Activated platelets release large lipid-protein complexes termed microparticles. These platelet microparticles (PMP) are composed of vesicular fragments of the plasma membrane and alpha-granules. PMP facilitate coagulation, promote platelet and leukocyte adhesion to the subendothelial matrix, support angiogenesis and stimulate vascular smooth muscle proliferation.

OBJECTIVE

PMP were separated into 4 size classes to facilitate identification of active protein and lipid components. PMP were obtained from activated human platelets and separated into 4 size classes by gel filtration chromatography. Proteins were identified using 2-dimensional, liquid chromatography tandem mass spectrometry. Functional effects on platelets were determined using the PFA-100 and on endothelial cells by measuring transendothelial cell electrical resistance. PMP size classes differed significantly in their contents of plasma membrane receptors and adhesion molecules, chemokines, growth factors and protease inhibitors. The two smallest size classes (3 and 4) inhibited collagen/adenosine-diphosphate-mediated platelet thrombus formation, while fractions 2 and 4 stimulated barrier formation by endothelial cells. Heat denaturation blocked the effect of fraction 4 on endothelial cell function, but not fraction 2 implying that the active component in fraction 4 is a protein and in fraction 2 is a heat-stable protein or lipid but not sphingosine-1-phosphate. Proteomic and functional analysis of PMP size fractions has shown that PMP can be separated into different size classes that differ in protein components, protein/lipid ratio, and functional effects on platelets and endothelial cells. This analysis will facilitate identification of active components in the PMP and clarify their involvement in diseases such as atherosclerosis and cancer.

When nucleosomal core histones were isolated from rat liver nuclei incubated with [14C]NAD+ and fractionated into the individual components (H2A, H2B, H3, and H4), [14C]adenosine diphosphate ribose (ADP-Rib) was found to be associated with all of them. However, while about 15% of the H2B molecules were modified, less than 2% of the other fractions contained radioactive ADP-Rib. The nucleotide attached to H2B was identified as a single monomer of ADP-Rib. On subjectint H2B to electrophoresis in polyacrylamide gels containing 2.5 M urea and 0.9 N acetic acid, one single band of H2B with 5% less mobility than the unomdified control was obtained. The linkage between H2B and ADP-Rib was rapidly hydrolyzed with 0.1 N NaOH or with 1 M neutral hydroxylamine. Hydrolysis of ADP-ribosylated H2B with trypsin generated a single peptide linked to ADP-Rib, which corresponded to the sequence Pro-Glu-Pro-Ala-Lys. We were able to dansylate the NH2-terminal proline, which proved that the imino group of this amino acid was not substituted. These findings, together with the chemical properties of the linkage, which were typical of those of an ester-like bond, strongly suggest that the ADP-Rib residue was linked to the gamma-COOH group of the glutamic acid in position 2 of H2B.

OBJECTIVE

The aim of this study was to assess the effects of different proton pump inhibitors (PPIs) on the steady-state pharmacokinetics and pharmacodynamics of clopidogrel.

BACKGROUND

Metabolism of clopidogrel requires cytochrome P450s (CYPs), including CYP2C19. However, PPIs may inhibit CYP2C19, potentially reducing the effectiveness of clopidogrel.

METHODS

A randomized, open-label, 2-period, crossover study of healthy subjects (n = 160, age 18 to 55 years, homozygous for CYP2C19 extensive metabolizer genotype, confined, standardized diet) was conducted. Clopidogrel 75 mg with or without a PPI (dexlansoprazole 60 mg, lansoprazole 30 mg, esomeprazole 40 mg, or, as a positive control to maximize potential interaction and demonstrate assay sensitivity, omeprazole 80 mg) was given daily for 9 days. Pharmacokinetics and pharmacodynamics were assessed on days 9 and 10. Pharmacodynamic end-points were vasodilator-stimulated phosphoprotein P2Y(12) platelet reactivity index, maximal platelet aggregation to 5 and 20 μmol/l adenosine diphosphate, and VerifyNow P2Y12 platelet response units.

