OBJECTIVE

To investigate the ability of resolvin E1 (RvE1) to regulate adenosine diphosphate (ADP) activation of platelets via specific receptors because RvE1 reduces platelet aggregation with certain agonists, including ADP.

RESULTS

RvE1 is an eicosapentaenoic acid-derived specialized proresolving mediator generated during the resolution of acute inflammation. RvE1 exhibits potent organ-protective actions in vivo and acts on specific cell types, including platelets. RvE1, 0.1 to 100 nmol/L, incubated with platelets gave reduced ADP-stimulated P-selectin mobilization (IC(50), approximately 1.6×10(-12) mol/L) and polymerized actin content compared with control platelets. RvE1, 1 to 100 nmol/L, did not stimulate or block intracellular Ca(2+) mobilization. By using a new P2Y(12)-β-arrestin-coupled cell system, ADP-activated P2Y(12) with an EC(50) of 5×10(-6) mol/L and RvE1 did not directly stimulate P2Y(12) or block the ADP-P2Y(12) signals. In this system, another eicosanoid, leukotriene E(4) (LTE(4)) (EC(50), 1.3×10(-11) mol/L), dose dependently activated P2Y(12). When recombinant P2Y(12)-expressing cells were transiently transfected with an RvE1 receptor, human ChemR23 (present on human platelets), with the addition of RvE1 (0.1-10.0 nmol/L), blocked ADP signals (IC(50), approximately 1.6×10(-11) mol/L) in P2Y(12)-ChemR23-expressing cells compared with mock transfections.

CONCLUSIONS

RvE1's regulatory actions (ie, reducing ADP-stimulated P-selectin mobilization and actin polymerization) are human (h)ChemR23-dependent. Moreover, specific platelet actions of RvE1 selectively engaged with ADP-activated platelets that illuminate a new cellular mechanism and affect ω-3 eicosapentaenoic acid, which may contribute to both resolution of vascular inflammation and ADP-dependent platelet activation relevant in pathological cardiovascular events.

Cyclic adenosine diphosphate ribose (cADPR) is a potent endogenous calcium-mobilizing agent synthesized from beta-NAD+ by ADP-ribosyl cyclases in sea urchin eggs and in several mammalian cells (Galione, A., and White, A. (1994) Trends Cell Biol. 4, 431 436). Pharmacological studies suggest that cADPR is an endogenous modulator of Ca2+-induced Ca2+ release mediated by ryanodine-sensitive Ca2+ release channels. An unresolved question is whether cADPR can act as a Ca2+-mobilizing intracellular messenger. We show that exogenous application of nitric oxide (NO) mobilizes Ca2+ from intracellular stores in intact sea urchin eggs and that it releases Ca2+ and elevates cADPR levels in egg homogenates. 8-Amino-cADPR, a selective competitive antagonist of cADPR-mediated Ca2+ release, and nicotinamide, an inhibitor of ADP-ribosyl cyclase, inhibit the Ca2+-mobilizing actions of NO, while, heparin, a competitive antagonist of the inositol 1,4,5-trisphosphate receptor, did not affect NO-induced Ca2+ release. Since the Ca2+-mobilizing effects of NO can be mimicked by cGMP, are inhibited by the cGMP-dependent-protein kinase inhibitor, Rp-8-pCPT-cGMPS, and in egg homogenates show a requirement for the guanylyl cyclase substrate, GTP, we suggest a novel action of NO in mobilizing intracellular calcium from microsomal stores via a signaling pathway involving cGMP and cADPR. These results suggest that cADPR has the capacity to act as a Ca2+-mobilizing intracellular messenger.

