The binding of cytosolic coat proteins to organelles may regulate membrane structure and traffic. Evidence is presented that a small guanosine triphosphate (GTP)-binding protein, the adenosine diphosphate ribosylation factor (ARF), reversibly associates with the Golgi apparatus in an energy, GTP, and fungal metabolite brefeldin A (BFA)-sensitive manner similar to, but distinguishable from, the 110-kilodalton cytosolic coat protein beta-COP. Addition of beta gamma subunits of G proteins inhibited the association of both ARF and beta-COP with Golgi membranes that occurred upon incubation with guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S). Thus, heterotrimeric G proteins may function to regulate the assembly of coat proteins onto the Golgi membrane.

Nuclear magnetic resonance (NMR) spectroscopy was used to monitor, on a beat-to-beat basis, the concentration of creatine phosphate and adenosine triphosphate during alterations in the work output of canine hearts in vivo. Over a wide range of rate-pressure products (5,000 to 25,000 mmHg/min), the relative amounts of creatine phosphate and adenosine triphosphate within the heart remained constant. The relative concentration of free adenosine diphosphate was calculated under the reasonable assumption that the creatine kinase-catalyzed reaction is near equilibrium in this tissue. The free concentration of adenosine diphosphate also did not change over this range of rate-pressure products. The data demonstrate that the concentration of these compounds is highly regulated in vivo and suggest that factors other than their concentration may be involved in the modulation of steady-state myocardial work output with oxygen consumption.

1. A new simple method is described for quantitating the adhesiveness of circulating polymorphonuclear leucocytes, or granulocytes, to the walls of blood vessels. The cheek pouch of anaesthetized hamsters or a small part of the mesentery of anaesthetized mice were prepared for continuous microscopic observation of selected venules. Those granulocytes which moved sufficiently slowly to be individually visible were counted for 1 or 2 min periods as they rolled past a selected point on one side of a vessel. The velocity distribution of these cells was determined by analysing films. Films were used also to measure mean blood flow velocity in the venules by observing embolizing platelet thrombi induced by the iontophoretic application of adenosine diphosphate. Emigration of granulocytes into the tissues was quantitated by enumerating them in standard areas of stained histological sections.2. In control experiments with hamster cheek pouch venules, the rolling granulocyte count usually passed through a maximum shortly after the preparation was set up and then fell to a low constant value. In mouse mesentery venules the count remained at a low approximately constant value from the beginning for at least 3 hr.3. The mean velocity of blood flow in the venules was between 900 and 200 mu/sec. All rolling granulocytes moved much more slowly; in hamster cheek pouch venules the mean velocity was about 20 mu/sec and in mouse mesentery venules about 10 mu/sec. Around these means the velocity distribution of individual cells was narrow.4. Rolling of granulocytes was abolished by superfusing ethylenediamine tetra-acetate (EDTA, 0.1 M) suggesting that the phenomenon depends on calcium or magnesium ions.5. Agents were applied locally to the observed venules. Human serum albumin, trypsin or histamine in high concentrations did not affect the rolling granulocyte count.6. The rolling granulocyte count was increased during the application of Hammarsten casein or Escherichia coli culture filtrate which are chemotactic to granulocytes in vitro. These agents did not cause alterations in mean blood flow velocity in the observed venules which might have accounted for the effect on the rolling granulocyte counts. When E. coli culture filtrate was perfused through mouse intestine the increase in rolling granulocyte count in the draining venous blood was proportional to the logarithm of the concentration of filtrate.7. The rolling granulocyte count was also increased by the local application of plasma globulin permeability factor or lymph node permeability factor.8. Granulocyte counts in standard histological sections showed no significant increases in control preparations but considerable increases following the application of Hammarsten casein.

Niraparib, an inhibitor of poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP), has been associated with significantly increased progression-free survival among patients with recurrent ovarian cancer after platinum-based chemotherapy, regardless of the presence or absence of BRCA mutations. The efficacy of niraparib in patients with newly diagnosed advanced ovarian cancer after a response to first-line platinum-based chemotherapy is unknown.

