons, as compared with the cells treated with ATP alone.

BACKGROUND

Some patients evaluated for chest pain with angiographically normal coronary arteries show coronary slow flow phenomenon (CSFP) on angiography. Slow flow of dye in epicardial coronary arteries is also not an infrequent finding in patients during routine coronary angiography. The precise pathophysiology of CSFP is not known yet.

OBJECTIVE

This study investigates the presence of platelet function disorders in patients with CSFP.

METHODS

The patient group included 24 patients with CSFP detected by coronary angiography via the TIMI "frame count" method, and a control group included 23 patients with normal coronary flow. Platelet aggregability induced by use of ristocetin, collagen, and adenosine diphosphate (ADP), was measured from all blood samples in both control and patient groups.

RESULTS

The ratio of platelet aggregability increased significantly in patients with CSFP compared with patients with normal coronary flow (ristocetin 57.6 +/- 15 vs. 45.4 +/- 17.1, collagen 62.9 +/- 16.4 vs. 48.9 +/- 25.3, ADP 59.4 +/- 18 vs. 42.4 +/- 15.2, p < 0.05).

CONCLUSIONS

Platelet aggregability is increased in patients with CSFP.

The involvement of metabolites of arachidonic acid in platelet-dense granule secretion and secondary platelet-platelet interactions is well characterized. However, their role in heterotypic interactions dependent on alpha-granule secretion is less well understood. Using platelet-surface expression of P-selectin as a marker of alpha-granule secretion, we have shown that: (1) aspirin treatment of platelets at doses that block dense granule secretion does not inhibit alpha-granule secretion to adenosine diphosphate (ADP); (2) synergism between epinephrine and ADP in the induction of P-selectin expression is similarly unaffected by aspirin; and (3) the ability of P-selectin to mediate adhesion of activated platelets to monocytes and polymorphonuclear lymphocytes in whole blood is also unchanged by aspirin treatment. To further explore the mechanisms responsible for platelet alpha-granule secretion, we have shown that inhibition of Na+/H+ exchange by either acidification of the extracellular medium or amiloride treatment blocked ADP-induced P-selectin expression. In contrast, incubation with the platelet lipoxygenase inhibitor 5,8,11-eicosatrynoic acid, by itself and with aspirin, did not decrease ADP-induced P-selectin expression. We conclude that platelet alpha-granule secretion in response to ADP is dependent on intact Na+/H+ exchange but is independent of the lipoxygenase- and cyclooxygenase-dependent metabolites of arachidonic acid.

Stroking the mucosal lining of the guinea pig colon with a brush elicits an intestinal neural reflex, and an increase in short-circuit current (Isc) indicative of chloride secretion. We tested whether endogenous and exogenous nucleotides are physiologic regulators of mucosal reflexes that modulate chloride secretion. The basal Isc was augmented by 6-N,N-diethyl-beta,gamma-dibromomethylene-D-adenosine-5'-triphosphate (ARL67156) inhibition of nucleotide breakdown or adenosine A1 receptor blockade and reduced by apyrase inactivation of nucleotidases, P2 receptor antagonists, tetrodotoxin (TTX), or piroxicam. ARL67156 augmented, and apyrase inhibited, stroking-evoked Isc responses. TTX and atropine inhibited nucleotide-evoked Isc responses. The agonist potency profile for Isc, 2-methylthioadenosine-diphosphate (2MeSADP) = 2-methioadenosine-triphosphate>>) 5'adenosine-triphosphate (ATP)>> or = 5'adenosine-diphosphate>> 5'uridine-triphosphate>> or = 5'uridine-diphosphate, supports a P2Y1 receptor (R). The P2 receptor antagonists suramin and pyridoxalphosphate-6-azophenyl-2'4'-disulfonic acid, reduced stroking responses (36%) and their effects were additive. The selective P2Y1 R antagonist, 2'deoxy-N6-methyl adenosine 3',5'-diphosphate diammonium salt, reduced stroking (54%) and 2MeSADP (70%) responses at P2Y1 Rs. The P2X1/3 R agonist, alpha,betaMeATP, increased Isc. A desensitizing dose of alpha,betaMeATP reduced stroking Isc responses but did not prevent the 2MeSADP-evoked Isc response. Reverse transcriptase polymerase chain reaction analysis revealed mRNAs for P2Y1 R, P2Y2 R, P2Y4 R, P2Y6 R, and P2Y12 R in submucosa. The expression of P2Y R immunoreactivity (ir) in cell bodies of submucous neurons followed the order of P2Y1 = P2Y2>>) P2Y4 R ir; P2Y1 Rs and P2Y2 R ir were abundant (21-50% of neurons). P2Y1 R ir was abundant in cholinergic secretomotor neurons and fewer than 2% of neuropeptide Y (NPY)/choline acetyltransferase secretomotor neurons, and P2Y2 R ir was expressed in virtually all NPY secretomotor neurons and approximately 30% of calbindin/intrinsic primary afferent neurons. P2Y4 R ir was present in NPY-positive neurons. P2Y ir was rare or absent in varicose nerve fibers. The functional data support the hypothesis that mechanical stimulation with a brush releases nucleotides that act predominantly at P2Y1 Rs and to a lesser extent on P2X1/3 Rs to mediate reflex chloride secretion. A separate P2Y2 R neural circuit pathway exists that is not activated by mechanical forces. Other receptors including P2Y4, P2Y6, P2Y12, or P4 Rs cannot be excluded.