Abnormal mitochondria are an increasingly recognized cause of neuromuscular disease. We have used phosphorus magnetic resonance spectroscopy to monitor noninvasively the metabolism of high-energy phosphates and the intracellular pH of human skeletal muscle in vivo in 12 patients with mitochondrial myopathy. At rest, an abnormality could be demonstrated in 11 of 12 patients. Ten patients had evidence of a reduced muscle energy state with at least one of the following abnormalities: low phosphorylation potential, low phosphocreatine concentration, high adenosine diphosphate concentration, or high inorganic phosphate concentration. Two patients had abnormal resting muscle intracellular pH. In some patients phosphocreatine concentration decreased to low values during exercise despite limited work output. This was not accompanied by particularly severe intracellular acidosis. Evidence of impaired rephosphorylation of adenosine diphosphate to adenosine triphosphate during recovery from exercise was found in approximately half the patients. Phosphorus magnetic resonance spectroscopy is useful in the noninvasive diagnosis of mitochondrial myopathies and in defining the pathophysiological basis of these disorders.

Natural killer (NK) cells play crucial roles in innate immunity and express CD39 (Ecto-nucleoside triphosphate diphosphohydrolase 1 [E-NTPD1]), a rate-limiting ectonucleotidase in the phosphohydrolysis of extracellular nucleotides to adenosine. We have studied the effects of CD39 gene deletion on NK cells in dictating outcomes after partial hepatic ischemia/reperfusion injury (IRI). We show in mice that gene deletion of CD39 is associated with marked decreases in phosphohydrolysis of adenosine triphosphate (ATP) and adenosine diphosphate to adenosine monophosphate on NK cells, thereby modulating the type-2 purinergic (P2) receptors demonstrated on these cells. We note that CD39-null mice are protected from acute vascular injury after single-lobe warm IRI, and, relative to control wild-type mice, display significantly less elevation of aminotransferases with less pronounced histopathological changes associated with IRI. Selective adoptive transfers of immune cells into Rag2/common gamma null mice (deficient in T cells, B cells, and NK/NKT cells) suggest that it is CD39 deletion on NK cells that provides end-organ protection, which is comparable to that seen in the absence of interferon gamma. Indeed, NK effector mechanisms such as interferon gamma secretion are inhibited by P2 receptor activation in vitro. Specifically, ATPgammaS (a nonhydrolyzable ATP analog) inhibits secretion of interferon gamma by NK cells in response to interleukin-12 and interleukin-18, providing a mechanistic link between CD39 deletion and altered cytokine secretion.

CONCLUSIONS

We propose that CD39 deficiency and changes in P2 receptor activation abrogate secretion of interferon gamma by NK cells in response to inflammatory mediators, thereby limiting tissue damage mediated by these innate immune cells during IRI.

The involvement of P2Y receptors, which are activated by extracellular nucleotides, in proliferative regulation of human lung epithelial cells is unclear. Here we show that extracellular ATP and UTP stimulate bromodeoxyuridine (BrdU) incorporation into epithelial cell lines. The nucleotide efficacy profile [ATP = ADP>> UDP>>or= UTP>> adenosine>>or= 2-methylthioadenosine-5'-diphosphate, with alpha,beta-methylene adenosine 5'-triphosphate, 2',3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate, AMP, UMP, and ATPalphaS inactive] and PCR analysis indicate involvement of P2Y2 and P2Y6 receptors. The signal transduction pathway, which, via the P2Y2 receptor, transmits the proliferative activity of ATP or UTP in A549 cells downstream of phospholipase C, depends on Ca2+/calmodulin-dependent protein kinase II and nuclear factor-kappaB, but not on protein kinase C. Signaling does not involve the mitogen-activated protein kinases extracellular signal-regulated kinases-1 and -2, the phosphatidylinositol 3-kinase pathway, or Src kinases. Thus nucleotides regulate proliferation of human lung epithelial cells by a novel pathway. The stimulatory effect of UTP, but not ATP, in A549 cells is attenuated by preincubation with interleukin-1beta and interleukin-6, but not tumor necrosis factor-alpha. This indicates an important role for the pyrimidine-activated P2Y receptor in the inflammatory response of lung epithelia. ATP antagonizes the antiproliferative effect of the anticancer drugs paclitaxel and etoposide, whereas it enhances the activity of cisplatin about fourfold. Thus pathways activated by extracellular nucleotides differentially control proliferation of lung epithelial tumor cells.