In this randomized, double-blind, phase 3 trial, we randomly assigned patients with newly diagnosed advanced ovarian cancer in a 2:1 ratio to receive niraparib or placebo once daily after a response to platinum-based chemotherapy. The primary end point was progression-free survival in patients who had tumors with homologous-recombination deficiency and in those in the overall population, as determined on hierarchical testing. A prespecified interim analysis for overall survival was conducted at the time of the primary analysis of progression-free survival.Of the 733 patients who underwent randomization, 373 (50.9%) had tumors with homologous-recombination deficiency. Among the patients in this category, the median progression-free survival was significantly longer in the niraparib group than in the placebo group (21.9 months vs. 10.4 months; hazard ratio for disease progression or death, 0.43; 95% confidence interval [CI], 0.31 to 0.59; P<0.001). In the overall population, the corresponding progression-free survival was 13.8 months and 8.2 months (hazard ratio, 0.62; 95% CI, 0.50 to 0.76; P<0.001). At the 24-month interim analysis, the rate of overall survival was 84% in the niraparib group and 77% in the placebo group (hazard ratio, 0.70; 95% CI, 0.44 to 1.11). The most common adverse events of grade 3 or higher were anemia (in 31.0% of the patients), thrombocytopenia (in 28.7%), and neutropenia (in 12.8%). No treatment-related deaths occurred.Among patients with newly diagnosed advanced ovarian cancer who had a response to platinum-based chemotherapy, those who received niraparib had significantly longer progression-free survival than those who received placebo, regardless of the presence or absence of homologous-recombination deficiency. (Funded by GlaxoSmithKline; PRIMA/ENGOT-OV26/GOG-3012 ClinicalTrials.gov number, NCT02655016.).

In the absence of the hematopoietic transcription factor GATA-1, mice develop thrombocytopenia and an increased number of megakaryocytes characterized by marked ultrastructural abnormalities. These observations establish a critical role for GATA-1 in megakaryopoiesis and raise the question as to how GATA-1 influences megakaryocyte maturation and platelet production. To begin to address this, we have performed a more detailed examination of the megakaryocytes and platelets produced in mice that lack GATA-1 in this lineage. Our analysis demonstrates that compared with their normal counterparts, GATA-1-deficient primary megakaryocytes exhibit significant hyperproliferation in liquid culture, suggesting that the megakaryocytosis seen in animals is nonreactive. Morphologically, these mutant megakaryocytes are small and show evidence of retarded nuclear and cytoplasmic development. A significant proportion of these cells do not undergo endomitosis and express markedly lower levels of mRNA of all megakaryocyte-associated genes tested, including GPIbalpha, GPIbbeta, platelet factor 4 (PF4), c-mpl, and p45 NF-E2. These results are consistent with regulation of a program of megakaryocytic differentiation by GATA-1. Bleeding times are significantly prolonged in mutant animals. GATA-1-deficient platelets show abnormal ultrastructure, reminiscent of the megakaryocytes from which they are derived, and exhibit modest but selective defects in platelet activation in response to thrombin or to the combination of adenosine diphosphate (ADP) and epinephrine. Our findings indicate that GATA-1 serves multiple functions in megakaryocyte development, influencing both cellular growth and maturation.

OBJECTIVE

The aim of this prospective trial was to assess whether platelet reactivity to clopidogrel assessed with multiple electrode platelet aggregometry (MEA) correlates with the risk of early drug-eluting stent thrombosis (ST).

BACKGROUND

Studies using light transmission aggregometry (LTA) have shown that insufficient suppression of platelet reactivity to adenosine diphosphate (ADP) after clopidogrel treatment is associated with an increased risk of adverse cardiovascular events after percutaneous coronary intervention (PCI). However, LTA is time- and labor-intensive and inconvenient for the routine. A point-of-care assay with similar predictive power would be of great value.

METHODS

Between February 2007 and April 2008, a total of 1,608 consecutive patients with coronary artery disease and planned drug-eluting stent implantation were enrolled. Before PCI, all patients received 600 mg clopidogrel. Blood was obtained directly before PCI. The ADP-induced platelet aggregation was assessed in whole blood with MEA on a Multiplate analyzer (Dynabyte, Munich, Germany). The primary end point was definite ST at 30 days.