RESULTS

Pharmacokinetic and pharmacodynamic responses with omeprazole demonstrated assay sensitivity. The area under the curve for clopidogrel active metabolite decreased significantly with esomeprazole but not with dexlansoprazole or lansoprazole. Similarly, esomeprazole but not dexlansoprazole or lansoprazole significantly reduced the effect of clopidogrel on vasodilator-stimulated phosphoprotein platelet reactivity index. All PPIs decreased the peak plasma concentration of clopidogrel active metabolite (omeprazole>> esomeprazole>> lansoprazole>> dexlansoprazole) and showed a corresponding order of potency for effects on maximal platelet aggregation and platelet response units.

CONCLUSIONS

Generation of clopidogrel active metabolite and inhibition of platelet function were reduced less by the coadministration of dexlansoprazole or lansoprazole with clopidogrel than by the coadministration of esomeprazole or omeprazole. These results suggest that the potential of PPIs to attenuate the efficacy of clopidogrel could be minimized by the use of dexlansoprazole or lansoprazole rather than esomeprazole or omeprazole.

The kinetics of binding of the nonhydrolyzable nucleotides adenosine 5'-diphosphate (ADP) and adenosine 5'-(beta, gamma-imidotriphosphate) (AMP-PNP) to myosin subfragment 1 (SF-1) and actosubfragment 1 (acto-SF-1) were reinvestigated. The binding of these ligands to SF-1 can be described by (Formula: see text). The nucleotide binds in a rapid equilibrium step (K0), followed by two first-order fluorescence transitions with k1 + k-1 much greater than k2 + k-2. The rates and amplitudes of the fluorescence transitions are different for ADP and AMP-PNP and in turn can be distinguished from the corresponding steps involved in adenosine 5'-triphosphate (ATP) binding. The similarity in the maximum rate of the observed fluorescence signal for ADP and ATP binding to SF-1 in 0.1 M KCl is fortuitous as the maximum rates differ greatly at higher ionic strength. Under favorable conditions of high ionic strength where the amplitude of the fluorescence enhancement is large, the binding of AMP-PNP to acto-SF-1 gave a fluorescence change prior to dissociation, followed by a second fluorescence transition at the same rate as the dissociation of the proteins. Thus a conformation change precedes the nucleotide-induced dissociation of actomyosin. At least three acto-SF-1-nucleotide complexes are necessary to explain the kinetic behavior.

In Saccharomyces cerevisiae, the product of the CDC25 gene controls the RAS-mediated production of adenosine 3',5'-monophosphate (cAMP). In vivo the carboxyl-terminal third of the CDC25 gene product is sufficient for the activation of adenylate cyclase. The 3'-terminal part of SCD25, a gene of S. cerevisiae structurally related to CDC25, can suppress the requirement for CDC25. Partially purified preparations of the carboxy-terminal domain of the SCD25 gene product enhanced the exchange rate of guanosine diphosphate (GDP) to guanosine triphosphate (GTP) of pure RAS2 protein by stimulating the release of GDP. This protein fragment had a similar effect on the human c-H-ras-encoded p21 protein. Thus, the SCD25 carboxyl-terminal domain can enhance the regeneration of the active form of RAS proteins.

Observations on the relationship between cardiac work rate and the levels of energy metabolites adenosine triphosphate (ATP), adenosine diphosphate (ADP), and phosphocreatine (CrP) have not been satisfactorily explained by theoretical models of cardiac energy metabolism. Specifically, the in vivo stability of ATP, ADP, and CrP levels in response to changes in work and respiratory rate has eluded explanation. Here a previously developed model of mitochondrial oxidative phosphorylation, which was developed based on data obtained from isolated cardiac mitochondria, is integrated with a spatially distributed model of oxygen transport in the myocardium to analyze data obtained from several laboratories over the past two decades. The model includes the components of the respiratory chain, the F0F1-ATPase, adenine nucleotide translocase, and the mitochondrial phosphate transporter at the mitochondrial level; adenylate kinase, creatine kinase, and ATP consumption in the cytoplasm; and oxygen transport between capillaries, interstitial fluid, and cardiomyocytes. The integrated model is able to reproduce experimental observations on ATP, ADP, CrP, and inorganic phosphate levels in canine hearts over a range of workload and during coronary hypoperfusion and predicts that cytoplasmic inorganic phosphate level is a key regulator of the rate of mitochondrial respiration at workloads for which the rate of cardiac oxygen consumption is less than or equal to approximately 12 mumol per minute per gram of tissue. At work rates corresponding to oxygen consumption higher than 12 mumol min(-1) g(-1), model predictions deviate from the experimental data, indicating that at high work rates, additional regulatory mechanisms that are not currently incorporated into the model may be important. Nevertheless, the integrated model explains metabolite levels observed at low to moderate workloads and the changes in metabolite levels and tissue oxygenation observed during graded hypoperfusion. These findings suggest that the observed stability of energy metabolites emerges as a property of a properly constructed model of cardiac substrate transport and mitochondrial metabolism. In addition, the validated model provides quantitative predictions of changes in phosphate metabolites during cardiac ischemia.