We found that the interaction of platelets with immobilized von Willebrand factor (VWF) under flow induces distinct elevations of cytosolic Ca(++) concentration ([Ca(++)](i)) that are associated with sequential stages of integrin alpha(IIb)beta(3) activation. Fluid-dynamic conditions that are compatible with the existence of tensile stress on the bonds between glycoprotein Ibalpha (GPIbalpha) and the VWF A1 domain led to Ca(++) release from intracellular stores (type alpha/beta peaks), which preceded stationary platelet adhesion. Raised levels of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate, as well as membrane-permeable calcium chelators, inhibited these [Ca(++)](i) oscillations and prevented stable adhesion without affecting the dynamic characteristics of the typical platelet translocation on VWF mediated by GPIbalpha. Once adhesion was established through the integrin alpha(IIb)beta(3), new [Ca(++)](i) oscillations (type gamma) of greater amplitude and duration, and involving a transmembrane ion flux, developed in association with the recruitment of additional platelets into aggregates. Degradation of released adenosine diphosphate (ADP) to AMP or inhibition of phosphatidylinositol 3-kinase (PI3-K) prevented this response without affecting stationary adhesion and blocked aggregation. These findings indicate that an initial signal induced by stressed GPIbalpha-VWF bonds leads to alpha(IIb)beta(3) activation sufficient to support localized platelet adhesion. Then, additional signals from ADP receptors and possibly ligand-occupied alpha(IIb)beta(3), with the contribution of a pathway involving PI3-K, amplify platelet activation to the level required for aggregation. Our conclusions modify those proposed by others regarding the mechanisms that regulate signaling between GPIbalpha and alpha(IIb)beta(3) and lead to platelet adhesion and aggregation on immobilized VWF.

In an attempt to further define the site of myocardial adenosine formation, isolated guinea pig hearts were perfused with potent inhibitors of 5'-nucleotidase [alpha, beta-methylene adenosine 5'-diphosphate (AOPCP)] and of nucleoside transport [4-nitrobenzyl thioinosine (NBMPR)]. AOPCP (50 microM) inhibited the activity of cardiac ecto-5'-nucleotidase by 85% but did not influence the release of adenosine, inosine, and hypoxanthine formed at an accelerated rate by the heart during hypoxic perfusion (30% O2). In contrast, NBMPR (5 microM) diminished the hypoxia-induced release of adenosine and its degradatives and greatly potentiated the increase of myocardial tissue levels of respective purine compounds. Studies carried out with 5'-deoxyadenosine, an adenosine derivative that is not metabolized, indicate NBMPR to inhibit both uptake and release of adenosine in the isolated heart and in human erythrocytes. Cell fractionation studies on guinea pig ventricular muscle revealed that 5'-nucleotidase, though mainly associated with the membrane fraction, is also found in the cardiac cytosol (200,000-g supernatant), exhibiting a different substrate specificity. Furthermore, S-adenosylhomocysteine hydrolase as well as adenosine kinase and adenosine deaminase proved to be exclusively present in the cytosolic fraction. Our findings suggest that in the hypoxic heart a) ecto-5'-nucleotidase most likely is not involved in the formation of adenosine, b) release of adenosine from the heart requires adenosine to be transported across the sarcolemma membrane, and c) adenosine is predominantly formed intracellularly, a process involving cytosolic 5'-nucleotidase and/or S-adenosylhomocysteine hydrolase.