RESULTS

The upper quintile of patients according to MEA measurements (n = 323) was defined as clopidogrel low responders. Compared with normal responders (n = 1,285), low responders had a significantly higher risk of definite ST within 30 days (2.2% vs. 0.2%; odds ratio [OR]: 9.4; 95% confidence interval [CI]: 3.1 to 28.4; p < 0.0001). Mortality rates were 1.2% in low versus 0.4% in normal responders (OR: 3.2; 95% CI: 0.9 to 11.1; p = 0.07). The composite of death or ST was higher in low versus normal responders (3.1% vs. 0.6%; OR: 5.1; 95% CI: 2.2 to 11.6; p < 0.001).

CONCLUSIONS

Low response to clopidogrel assessed with MEA is significantly associated with an increased risk of ST. Further studies are warranted to evaluate the ability of MEA to guide antiplatelet therapy in patients undergoing PCI.

Activation of death receptors and mitochondrial damage are well-described common apoptotic pathways. Recently, a novel pathway via endoplasmic reticulum (ER) stress has been reported. We assessed the role of tauroursodeoxycholic acid (TUDCA) in inhibition of caspase-12 activation and its effect on calcium homeostasis in an ER stress-induced model of apoptosis. The human liver-derived cell line, Huh7, was treated with thapsigargin (TG) to induce ER stress. Typical morphologic changes of ER stress preceded development of apoptotic changes, including DNA fragmentation and cleavage of poly (adenosine diphosphate-ribose) polymerase (PARP), as well as activation of caspase-3 and -7. Elevation of intracellular calcium levels without loss of mitochondrial membrane potential (MMP) was shown using Fluo-3/Fura-red labeling and flow cytometry, and confirmed by induction of Bip/GRP78, a calcium-dependent chaperon of ER lumen. These changes were accompanied by procaspase-12 processing. TUDCA abolished TG-induced markers of ER stress; reduced calcium efflux, induction of Bip/GRP78, and caspase-12 activation; and subsequently inhibited activation of effector caspases and apoptosis. In conclusion, we propose that mitochondria play a secondary role in ER-mediated apoptosis and that TUDCA prevents apoptosis by blocking a calcium-mediated apoptotic pathway as well as caspase-12 activation. This novel mechanism of TUDCA action suggests new intervention methods for ER stress-induced liver disease.

Ingestion of 1.5 g of aspirin, but not of sodium salicylate, produced a significant prolongation of the bleeding time in six normal male subjects when compared with the effects of a placebo. Similar differences in the effect of the two drugs on platelets was also observed. Aspirin ingestion resulted in impaired platelet aggregation by connective tissue and was associated with a decreased release of platelet adenosine diphosphate (ADP); sodium salicylate had no effect on these values. In vitro, incubation of platelet-rich plasma with an optimum aspirin concentration of 0.50 mmole/liter (0.045 mg/ml) inhibited both the adhesion of platelets to connective tissue and the release of ADP as well as the secondary wave of platelet aggregation produced with ADP or epinephrine; sodium salicylate had no effect on these reactions, which were also normal in patients with von Willebrand's disease. The inhibitory effect produced by ingesting a single 1.8 g dose of aspirin was detectable for 4-7 days at which time salicylate was no longer detectable in the blood, which suggested an irreversible effect on the platelet. Aspirin also inhibited the release of platelet adenosine triphosphate (ATP), but had no effect on the platelet surface charge, available platelet ATP or ADP, or the destruction of ADP by plasma ADPase. These studies lend further support to the hypothesis that ingestion of aspirin, in contrast to sodium salicylate, prolongs the bleeding time by inhibiting the release of platelet ADP, perhaps reflecting the findings in other cell systems which suggest that aspirin alters membrane permeability.