Pertussis toxin (PTX) is a major virulence factor in whooping cough and can elicit protective antibodies. Amino acid residues 8 to 15 of PTX subunit S1 are important for the adenosine diphosphate-ribosyltransferase activity associated with the pathobiological effects of PTX. Furthermore, this region contains at least a portion of an epitope that elicits both toxin-neutralizing and protective antibody responses in mice. The gene encoding the S1 subunit was subjected to site-specific mutagenesis in this critical region. A mutant containing a single amino acid substitution (Arg9----Lys) had reduced enzymatic activity (approximately 0.02% of control) while retaining the protective epitope. This analog S1 molecule may provide the basis for a genetically detoxified PTX with potential for use as a component of an acellular vaccine against whooping cough.

OBJECTIVE

Mitochondrial dysfunctions have been associated with the pathogenesis of sepsis. A systematic survey of mitochondrial function in brain tissues during sepsis is lacking. In the present work, we investigate brain mitochondrial function in a septic mouse model.

METHODS

Prospective animal study.

METHODS

University research laboratory.

METHODS

Male Swiss mice, aged 6-8 wks.

METHODS

Mice were subjected to cecal ligation and perforation (sepsis group) with saline resuscitation or to sham operation (control group).

RESULTS

Oxygen consumption was measured polarographically in an oximeter. Brain homogenates from septic animals presented higher oxygen consumption in the absence of adenosine 5'-diphosphate (state 4) compared with control animals. The increase in state 4 respiration in animals in the cecal ligation and perforation group resulted in a drastic decrease in both respiratory control and adenosine 5'-diphosphate/oxygen ratios, indicating a reduction in the oxidative phosphorylation efficiency. Septic animals presented a significant increase in the recovery time of mitochondrial membrane potential on adenosine 5'-diphosphate addition compared with control animals, suggesting a proton leak through the inner mitochondrial membrane. The septic group presented a general reduction in the content of cytochromes. Moreover, the activity of cytochrome c oxidase was specifically and significantly decreased in the brain during sepsis. Hydrogen peroxide generation by brain mitochondria from septic mice did not respond to substrates of electron transport chain or to adenosine 5'-diphosphate, showing that mitochondrial function may be compromised in a critical level in the brain during sepsis.

CONCLUSIONS

The mitochondrial dysfunctions demonstrated here indicate that uncoupling of oxidative phosphorylation takes place in the brain of septic mice, compromising tissue bioenergetic efficiency.

OBJECTIVE

To examine the extent of platelet inhibition by prasugrel vs. clopidogrel in a TRITON-TIMI 38 substudy.

RESULTS

TRITON-TIMI 38 randomized acute coronary syndrome (ACS) patients undergoing percutaneous coronary intervention (PCI) to prasugrel or standard dose clopidogrel. Selected sites prospectively enrolled TRITON-TIMI 38 patients to evaluate adenosine diphosphate (ADP)-attenuated phosphorylation of platelet vasodilator-stimulated phosphoprotein (VASP) (n = 125 patients) and, in a subset (n = 31 patients), ADP-stimulated platelet aggregation. VASP platelet reactivity index (PRI) was lower in prasugrel-treated patients than in clopidogrel-treated patients at 1-2 h post-PCI >>or=1 h after loading dose) (P < 0.001) and at 30 days (P < 0.001). Maximal platelet aggregation to 20 microM ADP was lower in prasugrel-treated patients than in clopidogrel-treated patients at 1-2 h (P = 0.004) and 30 days (P = 0.03). Results were similar with 5 microM ADP. Thienopyridine hyporesponsiveness, prespecified as VASP PRI >50%, was more frequent in clopidogrel-treated patients than in prasugrel-treated patients at 1-2 h (P < 0.001) and 30 days (P = 0.03).