Adenosine 5'-triphosphate is known to function as a potent extracellular messenger producing its effects via a distinct family of cell surface receptors. Different receptor subtypes have been shown to modulate different cellular functions such as proliferation, differentiation and apoptosis. We investigated the functional expression and proliferative action of metabotropic P2Y receptors in human melanoma tissue and cells. Expression of functional P2Y1, P2Y2 and P2Y6 receptor subtypes was established by reverse transcriptase polymerase chain reaction, immunohistochemistry and intracellular calcium measurements using a Fluorometric Imaging Plate Reader. Incubation of A375 melanoma cells with the P2Y1 receptor-selective agonist 2-methylthioadenosine-5-diphosphate caused a decrease in cell number which was dose-dependent, whereas incubation with the P2Y2 receptor agonist uridine triphosphate caused a dose-dependent increase in cell number. The action of extracellular nucleotides on P2Y receptors was shown to mediate the growth of melanomas and the P2Y1 receptor is a putative target for melanoma therapy.

Monocyte-derived dendritic cells (Mo-DC) express functional P2X7 receptors; however, the expression of these receptors on tissue-derived dendritic cells including epidermal Langerhans cells (LC) is unknown. Using immunolabeling and flow cytometry, we demonstrated that P2X7 was present on both human epidermal LC and monocyte-derived LC (Mo-LC), as well as on human keratinocytes. The ecto-ATPDase (CD39) was also present on LC, but not keratinocytes. The P2X7 agonists, 2'- and 3'-0(4-benzoylbenzoyl) adenosine 5'-triphosphate (BzATP) or ATP, but neither adenosine 5'-diphosphate (ADP) nor uridine 5'-triphosphate (UTP), induced ethidium+ uptake into these cells. Furthermore, ATP-induced ethidium+ uptake into epidermal LC, Mo-LC and keratinocytes was inhibited by the specific P2X7 antagonist, KN-62 (1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine). ATP-induced ethidium+ uptake into Mo-LC and Mo-DC was 2- and 3-fold greater, respectively, than that for fresh monocytes. P2X7 activation on LC induced downstream signaling events, as BzATP or ATP, but neither ADP nor UTP, induced shedding of the low-affinity receptor for IgE (CD23) from Mo-LC. This process was inhibited by KN-62. Finally, ATP-induced ethidium+ uptake and CD23 shedding were impaired in Mo-LC obtained from subjects homozygous for the loss-of-function Glu-496 to Ala polymorphism in the P2X7 receptor. These results demonstrate that human LC express functional P2X7 receptors, and suggest a role for this receptor in the skin immune system.

A general network theory for the molecular motor kinesin is developed that is based on the distinct chemical states of the motor and on recent observations about its mechanical steps. For small concentrations of adenosine diphosphate (ADP), the motor's behavior is governed by the competition of two chemomechanical motor cycles which determine the motor's stall force. A third cycle becomes important for large ADP concentrations. The theory provides a quantitative description for the functional dependencies of different motor properties as observed in single molecule experiments.

BACKGROUND

Evidence suggests the wide variation in platelet response within the population is genetically controlled. Unraveling the complex relationship between sequence variation and platelet phenotype requires accurate and reproducible measurement of platelet response.

OBJECTIVE

To develop a methodology suitable for measuring signaling pathway-specific platelet phenotype, to use this to measure platelet response in a large cohort, and to demonstrate the effect size of sequence variation in a relevant model gene.