Achieving hemostasis following vascular injury requires the rapid accumulation of platelets and fibrin. Here we used a combination of confocal intravital imaging, genetically engineered mice, and antiplatelet agents to determine how variations in the extent of platelet activation following vascular injury arise from the integration of different elements of the platelet-signaling network. Two forms of penetrating injury were used to evoke the hemostatic response. Both produced a hierarchically organized structure in which a core of fully activated platelets was overlaid with an unstable shell of less-activated platelets. This structure emerged as hemostasis was achieved and persisted for at least 60 minutes following injury, its organization at least partly reflecting agonist concentration gradients. Thrombin activity and fibrin formation were found primarily in the innermost core. As proposed previously, greater packing density in the core facilitated contact-dependent signaling and limited entry of plasma-borne molecules visualized with fluorophores coupled to dextran and albumin. Blocking contact-dependent signaling or inhibiting thrombin reduced the size of the core, while the shell was heavily influenced by adenosine 5'-diphosphate and regulators of Gi2-mediated signaling. Thus, the hemostatic response is shown to produce a hierarchical structure arising, in part, from distinct elements of the platelet-signaling network.

Ecto-ATPases are ubiquitous in eukaryotic cells. They hydrolyze extracellular nucleoside tri- and/or diphosphates, and, when isolated, they exhibit E-type ATPase activity, (that is, the activity is dependent on Ca2+ or Mg2+, and it is insensitive to specific inhibitors of P-type, F-type, and V-type ATPases; in addition, several nucleotide tri- and/or diphosphates are hydrolysed, but nucleoside monophosphates and nonnucleoside phosphates are not substrates). Ecto-ATPases are glycoproteins; they do not form a phosphorylated intermediate during the catalytic cycle; they seem to have an extremely high turnover number; and they present specific experimental problems during solubilization and purification. The T-tubule Mg2+-ATPase belongs to this group of enzymes, which may serve at least two major roles: they terminate ATP/ADP-induced signal transduction and participate in adenosine recycling. Several other functions have been discussed and identity to certain cell adhesion molecules and the bile acid transport protein was suggested on the basis of cDNA clone isolation and immunological work.

OBJECTIVE

Our goal was to compare the safety and initial efficacy of AZD6140, the first reversible oral adenosine diphosphate receptor antagonist, with clopidogrel in patients with non-ST-segment elevation acute coronary syndromes (NSTE-ACS).

BACKGROUND

AZD6140 achieves higher mean levels of platelet inhibition than clopidogrel in patients with stable coronary artery disease.

METHODS

A total of 990 patients with NSTE-ACS, treated with aspirin and standard therapy for ACS, were randomized in a 1:1:1 double-blind fashion to receive either twice-daily AZD6140 90 mg, AZD6140 180 mg, or clopidogrel 300-mg loading dose plus 75 mg once daily for up to 12 weeks.

RESULTS

The primary end point, the Kaplan-Meier rate of major or minor bleeding through 4 weeks, was 8.1% in the clopidogrel group, 9.8% in the AZD6140 90-mg group, and 8.0% in the AZD6140 180-mg group (p = 0.43 and p = 0.96, respectively, vs. clopidogrel); the major bleeding rates were 6.9%, 7.1%, and 5.1%, respectively (p = 0.91 and p = 0.35, respectively, vs. clopidogrel). Although not statistically significant, favorable trends were seen in the Kaplan-Meier rates of myocardial infarction (MI) over the entire study period (MI: 5.6%, 3.8%, and 2.5%, respectively; p = 0.41 and p = 0.06, respectively, vs. clopidogrel). In a post-hoc analysis of continuous electrocardiograms, mostly asymptomatic ventricular pauses >2.5 s were more common, especially in the AZD6140 180-mg group (4.3%, 5.5%, and 9.9%, respectively; p = 0.58 and p = 0.01, respectively, vs. clopidogrel).

CONCLUSIONS

This initial experience with AZD6140 in patients with ACS showed no difference in major bleeding but an increase in minor bleeding at the higher dose with encouraging results on the secondary end point of MI. This agent is currently being studied in a large outcomes trial in 18,000 patients with ACS.