CONCLUSIONS

The TRITON-TIMI 38 platelet substudy shows that prasugrel results in greater inhibition of ADP-mediated platelet function in ACS patients than clopidogrel, supporting the hypothesis that greater platelet inhibition leads to a lower incidence of ischaemic events and more bleeding both early and late following PCI.

Cytosolic phospholipase A2alpha (cPLA2alpha) hydrolyzes arachidonic acid from cellular membrane phospholipids, thereby providing enzymatic substrates for the synthesis of eicosanoids, such as prostaglandins and leukotrienes. Considerable understanding of cPLA2alpha function has been derived from investigations of the enzyme and from cPLA2alpha-null mice, but knowledge of discrete roles for this enzyme in humans is limited. We investigated a patient hypothesized to have an inherited prostanoid biosynthesis deficiency due to his multiple, complicated small intestinal ulcers despite no use of cyclooxygenase inhibitors. Levels of thromboxane B2 and 12-hydroxyeicosatetraenoic acid produced by platelets and leukotriene B4 released from calcium ionophore-activated blood were markedly reduced, indicating defective enzymatic release of the arachidonic acid substrate for the corresponding cyclooxygenase and lipoxygenases. Platelet aggregation and degranulation induced by adenosine diphosphate or collagen were diminished but were normal in response to arachidonic acid. Two heterozygous single base pair mutations and a known SNP were found in the coding regions of the patient's cPLA2alpha genes (p.[Ser111Pro]+[Arg485His; Lys651Arg]). The total PLA2 activity in sonicated platelets was diminished, and the urinary metabolites of prostacyclin, prostaglandin E2, prostaglandin D2, and thromboxane A2 were also reduced. These findings characterize what we believe is a novel inherited deficiency of cPLA2.

P2Y purinergic receptors previously have been shown to couple either to activation of phospholipase C through a pertussis toxin-insensitive mechanism or to inhibition of adenylyl cyclase through pertussis toxin-sensitive members of the G1 family of G proteins. These and other pharmacological data strongly suggest that multiple P2Y purinergic receptors exist. Webb et al. [FEBS Lett. 324:219-225 (1993)] cloned a cDNA that, when expressed in frog oocytes, displayed the general pharmacological characteristics of a P2Y purinergic receptor but whose second messenger linkage was not resolved. We have now cloned the meleagrid (turkey) homologue of the previously cloned chick P2Y purinergic receptor and have stably expressed it in a heterologous human cell line (1321N1 astrocytoma cells) to establish its signaling properties. The purinergic receptor agonist 2-methylthio-ATP (2MeSATP) stimulated a marked activation of phospholipase C in 1321N1 cells stably expressing the meleagrid receptor. The order of potency of a series of analogues of ATP and ADP for stimulation of phospholipase C by the receptor expressed in 1321N1 cells [2MeSATP = 2-methylthio-ADP>> adenosine 5'-O-(2-thio)diphosphate>> ADP>> 2-chloro-ATP = adenosine 5'-O-(3-thio)triphosphate>> or = ATP>> adenylyl-imidodiphosphate>> UTP] was similar to that observed for P2Y purinergic receptors in turkey erythrocytes and many other tissues and was markedly different from those of the P2U and P2X purinergic receptor subtypes. Stimulation of inositol lipid hydrolysis by P2Y purinergic agonists was not affected by preincubation of cells with pertussis toxin. In contrast to its marked effects on phospholipase C activity, 2MeSATP caused only a small and variable inhibition of cAMP accumulation. Ribonuclease protection analysis of turkey tissues showed that this P2Y purinergic receptor is most highly expressed in blood and brain. Taken together, these results indicate that a phospholipase-C-activating P2Y purinergic receptor has been cloned and stably expressed in 1321N1 astrocytoma cells.