3-Aminobenzamide and benzamide, purported to be specific inhibitors of the synthesis of poly(adenosine diphosphate-ribose), were used to elucidate possible functions of this biopolymer. These compounds, at frequently used experimental concentrations, not only inhibited the action of poly(adenosine diphosphate-ribose) synthetase but also affected cell viability, glucose metabolism, and DNA synthesis. Thus, the usefulness of 3-aminobenzamide and benzamide may be severely restricted by the difficulty of finding a dose small enough to inhibit the synthetase without producing additional metabolic effects.

The kinetic properties of pyruvate kinase (ATP:pyruvate-phosphotransferase, EC 2.7.1.40) from Streptococcus lactis have been investigated. Positive homotropic kinetics were observed with phosphoenolpyruvate and adenosine 5'-diphosphate, resulting in a sigmoid relationship between reaction velocity and substrate concentrations. This relationship was abolished with an excess of the heterotropic effector fructose-1,6-diphosphate, giving a typical Michaelis-Menten relationship. Increasing the concentration of fructose-1,6-diphosphate increased the apparent V(max) values and decreased the K(m) values for both substrates. Catalysis by pyruvate kinase proceeded optimally at pH 6.9 to 7.5 and was markedly inhibited by inorganic phosphate and sulfate ions. Under certain conditions adenosine 5'-triphosphate also caused inhibition. The K(m) values for phosphoenolpyruvate and adenosine 5'-diphosphate in the presence of 2 mM fructose-1,6-diphosphate were 0.17 mM and 1 mM, respectively. The concentration of fructose-1,6-diphosphate giving one-half maximal velocity with 2 mM phosphoenolpyruvate and 5 mM adenosine 5'-diphosphate was 0.07 mM. The intracellular concentrations of these metabolites (0.8 mM phosphoenolpyruvate, 2.4 mM adenosine 5'-diphosphate, and 18 mM fructose-1,6-diphosphate) suggest that the pyruvate kinase in S. lactis approaches maximal activity in exponentially growing cells. The role of pyruvate kinase in the regulation of the glycolytic pathway in lactic streptococci is discussed.

Mouse bone marrow mast cells sensitized with monoclonal IgE and activated with specific antigen released 2.8 +/- 0.5 ng of platelet-activating factor (1-0-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine) (PAF-acether)/ 10(6) cells. The PAF-acether was identified by its ability to aggregate fully aspirin-treated washed rabbit platelets in the presence of an adenosine diphosphate (ADP)-scavenger complex, by its co-chromatography with [3H]-labeled semi-synthetic PAF-acether and synthetic 1-0-octadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine, and by its inactivation by phospholipases A2, C, and D and not by lipase A1. The antigen-initiated release of PAF-acether, leukotriene C4 (LTC4), and leukotriene B4 (LTB4), and the secretion of the granule marker beta-hexosaminidase were not diminished by washing the cells before challenge, indicating that they were due to the interaction of antigen with the IgE fixed on the cell membrane and not to phagocytosis of immune complexes formed in the fluid phase. The parallel antigen-induced dose-response relationship, along with the superimposable time-course of the extracellular appearance, of beta-hexosaminidase, PAF-acether, and both leukotrienes indicated that the origin of these diverse mediators was from a common cell type with IgE-Fc receptors. Ethanol extraction of antigen-stimulated bone marrow-derived mast cells revealed the early transient appearance of a cell-associated platelet-aggregating activity, the action of which on platelets, like PAF-acether, was independent of ADP and arachidonic acid metabolism. The cell-associated activity contained a novel product that eluted at 13 min during high performance liquid chromatography (HPLC) (solvent hexane:n-propanol:water, 46:46:8), permitting resolution from PAF-acether and lyso-PAF-acether (1-O-alkyl-sn-glyceryl-3-phosphorylcholine), which eluted at 29 min and 30 min, respectively. The cell-associated material, which differs from lyso-PAF-acether, the putative precursor of PAF-acether, in being active in the bioassay on platelets may represent a newly recognized intermediate in the generation of PAF-acether. As the transiently present cell-associated intermediate has not been previously recognized, its detection may depend upon the relatively unique properties of the bone marrow-derived mast cell system in which IgE-dependent activation leading to product generation is complete within 5 min.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine 5'-triphosphate or ATP is the primary energy source within the cell, releasing its energy via hydrolysis into adenosine 5'-diphosphate or ADP. Actin is an important ATPase involved in many aspects of cellular function, and the binding and hydrolysis of ATP regulates its polymerization into actin filaments as well as its interaction with a host of actin-associated proteins. Here we study the dynamics of monomeric actin in ATP, ADP-Pi, and ADP states via molecular dynamics simulations. As observed in some crystal structures we see that the DNase-I loop is an alpha-helix in the ADP state but forms an unstructured coil domain in the ADP-Pi and ATP states. We also find that this secondary structure change is reversible, and by mimicking nucleotide exchange we can observe the transition between the helical and coil states. Apart from the DNase-I loop, we also see several key structural differences in the nucleotide binding cleft as well as in the hydrophobic cleft between subdomains 1 and 3 where WH2-containing proteins have been shown to interact. These differences provide a structural basis for understanding the observed differences between the various nucleotide states of actin and provide some insight into how ATP regulates the interaction of actin with itself and other proteins.