To determine half-life and turnover of plasma adenosine, heparinized blood from healthy volunteers was incubated with radiolabeled adenosine in the physiological concentration range of 0.1-1 microM. Plasma levels of adenosine in vitro were 82 +/- 14 nM and were similar to those determined immediately after blood collection with a "stopping solution." Dipyridamole (83 microM) and erythro-9(2-hydroxynon-3yl)-adenine (EHNA) (8 microM) did not measurably alter basal adenosine levels but completely blocked the uptake of added adenosine. Inhibition of ecto-5'-nucleotidase with 100 microM alpha, beta-methyleneadenosine 5'-diphosphate (AOPCP) reduced plasma adenosine to 22 +/- 6 nM. For the determination of adenosine turnover, the decrease in specific radioactivity of added [3H]adenosine was measured using a dipyridamole-containing stopping solution. Without altering basal adenosine levels, the half-life was estimated to be 0.6 s. Similar experiments were carried out with washed erythrocytes or in the presence of AOPCP, yielding half-lives of 0.7 and 0.9 s, respectively. When the initial adenosine concentration was 1 microM, its specific activity decreased by only 11% within 5 s, whereas total plasma adenosine exponentially decreased with a half-life of 1.5 s. Venous plasma concentrations were measured after relief of a 3-min forearm ischemia. Changes in plasma adenosine did not correlate well with changes in blood flow but were augmented in the presence of dipyridamole.(ABSTRACT TRUNCATED AT 250 WORDS)

F-actin is the major component of muscle thin filaments and, more generally, of the microfilaments of the dynamic, multifunctional cytoskeletal systems of nonmuscle eukaryotic cells. Polymeric F-actin is formed by reversible noncovalent self-association of monomeric G-actin. To understand the dynamics of microfilament systems in cells, the dynamics of polymerization of pure actin must be understood. The following model has emerged from recent work. During the polymerization process, adenosine 5'-triphosphate (ATP) that is bound to G-actin is hydrolyzed to adenosine 5'-diphosphate (ADP) that is bound to F-actin. The hydrolysis reaction occurs on the F-actin subsequent to the polymerization reaction in two steps: cleavage of ATP followed by the slower release of inorganic phosphate (Pi). As a result, at high rates of filament growth a transient cap of ATP-actin subunits exists at the ends of elongating filaments, and at steady state a stabilizing cap of ADP.Pi-actin subunits exists at the barbed ends of filaments. Cleavage of ATP results in a highly stable filament with bound ADP.Pi, and release of Pi destabilizes the filament. Thus these two steps of the hydrolytic reaction provide potential mechanisms for regulating the monomer-polymer transition.

Salmonella typhimurium LT2 induces a set of heat-shock proteins analogous to those found previously in Escherichia coli. These are virtually the only proteins synthesized after a temperature shift from 28 degrees C to 50 degrees C. Using a two-dimensional thin-layer chromatographic system developed to resolve adenylylated nucleotides, we have found that S. typhimurium and E. coli accumulate P1,P4-diadenosine-5'-tetraphosphate (AppppA), P1-(adenosine-5')-P3-(guanosine-3'-diphosphate-5')-triphosphate (ApppGpp), P1-(adenosine-5')-P4-(guanosine-5')-tetraphosphate (AppppG), P1-(adenosine-5')-P3-(guanosine-5')-triphosphate (ApppG), and P1,P3-diadenosine-5'-triphosphate (ApppA) after heat shock. These same adenylylated nucleotides accumulate after exposure to ethanol, an agent also known to induce the heat-shock response in a variety of cells. AppppA, ApppGpp, AppppG, ApppG, and ApppA were previously shown to accumulate under conditions of oxidation stress. We proposed that these adenylylated nucleotides may be alarmones--i.e., regulatory molecules, alerting cells to the onset of oxidation stress. The finding that these dinucleotides accumulate in response to heat shock suggests that oxidation and heat shock have a common physiological effect on cells. We hypothesize that these dinucleotides signal the onset of these stresses and trigger the "heat-shock response."