Adenosine diphosphate-ribosylation factor (Arf) proteins are members of the Arf arm of the Ras superfamily of guanosine triphosphate (GTP)-binding proteins. Arfs are named for their activity as cofactors for cholera toxin-catalyzed adenosine diphosphate-ribosylation of the heterotrimeric G protein Gs. Physiologically, Arfs regulate membrane traffic and the actin cytoskeleton. Arfs function both constitutively within the secretory pathway and as targets of signal transduction in the cell periphery. In each case, the controlled binding and hydrolysis of GTP is critical to Arf function. The activities of some guanine nucleotide exchange factors (GEFs) and guanosine triphosphatase (GTPase)-activating proteins (GAPs) are stimulated by phosphoinositides, including phosphatidylinositol 3,4,5-trisphosphate (PIP3) and phosphatidylinositol 4,5-bisphosphate (PIP2), and phosphatidic acid (PA), likely providing both a means to respond to regulatory signals and a mechanism to coordinate GTP binding and hydrolysis. Arfs affect membrane traffic in part by recruiting coat proteins, including COPI and clathrin adaptor complexes, to membranes. However, Arf function likely involves many additional biochemical activities. Arf activates phospholipase D and phosphatidylinositol 4-phosphate 5-kinase with the consequent production of PA and PIP2, respectively. In addition to mediating Arf's effects on membrane traffic and the actin cytoskeleton, PA and PIP2 are involved in the regulation of Arf. Arf also works with Rho family proteins to affect the actin cytoskeleton. Several Arf-binding proteins suspected to be effectors have been identified in two-hybrid screens. Arf-dependent biochemical activities, actin cytoskeleton changes, and membrane trafficking may be integrally related. Understanding Arf's role in complex cellular functions such as protein secretion or cell movement will involve a description of the temporal and spatial coordination of these multiple Arf-dependent events.

OBJECTIVE

Our goal was to identify the role of oxidative stress via activation of NAD(P)H oxidase in cerebrovascular dysfunction in aged rats.

METHODS

We examined the reactivity of cerebral arterioles in adult and aged Fisher-344 rats to endothelial nitric oxide synthase (eNOS)-dependent (acetylcholine and adenosine diphosphate [ADP]) and-independent (nitroglycerin) agonists before and during application of tempol, apocynin, and diphenyleneiodonium chloride (DPI). We used Western blot to examine subunits of NAD(P)H oxidase, eNOS, and superoxide dismutase (SOD-1) in cerebral microvessels and parietal cortex. Finally, we measured superoxide production by cortex tissue in adult and aged rats.

RESULTS

Acetylcholine-and ADP-induced, but not nitroglycerin-induced, dilatation of cerebral arterioles was impaired in aged compared to adult rats. While tempol, apocynin, and DPI did not alter responses in adults, they alleviated impaired eNOS-dependent vasodilatation in aged rats, without influencing responses to nitroglycerin. eNOS and p67phox proteins were increased in cerebral microvessels from aged compared to adult rats. Further, p67phox and gp91phox proteins were increased, but SOD-1 protein was decreased, in cortex tissue of aged rats. Basal and agonist-induced production of superoxide was elevated in aged rats.

CONCLUSIONS

Aging impairs eNOS-dependent reactivity of cerebral arterioles via an increase in superoxide produced by activation of NAD(P)H oxidase.

Clostridium difficile is an important nosocomial pathogen and the most frequently diagnosed cause of infectious hospital-acquired diarrhoea. Toxigenic strains usually produce toxin A and toxin B, which are the primary virulence factors of C. difficile. Some recently described strains produce an additional toxin, an adenosine-diphosphate ribosyltransferase known as binary toxin, the role of which in pathogenicity is unknown. There has been concern about the emergence of a hypervirulent fluoroquinolone-resistant strain of C. difficile in North America and Europe. The use of fluoroquinolone antimicrobials appears to be acting as a selective pressure in the emergence of this strain. In this review, we describe the current state of knowledge about C. difficile as a cause of diarrhoeal illness.