The imidazole derivative UK-37 248, a thromboxane synthetase inhibitor, reduces the in-vitro formation of thromboxane B2 and hydroxyheptadecatrienoic acid by washed platelets, and this is compensated for by an increased production of prostaglandins E2 and F2 alpha; arachidonic acid challenged platelets pretreated with UK-37 248 also stimulate the production of prostacyclin by aspirin pretreated cultured endothelial cells. In a double-blind placebo controlled study to examine the in vivo properties of UK-37 248, human volunteers ingested 200 mg of the compound. Their serum thromboxane B2 levels dropped and their plasma 6-keto-prostaglandin F1 alpha values rose. Arachidonic acid induced platelet aggregation was completely inhibited whereas that elicited by adenosine-5'-diphosphate was unaffected. By reducing formation of pro-aggregatory tromboxane A2 and increasing production of anti-aggregatory prostacyclin, thromboxane synthetase inhibitors may be better than aspirin as antithrombotic agents.

BACKGROUND

Generation of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) may contribute to renal ischemia/reperfusion (I/R) injury. The aim of this study was to investigate the effects of GW274150, a novel, highly selective, potent and long-acting inhibitor of iNOS activity in rat and mouse models of renal I/R.

METHODS

Rats were administered GW274150 (5 mg/kg intravenous bolus administered 30 minutes prior to I/R) and subjected to bilateral renal ischemia (45 minutes) followed by reperfusion (6 hours). Serum and urinary indicators of renal dysfunction, tubular and reperfusion injury were measured, specifically, serum urea, creatinine, aspartate aminotransferase (AST) and N-acetyl-beta-d-glucosaminidase (NAG) enzymuria. In addition, renal sections were used for histologic scoring of renal injury and for immunologic evidence of nitrotyrosine formation and poly [adenosine diphosphate (ADP)-ribose] (PAR). Nitrate levels were measured in rat plasma using the Griess assay. Mice (wild-type, administered 5 mg/kg GW274150, and iNOS-/-) were subjected to bilateral renal ischemia (30 minutes) followed by reperfusion (24 hours) after which renal dysfunction (serum urea, creatinine), renal myeloperoxidase (MPO) activity and malondialdehyde (MDA) levels were measured.