1. Patch-clamp techniques were used to study adenosine-5'-triphosphate (ATP)-dependent K+ channels in sarcolemmal vesicles from frog skeletal muscle. In addition to its ATP dependence, opening of these channels was voltage dependent, the open-state probability (P open) increasing with depolarization. 2. The reversal potential of unitary currents changed with external K+ concentration, [K+]o, as expected if the Na-K permeability ration (pNa/pK) equals 0.015. Unitary conductance increased with increasing [K+]o from 14.8 +/- 0.5 pS (n = 5) in 2.5 mM-K+ to 42.3 +/- 1.0 pS (n = 8) in 60 mM-K+. This increase was less than that expected from independence. 3. Replacement of 60 mM-external K+ by 60 mM-external Rb+ shifted the reversal potential of unitary currents by -6.7 mV, suggesting that Rb+ enters channels nearly as easily as does K+ (Rb-K permeability ration, pRb/pK = 0.76). Unitary currents were much smaller in Rb+, consistent with Rb+ binding within the channel. 4. The ATP-regulated K+ channel was blocked by both internal and external tetraethylammonium ions (TEA+). 2 mM-TEA+, applied to the cytoplasmic face of membrane patches, interrupted channel openings. Higher concentrations reduced unitary current amplitude, suggesting an increase in the rapidity of TEA+ block. 5. The reduction in P open by ATP was consistent with 1:1 binding and a dissociation constant of 0.135 mM. ATP appeared not to be hydrolysed to close channels. Adenosine 5'-diphosphate (ADP) and adenosine 5'-monophosphate (AMP) were less effective than ATP, but retained channel closing properties. Substitution of adenine with other purines or with pyrimidine bases substantially reduced activity, as did substitution of ribose by 2'-deoxyribose or by ribose 2',3'-dialdehyde. 6. Sarcoplasmic Ca2+ did not influence P open. 7. Myotubes, grown from thigh muscles of new-born rats, appeared to lack ATP-dependent K+ channels. Adult frog muscle appeared to lack high-conductance Ca2+-dependent K+ channels, at least in the surface membrane. Such channels were found in myotube membranes. 8. Open- and closed-time histograms were constructed and were consistent with at least two open and at least three closed states. Channel openings were grouped in bursts. Open times, burst lengths and the number of openings per burst were reduced by ATP. 9. The effects of [K+]o on unitary conductance and of K+ replacement with Rb+ are discussed in terms of a simple Eyring rate theory formulation.(ABSTRACT TRUNCATED AT 400 WORDS)

The Golgi-localized, gamma-ear-containing, adenosine diphosphate ribosylation factor-binding proteins (GGAs) are multidomain proteins that bind mannose 6-phosphate receptors (MPRs) in the Golgi and have an essential role in lysosomal enzyme sorting. Here the GGAs and the coat protein adaptor protein-1 (AP-1) were shown to colocalize in clathrin-coated buds of the trans-Golgi networks of mouse L cells and human HeLa cells. Binding studies revealed a direct interaction between the hinge domains of the GGAs and the gamma-ear domain of AP-1. Further, AP-1 contained bound casein kinase-2 that phosphorylated GGA1 and GGA3, thereby causing autoinhibition. This could induce the directed transfer of the MPRs from GGAs to AP-1. MPRs that are defective in binding to GGAs are poorly incorporated into AP-1-containing clathrin-coated vesicles. Thus, the GGAs and AP-1 interact to package MPRs into AP-1-containing coated vesicles.

Oxysterol binding proteins (OSBPs) comprise a large conserved family of proteins in eukaryotes. Their ubiquity notwithstanding, the functional activities of these proteins remain unknown. Kes1p, one of seven members of the yeast OSBP family, negatively regulates Golgi complex secretory functions that are dependent on the action of the major yeast phosphatidylinositol/phosphatidylcholine Sec14p. We now demonstrate that Kes1p is a peripheral membrane protein of the yeast Golgi complex, that localization to the Golgi complex is required for Kes1p function in vivo, and that targeting of Kes1p to the Golgi complex requires binding to a phosphoinositide pool generated via the action of the Pik1p, but not the Stt4p, PtdIns 4-kinase. Localization of Kes1p to yeast Golgi region also requires function of a conserved motif found in all members of the OSBP family. Finally, we present evidence to suggest that Kes1p may regulate adenosine diphosphate-ribosylation factor (ARF) function in yeast, and that it may be through altered regulation of ARF that Kes1p interfaces with Sec14p in controlling Golgi region secretory function.