METHODS

Three established platelet assays were evaluated: mobilization of [Ca(2+)](i), aggregometry and flow cytometry, each in response to adenosine 5'-diphosphate (ADP) or the glycoprotein (GP) VI-specific crosslinked collagen-related peptide (CRP). Flow cytometric measurement of fibrinogen binding and P-selectin expression in response to a single, intermediate dose of each agonist gave the best combination of reproducibility and inter-individual variability and was used to measure the platelet response in 506 healthy volunteers. Pathway specificity was ensured by blocking the main subsidiary signaling pathways.

RESULTS

Individuals were identified who were hypo- or hyper-responders for both pathways, or who had differential responses to the two agonists, or between outcomes. 89 individuals, retested three months later using the same methodology, showed high concordance between the two visits in all four assays (r(2) = 0.872, 0.868, 0.766 and 0.549); all subjects retaining their phenotype at recall. The effect of sequence variation at the GP6 locus accounted for approximately 35% of the variation in the CRP-XL response.

CONCLUSIONS

Genotyping-phenotype association studies in a well-characterized, large cohort provides a powerful strategy to measure the effect of sequence variation in genes regulating the platelet response.

The frequencies of onset of myocardial infarction and sudden cardiac death are increased between 6 AM and 12 noon. Platelet aggregability, which may play a role in the cause of these disorders, has been observed to increase after the normal morning activities of awakening, arising, and ambulating. To determine which morning activity or activities are responsible for this aggregability increase, we measured platelet aggregation in 16 normal subjects on a control day of delayed arising (i.e., subjects remained supine until 12:30 PM) and on a day in which normal morning activities were divided into three isolated components of awakening (8 AM), assumption of upright posture (9:30 AM), and ambulating (11 AM). Blood samples to assess platelet aggregability were drawn at 8 AM before activity and 90 minutes after the initiation of each activity (i.e., at 9:30 AM, 11 AM, and 12:30 PM). For the group, in vitro platelet responsiveness to adenosine diphosphate and epinephrine increased only after assumption of the upright posture. The lowest concentration of agonist required to produce biphasic platelet aggregation decreased (aggregability increased) between 9:30 and 11 AM (90 minutes after assumption of the upright posture) from 3.3 +/- 0.3 to 2.4 +/- 0.2 microM for adenosine diphosphate (p less than 0.05) and from 2.1 +/- 0.5 to 1.0 +/- 0.4 microM for epinephrine (p less than 0.05). During the same interval, plasma epinephrine increased from 34 +/- 7 to 55 +/- 9 pg/ml (p less than 0.05), and plasma norepinephrine increased from 169 +/- 19 to 298 +/- 25 pg/ml (p less than 0.01). There was no significant change in aggregability or catecholamine concentrations on the control day.(ABSTRACT TRUNCATED AT 250 WORDS)

Antithrombotic therapy has become an important goal for the treatment of ischemic disorders such as cerebral ischemia. Our recent studies found that Z-ligustilide (LIG), a characterized 3-n-alkylphthalide constituent of Radix Angelica sinensis essential oil, exerted significant neuroprotection against cerebral ischemic damage in several animal models. The present study evaluated the antithrombotic activity of LIG and its effect on platelet aggregation and coagulation time. LIG (10 or 40 mg/kg) was intragastrically administered to rats once daily for 3 days. Our results showed that LIG significantly and dose-dependently reduced arterial thrombus weight in an arteriovenous shunt thrombosis in rats and platelet aggregation induced by adenosine diphosphate in rats ex vivo. Meanwhile, LIG at 10 or 40 mg/kg had no significant effect on coagulation time, including activated partial thromboplastin time and prothrombin time, in rats ex vivo. The present study demonstrated for the first time that LIG may exert efficient antithrombotic activity through inhibition of platelet aggregation, without effecting coagulation time of peripheral blood. These data, together with the previously reported neuroprotective effects of LIG on cerebral ischemia, suggest that the antithrombotic activity of LIG may contribute to its potential for the treatment of ischemic diseases, including ischemic stroke.

Pyruvate kinase (EC 2.7.1.40) from Azotobacter vinelandii responds sharply to the adenylate energy charge, with a decrease in activity at high values of charge, as expected for an enzyme of an adenosine triphosphate-regenerating sequence. Glycolytic intermediates, especially glucose 6-phosphate, fructose 6-phosphate, and fructose-1,6-diphosphate, strongly stimulate the reaction and overcome the inhibition caused by high values of energy charge. Thus, the properties of this enzyme depend on interaction between energy charge and the concentrations of hexose phosphates. The properties of pyruvate kinase, together with those of phosphoenolpyruvate carboxylase, aspartokinase, and citrate synthase, seem adapted to provide appropriate partitioning of phosphoenolpyruvate between competing pathways in response to metabolic need.