RESULTS

GW274150, administered prior to I/R, significantly reduced serum urea, serum creatinine, AST, and NAG indicating reduction of renal dysfunction and injury caused by I/R. GW274150 reduced histologic evidence of tubular injury and markedly reduced immunohistochemical evidence of nitrotyrosine and PAR formation, indicating reduced peroxynitrite formation and poly (ADP-ribose) polymerase (PARP) activation, respectively. GW274150 abolished the rise in the plasma levels of nitrate (indicating reduced NO production). GW274150 also reduced the renal dysfunction in wild-type mice to levels similar to that observed in iNOS-/- mice subjected to I/R. Renal MPO activity and MDA levels were significantly reduced in wild-type mice administered GW274150 and iNOS-/- mice subjected to renal I/R, indicating reduced polymorphonuclear leukocyte (PMN) infiltration and lipid peroxidation.

CONCLUSIONS

These results suggest that (1). an enhanced formation of NO by iNOS contributes to the pathophysiology of renal I/R injury and (2). GW274150 reduces I/R injury of the kidney. We propose that selective inhibitors of iNOS activity may be useful against renal dysfunction and injury associated with I/R of the kidney.

Triple-negative breast cancer (TNBC) is defined by the lack of immunohistochemical expression of the estrogen and progesterone receptors and human epidermal growth factor receptor 2 (EGFR2). Most TNBC has a basal-like molecular phenotype by gene expression profiling and shares clinical and pathological features with hereditary BRCA1 related breast cancers. This review evaluates the activity of available chemotherapy and targeted agents in TNBC. A systematic review of PubMed and conference databases was carried out to identify randomised clinical trials reporting outcomes in women with TNBC treated with chemotherapy and targeted agents. Our review identified TNBC studies of chemotherapy and targeted agents with different mechanisms of action, including induction of synthetic lethality and inhibition of angiogenesis, growth and survival pathways. TNBC is sensitive to taxanes and anthracyclins. Platinum agents are effective in TNBC patients with BRCA1 mutation, either alone or in combination with poly adenosine diphosphate polymerase 1 inhibitors. Combinations of ixabepilone and capecitabine have added to progression-free survival (PFS) without survival benefit in metastatic TNBC. Antiangiogenic agents, tyrosine kinase inhibitors and EGFR inhibitors in combination with chemotherapy produced only modest gains in PFS and had little impact on survival. TNBC subgroups respond differentially to specific targeted agents. In future, the treatment needs to be tailored for a specific patient, depending on the molecular characteristics of their malignancy. TNBC being a chemosensitive entity, combination with targeted agents have not produced substantial improvements in outcomes. Appropriate patient selection with rationale combinations of targeted agents is needed for success.

We tested whether rat liver preservation performed by machine perfusion (MP) at 20 degrees C can enhance the functional integrity of steatotic livers versus simple cold storage. We also compared MP at 20 degrees C with hypothermic MP at 8 degrees C, and 4 degrees C. Obese and lean male Zucker rats were used as liver donors. MP was performed for 6 hours with a glucose and N-acetylcysteine-supplemented Krebs-Henseleit solution. Both MP and cold storage preserved livers were reperfused with Krebs-Henseleit solution (2 hours at 37 degrees C). MP at 4 degrees C and 8 degrees C reduced the fatty liver necrosis compared with cold storage but we further protected the organs using MP at 20 degrees C. Necrosis did not differ in livers from lean animals submitted to the different procedures; the enzymes released in steatotic livers preserved by MP at 20 degrees C were similar to those showed in nonsteatotic organs. The adenosine triphosphate/adenosine diphosphate ratio and bile production were higher and the oxidative stress and biliary enzymes were lower in steatotic livers preserved by MP at 20 degrees C as compared with cold storage. In livers from lean rats, the adenosine triphosphate/adenosine diphosphate ratio appears better conserved by MP at 20 degrees C as compared with cold storage. In steatotic livers preserved by cold storage, a 2-fold increase in tumor necrosis factor-alpha levels and caspase-3 activity was observed as compared with organs preserved by MP at 20 degrees C. These data are substantiated by better morphology, higher glycogen content, and lower reactive oxygen species production by sinusoidal cells in steatotic liver submitted to MP at 20 degrees C versus cold storage. MP at 20 degrees C improves cell survival and leads to a marked improvement in hepatic preservation of steatotic livers as compared with cold storage.