Blood platelets play critical roles in hemostasis, providing rapid essential protection against bleeding and catalyzing the important slower formation of stable blood clots via the coagulation cascade. They are also involved in protection from infection by phagocytosis of pathogens and by secreting chemokines that attract leukocytes. Platelet function usually is activated by primary agonists such as adenosine diphosphate (ADP), thrombin, and collagen, whereas secondary agonists like adrenalin do not induce aggregation on their own but become highly effective in the presence of low levels of primary agonists. Current research has revealed that chemokines represent an important additional class of agonists capable of causing significant activation of platelet function. Early work on platelet alpha-granule proteins suggested that platelet factor 4, now known as CXCL4, modulated aggregation and secretion induced by low agonist levels. Subsequent reports revealed the presence in platelets of messenger RNA for several additional chemokines and chemokine receptors. Three chemokines in particular, CXCL12 (SDF-1), CCL17 (TARC), and CCL22 (MDC), recently have been shown to be strong and rapid activators of platelet aggregation and adhesion after their binding to platelet CXCR4 or CCR4, when acting in combination with low levels of primary agonists. CXCL12 can be secreted by endothelial cells and is present in atherosclerotic plaques, whereas CCL17 and CCL22 are secreted by monocytes and macrophages. Platelet activation leads to the release of alpha-granule chemokines, including CCL3 (MIP-1alpha), CCL5 (RANTES), CCL7 (MCP-3), CCL17, CXCL1 (growth-regulated oncogene-alpha), CXCL5 (ENA-78), and CXCL8 (IL-8), which attract leukocytes and further activate other platelets. These findings help to provide a direct linkage between hemostasis, infection, and inflammation and the development of atherosclerosis.

OBJECTIVE

In this study, we have investigated the effects of cannabidiol (CBD) on myocardial dysfunction, inflammation, oxidative/nitrative stress, cell death, and interrelated signaling pathways, using a mouse model of type I diabetic cardiomyopathy and primary human cardiomyocytes exposed to high glucose.

BACKGROUND

Cannabidiol, the most abundant nonpsychoactive constituent of Cannabis sativa (marijuana) plant, exerts anti-inflammatory effects in various disease models and alleviates pain and spasticity associated with multiple sclerosis in humans.

METHODS

Left ventricular function was measured by the pressure-volume system. Oxidative stress, cell death, and fibrosis markers were evaluated by molecular biology/biochemical techniques, electron spin resonance spectroscopy, and flow cytometry.

RESULTS

Diabetic cardiomyopathy was characterized by declined diastolic and systolic myocardial performance associated with increased oxidative-nitrative stress, nuclear factor-κB and mitogen-activated protein kinase (c-Jun N-terminal kinase, p-38, p38α) activation, enhanced expression of adhesion molecules (intercellular adhesion molecule-1, vascular cell adhesion molecule-1), tumor necrosis factor-α, markers of fibrosis (transforming growth factor-β, connective tissue growth factor, fibronectin, collagen-1, matrix metalloproteinase-2 and -9), enhanced cell death (caspase 3/7 and poly[adenosine diphosphate-ribose] polymerase activity, chromatin fragmentation, and terminal deoxynucleotidyl transferase dUTP nick end labeling), and diminished Akt phosphorylation. Remarkably, CBD attenuated myocardial dysfunction, cardiac fibrosis, oxidative/nitrative stress, inflammation, cell death, and interrelated signaling pathways. Furthermore, CBD also attenuated the high glucose-induced increased reactive oxygen species generation, nuclear factor-κB activation, and cell death in primary human cardiomyocytes.

CONCLUSIONS

Collectively, these results coupled with the excellent safety and tolerability profile of CBD in humans, strongly suggest that it may have great therapeutic potential in the treatment of diabetic complications, and perhaps other cardiovascular disorders, by attenuating oxidative/nitrative stress, inflammation, cell death and fibrosis.