Platelet integrin alpha IIb beta 3 (GPIIb/IIIa) plays a central role in the initiation of arterial thrombosis, but its contribution to disseminated microvascular thrombosis is less well defined. Therefore, wild-type mice (beta 3(+/+)), beta 3-integrin-deficient mice (beta 3(-/-)), and wild-type mice treated with a hamster monoclonal antibody (1B5) that blocks murine alpha IIb beta 3 function were tested in models of large-vessel and microvascular thrombosis. In the large-vessel model, ferric chloride was used to injure the carotid artery, and the time to thrombosis was measured. In beta 3(+/+) mice, the median time to occlusion was 6.7 minutes, whereas occlusion did not occur in any of the beta 3(-/-) mice tested (P <.001). Fab and F(ab')(2) fragments of 1B5 increased the median time to occlusion. To initiate systemic intravascular thrombosis, prothrombotic agents were administered intravenously, and platelet thrombus formation was monitored by the decrease in circulating platelet count. Three minutes after the injection of adenosine diphosphate (ADP), collagen + epinephrine, or tissue factor, the platelet counts in beta 3(+/+) mice decreased by 289, 424, and 429 x 10(3)/microL, respectively. beta 3(-/-) mice and wild-type mice pretreated with 1B5 Fab (1 mg/kg, IP) were nearly completely protected from the effects of ADP. In contrast, beta 3(-/-) mice were only partially protected from the effects of collagen + epinephrine and minimally protected from the effects of tissue factor. In all cases, less fibrin became deposited in the lungs of beta 3(-/-) mice than in wild-type mice. These results suggest that though alpha IIb beta 3 plays a dominant role in large-vessel thrombosis, it plays a variable role in systemic intravascular thrombosis. (Blood. 2001;98:1055-1062)

Resveratrol (trans-3,4,5-trihydroxystilbene) has received attention for its potential chemopreventive and antitumor effects in experimental systems. Recent evidence suggests that paclitaxel, alone or in combination with other drugs, can be effectively used in the treatment of non-Hodgkin's lymphoma (NHL) and multiple myeloma (MM). This study investigated whether resveratrol can sensitize NHL and MM cell lines to paclitaxel-mediated apoptosis and to delineate the underlying molecular mechanism of sensitization. Both resveratrol and paclitaxel negatively modulated tumor cell growth by arresting the cells at the G(2)-M phase of the cell cycle. Low concentrations of resveratrol exerted a sensitizing effect on drug-refractory NHL and MM cells to apoptosis induced by paclitaxel. Resveratrol selectively down-regulated the expression of antiapoptotic proteins Bcl-x(L) and myeloid cell differentiation factor-1 (Mcl-1) and up-regulated the expression of proapoptotic proteins Bax and apoptosis protease activating factor-1 (Apaf-1). Paclitaxel down-regulated the expression of Bcl-x(L), Mcl-1, and cellular inhibitor of apoptosis protein-1 antiapoptotic proteins and up-regulated Bid and Apaf-1. Combination treatment resulted in apoptosis through the formation of tBid, mitochondrial membrane depolarization, cytosolic release of cytochrome c and Smac/DIABLO, activation of the caspase cascade, and cleavage of poly(adenosine diphosphate-ribose) polymerase. Combination of resveratrol with paclitaxel had minimal cytotoxicity against quiescent and mitogenically stimulated human peripheral blood mononuclear cells. Inhibition of Bcl-x(L) expression by resveratrol was critical for chemosensitization and its functional impairment mimics resveratrol-mediated sensitization to paclitaxel-induced apoptosis. Inhibition of Bcl-x(L) expression by resveratrol was due to the inhibition of the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway and diminished activator protein-1-dependent Bcl-x(L) expression. The findings by resveratrol were corroborated with inhibitors of the ERK1/2 pathway. This study demonstrates that in resistant NHL and MM cell lines resveratrol and paclitaxel selectively modify the expression of regulatory proteins in the apoptotic signaling pathway and the combination, via functional complementation, results in synergistic apoptotic activity.