The binding of EF2 (elongation factor 2) and of ADP-ribosyl-EF 2 to rat liver ribosomes is inhibited by ricin. This result suggests that the native enzyme and its ADP-ribose derivative have the same or closely related binding sites on the ribosome. The inhibition by ricin of the binding of EF 2 to ribosomes is consistent with the previous observation that ricin affects EF 2-catalysed translocation during polypeptide chain elongation.

The properties of a form of ribulose diphosphate carboxylase having a high affinity for CO(2) have been studied. Its apparent Km(HCO(3) (-)) of 0.5 to 0.8 mm (pH 7.8) and calculated Km(CO(2)) of 11 to 18 mum are comparable to the values exhibited by intact chloroplasts during photosynthesis. This form of the enzyme was released from chloroplasts in hypotonic media and was unstable, rapidly converting to a form having a high Km(HCO(3) (-)) of 20 to 25 mm similar to that for the purified enzyme. Incubation of the enzyme with MgCl(2) and HCO(3) (-) yielded a third form with an intermediate Km(HCO(3) (-)) of 2.5 to 3.0 mm.The low Km form had sufficient activity both at air levels of CO(2) and at saturating CO(2) to account for the rates of photosynthesis by intact chloroplasts. The low Km form could be stabilized in the presence of ribose 5-phosphate, adenosine triphosphate, and MgCl(2), at low temperatures for up to 2 hours.

Böck, August (Purdue University, Lafayette, Ind.), and Frederick C. Neidhardt. Properties of a mutant of Escherichia coli with a temperature-sensitive fructose-1,6-diphosphate aldolase. J. Bacteriol. 92:470-476. 1966.-A mutant of Escherichia coli in which fructose-1,6-diphosphate aldolase functions at 30 C but not at 40 C was used to study the physiological effect of a specific block in the Embden-Meyerhof glycolytic pathway. Growth of the mutant at 40 C was found to be inhibited by the presence of glucose or certain related compounds in the medium. At 40 C, glucose was metabolized at 30 to 40% of the control rate and was abnormal in that glucose was converted into other six-carbon substances (probably gluconate, in large part) that were released into the culture medium. The inhibition was complete, but transient; its duration depended upon the initial amount of inhibitor added. The resumption of growth at 40 C was correlated with the further catabolism of the excreted compounds. When glycerol was used to grow the mutant at 40 C, the growth inhibition by glucose was accompanied by cessation of glycerol metabolism. Growth on alpha-glycerol phosphate was not inhibited under these conditions, implicating glycerol kinase as a possible site of inhibition; no inhibition of glycerol kinase by sugar phosphates, however, could be detected in vitro. The inhibitory effect of glucose on growth at 40 C is not caused by a deficit of intracellular adenosine triphosphate, but may be the result of a generalized poisoning of many cell processes by a greatly increased intracellular concentration of fructose-1,6-diphosphate, the substrate of the damaged enzyme.

BACKGROUND

Rac1 GTPase, a member of the Ras-related Rho GTPase family, is the major Rac isoform present in platelets and has been shown to be involved in cell actin cytoskeleton reorganization and adhesion. Agonists that induce platelet secretion and aggregation also activate Rac1 GTPase, raising the possibility that Rac1 GTPase may be involved in regulation of platelet function.

OBJECTIVE

To rigorously define the role of Rac1 in platelet regulation.

METHODS

We have used a dual approach of gene targeting in mice and pharmacologic inhibition of Rac1 by NSC23766, a rationally designed specific small molecule inhibitor, to study the role of Rac1 in platelet function.