We used phosphorus nuclear magnetic resonance spectroscopy (31P-NMR) to probe the cellular events in contracting muscle that initiate the reflex stimulation of sympathetic outflow during exercise. In conscious humans, we performed 31P-NMR on exercising forearm muscle and simultaneously recorded muscle sympathetic nerve activity (MSNA) with microelectrodes in the peroneal nerve to determine if the activation of MSNA is coupled to muscle pH, an index of glycolysis, or to the concentrations (II) of inorganic phosphate (Pi) and adenosine diphosphate (ADP) which are modulators of mitochondrial respiration. During both static and rhythmic handgrip, the onset of sympathetic activation in resting muscle coincided with the development of cellular acidification in active muscle. Furthermore, increases in MSNA were correlated closely with decreases in intracellular pH but dissociated from changes in phosphocreatine [( PCr]), [Pi], and [ADP]. The principal new conclusion is that activation of muscle sympathetic outflow during exercise in humans is coupled to the cellular accumulation of protons in contracting muscle.

OBJECTIVE

We investigated the relation of high ex vivo platelet reactivity, rapid fibrin generation, and high thrombin-induced clot strength to postdischarge ischemic events in patients undergoing percutaneous coronary intervention (PCI).

BACKGROUND

High platelet reactivity and rapid fibrin generation may affect the incidence of ischemic events after PCI. However, limited data is available to link these ex vivo markers to the occurrence of events.

METHODS

We measured platelet reactivity to adenosine diphosphate (ADP) by light transmittance aggregometry (LTA) in patients undergoing PCI (n = 192). Clot strength, a measure of thrombin-induced fibrin and platelet interactions, and the time to initial fibrin generation, a marker of thrombin activity, were measured by thrombelastography. The relation of these measurements to ischemic event occurrence was prospectively examined over six months.

RESULTS

A total of 100% and 84% of patients were on aspirin and clopidogrel therapy, respectively, at the time of the initial event. Posttreatment ADP-induced aggregation by LTA (63 +/- 12% vs. 56 +/- 15%, p = 0.02) and clot strength (MA) were higher (74 +/- 5 mm vs. 65 +/- 4 mm, p < 0.001) and time to initial fibrin generation was shorter (4.3 +/- 1.3 min vs. 5.9 +/- 1.5 min, p < 0.001) in patients with events (n = 38). The event rates in the highest quartiles of LTA and MA were 32% and 58%, respectively.

CONCLUSIONS

High platelet reactivity and clot strength, and rapid fibrin formation are novel risk factors for ischemic events after PCI. Clot strength is more predictive than ADP-induced platelet aggregation and may explain the occurrence of events despite treatment with cyclooxygenase-1 and P2Y12 inhibitors.

1. Conventional electrophysiological techniques were used to record from isolated rat phrenic nerve-hemidiaphragm preparations. After periods of rest (20 min) or nerve stimulation (7/sec for 20 min) the bathing medium of the preparation was removed and assayed for adenosine triphosphate (ATP) and adenosine diphosphate (ADP) using a sensitive modification of the firefly luciferase method (Silinsky, 1974). 2. In the presence of tubocurarine and normal (2 mM) calcium, fourteen periods of nerve stimulation (eight preparations) caused the appearance of ATP and/or ADP in amounts ranging from 28 to 641 p-mole. Experiments using carbachol (30 muM or 1 mM) suggested that this nucleotide efflux was not produced by a secondary action of released acetylcholine (ACh). 3. Stimulation of isolate phrenic nerve trunks at 7/sec for 20 min did not cause the efflux of ATP or ADP. 4. In solutions of normal osmotic pressure and reduced calcium concentrations (0-1 mM or 'calcium-free'), stimulation failed to release adenine nucleotide from non-contracting preparations. 5. Diaphragms were bathed in normal calcium and indirectly stimulated at 11/sec for 80-90 min in the presence of 5 times 10-minus 5 M hemicholinium-3. After all detectable signs of ACh release were eliminated, nerve stimulation failed to release ATP or ADP. 6. These results in conjunction with experiments on the hydrolysis of exogenous ATP suggest that ATP is released from the motor nerve ending and is subsequently degraded by enzymatic activity. It is also suggested that the released nucleotide may be derived from the cholinergic vesicle.