The BTK (Bruton's tyrosine kinase) inhibitor ibrutinib is associated with an increased risk of bleeding. A previous study reported defects in collagen- and adenosine diphosphate (ADP)-dependent platelet responses when ibrutinib was added ex vivo to patient samples. Whereas the collagen defect is expected given the central role of BTK in glycoprotein VI signaling, the ADP defect lacks a mechanistic explanation. In order to determine the real-life consequences of BTK platelet blockade, we performed light transmission aggregometry in 23 patients receiving ibrutinib treatment. All patients had reductions in collagen-mediated platelet aggregation, with a significant association between the degree of inhibition and the occurrence of clinical bleeding or bruising (P=0.044). This collagen defect was reversible on drug cessation. In contrast to the previous ex vivo report, we found no in vivo ADP defects in subjects receiving standard doses of ibrutinib. These results establish platelet light transmission aggregometry as a method for gauging, at least qualitatively, the severity of platelet impairment in patients receiving ibrutinib treatment.

Gene expression programs that regulate the abundance of the chaperone BiP adapt the endoplasmic reticulum (ER) to unfolded protein load. However, such programs are slow compared with physiological fluctuations in secreted protein synthesis. While searching for mechanisms that fill this temporal gap in coping with ER stress, we found elevated levels of adenosine diphosphate (ADP)-ribosylated BiP in the inactive pancreas of fasted mice and a rapid decline in this modification in the active fed state. ADP ribosylation mapped to Arg470 and Arg492 in the substrate-binding domain of hamster BiP. Mutations that mimic the negative charge of ADP-ribose destabilized substrate binding and interfered with interdomain allosteric coupling, marking ADP ribosylation as a rapid posttranslational mechanism for reversible inactivation of BiP. A kinetic model showed that buffering fluctuations in unfolded protein load with a recruitable pool of inactive chaperone is an efficient strategy to minimize both aggregation and costly degradation of unfolded proteins.

A new, improved procedure for isolating mitochondria from ascites tumor cells is described. The unique feature of this technique is the use of digitonin to make the cells susceptible to disruption by Teflon pestle/glass vessel homogenization. The yield and respiratory control ratios of mitochondria isolated by this method from murine Ehrlich ascites tumor cells and rat AS30-D ascites hepatoma cells are significantly better than those obtained for mitochondria isolated by the commonly employed Nagarse method, which involves the use of proteolytic enzymes. Moreover, mitochondria isolated by this new procedure from three different lines of tumors exhibit respiratory control ratios with both adenosine diphosphate and a respiratory uncoupler comparable to those obtained with mitochondria present in situ within digitonin-permeabilized tumor cells.

Three components of the chloroplast protein translocon, Tic110, Hsp93 (ClpC), and Tic40, have been shown to be important for protein translocation across the inner envelope membrane into the stroma. We show the molecular interactions among these three components that facilitate processing and translocation of precursor proteins. Transit-peptide binding by Tic110 recruits Tic40 binding to Tic110, which in turn causes the release of transit peptides from Tic110, freeing the transit peptides for processing. The Tic40 C-terminal domain, which is homologous to the C terminus of cochaperones Sti1p/Hop and Hip but with no known function, stimulates adenosine triphosphate hydrolysis by Hsp93. Hsp93 dissociates from Tic40 in the presence of adenosine diphosphate, suggesting that Tic40 functions as an adenosine triphosphatase activation protein for Hsp93. Our data suggest that chloroplasts have evolved the Tic40 cochaperone to increase the efficiency of precursor processing and translocation.

BACKGROUND

Blockade of platelet activation during primary percutaneous intervention for acute myocardial infarction is standard care to minimize stent thrombosis. To determine whether antiplatelet agents offer any direct cardioprotective effect, we tested whether they could modify infarction in a rabbit model of ischemia/reperfusion caused by reversible ligation of a coronary artery.