OBJECTIVE

To evaluate the levels of malondialdehyde (MDA) and total antioxidant capacity (TAC) in the blood and aqueous humor of glaucomatous and nonglaucomatous patients. To measure the adenosine triphosphate/adenosine diphosphate/adenosine monophosphate (ATP/ADP/AMP) concentration as a biomarker of the blood energy charge potential.

METHODS

We examined 40 consecutive patients with primary open-angle glaucoma scheduled for cataract surgery. Twenty-six age-matched subjects scheduled for cataract surgery were enrolled as a control group. Blood and aqueous humor samples were collected at the time of surgery. MDA concentrations and blood nucleotides were measured with high-performance liquid chromatography. The TAC of the samples was estimated with the oxygen-radical absorbance capacity method.

RESULTS

Blood and aqueous humor MDA levels in glaucoma patients (respectively, 0.976±0.370 and 0.145±0.065 μmol/ml) were significantly increased (p<0.001 for both) over those of the control group (respectively, 0.454±0.395 and 0.060±0.039 μmol/ml). In contrast, the control group presented significantly higher TACs than did the glaucoma group in both the blood (control: 2.681±1.101 and glaucoma: 1.617±0.674 μmol Trolox Equi/g; p<0.001) and aqueous humor (control: 0.963±0.302 and glaucoma: 0.788±0.346 μmol Trolox Equi/g; p=0.039). The control group (0.869±0.037) exhibited statistically significant (p<0.001) higher values of blood adenosine triphosphate/adenosine diphosphate (ATP-ADP) levels than did the glaucoma group (0.791±0.037).

CONCLUSIONS

Our data further support the hypothesis that oxidative stress and decreased antioxidant defenses are involved in glaucoma. High-performance liquid chromatography appears to be an effective and sensitive method to detect altered levels of oxidative stress markers in glaucoma patients.

OBJECTIVE

We sought to test the platelet inhibitory and anti-inflammatory effects of a higher statin dosage compared with combined treatment with ezetimibe plus a low statin dose.

BACKGROUND

Reducing the level of low-density lipoprotein cholesterol (LDL-C) with statins induces important pleiotropic effects such as platelet inhibition. An insufficient LDL-C reduction often is treated with ezetimibe, an intestinal cholesterol absorption inhibitor, in combination with a low statin dose. It is not known whether this combination therapy has the same pleiotropic effects as a statin monotherapy.

METHODS

Fifty-six patients with coronary artery disease were assigned randomly to receive either 40 mg/day of atorvastatin or 10 mg/day of ezetimibe plus 10 mg/day of atorvastatin for 4 weeks. The levels of LDL-C, platelet activation markers after stimulation, platelet aggregation, and plasma chemokine levels (i.e., regulated on activation normally T-cell expressed and secreted [RANTES]) were measured before and after changing lipid-lowering medication.

RESULTS

Platelet activation markers (P-selectin) after stimulation (adenosine diphosphate) were reduced by 40 mg/day of atorvastatin (-5.2 +/- 1.6 arbitrary units) but not by ezetimibe plus low-dose atorvastatin (2.1 +/- 1.8 arbitrary units; p < 0.005) despite a similar reduction of LDL-C (atorvastatin -1.01 +/- 0.18 mmol/l vs. ezetimibe plus atorvastatin -1.36 +/- 0.22 mmol/l, p = NS). Thrombin receptor-activating peptide-induced platelet aggregation as well as plasma RANTES levels were reduced by 40 mg/day of atorvastatin but not by ezetimibe plus low-dose atorvastatin.

CONCLUSIONS

Platelet reactivity and a proinflammatory chemokine were reduced more by the higher atorvastatin dose than by ezetimibe plus low-dose atorvastatin. In patients with coronary artery disease, it might be important to combine ezetimibe with higher statin dosages to benefit from cholesterol-independent pleiotropic effects.

We investigated 22 patients with migraine without aura, all drug-free and in headache-free periods, by means of 31P-magnetic resonance spectroscopy (MRS) of brain and muscle. Brain 31P-MRS showed significantly low phosphocreatine, increased adenosine diphosphate, and decreased phosphorylation potential. There was a slow rate of phosphocreatine recovery after exercise in the muscle of 12 of 22 patients. Energy metabolism is abnormal in migraine without aura, as previously demonstrated in patients with migraine stroke and migraine with aura.