RESULTS

The P2Y₁₂ (adenosine diphosphate) receptor blocker cangrelor administered shortly before reperfusion in rabbits undergoing 30-minute regional ischemia/3-hour reperfusion reduced infarction from 38% of ischemic zone in control hearts to only 19%. Protection was dose dependent and correlated with the degree of inhibition of platelet aggregation. Protection was comparable to that seen with ischemic postconditioning (IPOC). Cangrelor protection, but not its inhibition of platelet aggregation, was abolished by the same signaling inhibitors that block protection from IPOC suggesting protection resulted from protective signaling rather than anticoagulation. As with IPOC, protection was lost when cangrelor administration was delayed until 10 minutes after reperfusion and no added protection was seen when cangrelor and IPOC were combined. These findings suggest both IPOC and cangrelor may protect by the same mechanism. No protection was seen when cangrelor was used in crystalloid-perfused isolated hearts indicating some component in whole blood is required for protection. Clopidogrel had a very slow onset of action requiring 2 days of treatment before platelets were inhibited, and only then the hearts were protected. Signaling inhibitors given just prior to reperfusion blocked clopidogrel's protection. Neither aspirin nor heparin was protective.

CONCLUSIONS

Clopidogrel and cangrelor protected rabbit hearts against infarction. The mechanism appears to involve signal transduction during reperfusion rather than inhibition of intravascular coagulation. We hypothesize that both drugs protect by activating IPOC's protective signaling to prevent reperfusion injury. If true, patients receiving P2Y₁₂ inhibitors before percutaneous intervention may already be postconditioned thus explaining failure of recent clinical trials of postconditioning drugs.

Multidrug therapy increases the risk for drug-drug interactions. Clopidogrel, a prodrug, requires hepatic cytochrome P450 (CYP) metabolic activation to produce the active metabolite that inhibits the platelet P2Y₁₂ adenosine diphosphate (ADP) receptor, decreasing platelet activation and aggregation processes. Atorvastatin, omeprazole, and several other drugs have been shown in pharmacodynamic studies to competitively inhibit CYP activation of clopidogrel, reducing clopidogrel responsiveness. Conversely, other agents increase clopidogrel responsiveness by inducing CYP activity. The clinical implications of these pharmacodynamic interactions have raised concern because many of these drugs are coadministered to patients with coronary artery disease. There are multiple challenges in proving that a pharmacodynamic drug-drug interaction is clinically significant. To date, there is no consistent evidence that clopidogrel-drug interactions impact adverse cardiovascular events. Statins and proton pump inhibitors have been shown to decrease adverse clinical event rates and should not be withheld from patients with appropriate indications for therapy because of concern about potential clopidogrel-drug interactions. Clinicians concerned about clopidogrel-drug interactions have the option of prescribing either an alternative platelet P2Y₁₂ receptor inhibitor without known drug interactions, or statin and gastro-protective agents that do not interfere with clopidogrel metabolism.

BACKGROUND

A growing number of observational studies suggest that high on-clopidogrel platelet reactivity (HPR) is associated with recurrent thrombotic events after percutaneous coronary intervention. We aimed to perform an updated systematic review and meta-analysis on the clinical relevance of HPR to summarize the available evidence and to define more precise effect estimates.

METHODS

Relevant observational studies published between January 2003 and February 2010 were searched that presented intent-to-treat analyses on the clinical relevance of HPR measured with an adenosine diphosphate (ADP)-specific platelet function assay. The main outcome measures were cardiovascular (CV) death, definite/probable stent thrombosis (ST), nonfatal myocardial infarction (MI), and a composite end point of reported ischemic events. The outcome parameters were pooled with the random-effect model via generic inverse variance weighting.

RESULTS

Twenty studies comprising a total number of 9,187 patients qualified. High on-clopidogrel platelet reactivity appeared to be a strong predictor of MI, ST, and the composite end point of reported ischemic events (odds ratios [95% CI]: 3.00 [2.26-3.99], 4.14 [2.74-6.25], and 4.95 [3.34-7.34], respectively; P < .00001 for all cases). According to the meta-analysis, patients with HPR had a 3.4-fold higher risk for CV death compared with patients with normal ADP reactivity (odds ratio 3.35, 95% CI 2.39-4.70, P < .00001). Although there were large differences in the methodology as well as in the definition of HPR between studies, the predicted risk for CV death, MI, or ST was not heterogeneous (I(2): 0%, 0%, and 12%, respectively; P = not significant for all cases).

CONCLUSIONS

High on-clopidogrel platelet reactivity, measured by an ADP-specific platelet function assay, is a strong predictor of CV death, MI, and ST in patients after percutaneous coronary intervention.