RESULTS

Platelets from mice as well as human platelets treated with NSC23766 exhibited a significant decrease in: (i) active Rac1 species and phosphorylation of the Rac effector, p21-activated kinase; (ii) expression of P-selectin and secretion of adenosine triphosphate induced by thrombin or U46619; and (iii) aggregation induced by adenosine 5'-diphosphate, collagen, thrombin and U46619, a stable analog of thromboxane A(2). NSC23766 did not alter the cAMP or cGMP levels in platelets. Consistent with the requirement of Rac1 for normal platelet function, the bleeding times in Rac1(-/-) mice or mice given NSC23766 were significantly prolonged.

CONCLUSIONS

Our data show that deficiency or inhibition of Rac1 GTPase blocks platelet secretion. The inhibition of secretion, at least in part, is responsible for diminished platelet aggregation and prolonged bleeding times observed in Rac1 knockout or Rac1 inhibitor-treated mice.

Within the healthy population, there is substantial, heritable, and interindividual variability in the platelet response. We explored whether a proportion of this variability could be accounted for by interindividual variation in gene expression. Through a correlative analysis of genome-wide platelet RNA expression data from 37 subjects representing the normal range of platelet responsiveness within a cohort of 500 subjects, we identified 63 genes in which transcript levels correlated with variation in the platelet response to adenosine diphosphate and/or the collagen-mimetic peptide, cross-linked collagen-related peptide. Many of these encode proteins with no reported function in platelets. An association study of 6 of the 63 genes in 4235 cases and 6379 controls showed a putative association with myocardial infarction for COMMD7 (COMM domain-containing protein 7) and a major deviation from the null hypo thesis for LRRFIP1 [leucine-rich repeat (in FLII) interacting protein 1]. Morpholino-based silencing in Danio rerio identified a modest role for commd7 and a significant effect for lrrfip1 as positive regulators of thrombus formation. Proteomic analysis of human platelet LRRFIP1-interacting proteins indicated that LRRFIP1 functions as a component of the platelet cytoskeleton, where it interacts with the actin-remodeling proteins Flightless-1 and Drebrin. Taken together, these data reveal novel proteins regulating the platelet response.

Human platelets express 2 G protein-coupled nucleotide receptors: the platelet adenosine diphosphate (ADP) receptor coupled to stimulation of phospholipase C (P2Y(1)) via heterotrimeric guanosine 5-triphosphate (GTP)-binding protein G(q), and the platelet ADP receptor coupled to inhibition of adenylyl cyclase (P2Y(12)) via heterotrimeric GTP-binding protein G(i). Although these 2 receptors are encoded on the same chromosome and have similar pharmacologic profiles, they have different reactivities toward thiol reagents. The thiol agent p-chloromercuribenzene sulfonic acid (pCMBS) and the active metabolites from antiplatelet drugs, clopidogrel and CS-747, inactivate the P2Y(12) receptor and are predicted to interact with the extracellular cysteine residues on the P2Y(12) receptor. In this study we identified the reactive cysteine residues on the human P2Y(12) receptor by site-directed mutagenesis using pCMBS as the thiol reagent. Cys97Ser and Cys175Ser mutants of the P2Y(12) receptor did not express when transfected into Chinese hamster ovary (CHO-K1) cells, indicating the essential nature of a disulfide bridge between these residues. The Cys17Ser, Cys270Ser, and Cys17Ser/Cys270Ser double mutants had similar median effective concentration (EC(50)) values for ADP and 2-methylthio-ADP (2-MeSADP) when compared with the wild-type P2Y(12). Similarly, the median inhibitory concentration (IC(50)) values for BzATP (2',3'-O-(4- benzoylbenzoyl) adenosine 5'-triphosphate), an antagonist of the P2Y(12) receptor, also did not differ dramatically among these mutants and the wild-type P2Y(12) receptor. pCMBS inactivated the wild-type P2Y(12) receptor in a concentration-dependent manner, whereas it had no effect on the P2Y(1) receptor. Finally, pCMBS partially affected the G(i) coupling of Cys17Ser or Cys270Ser receptor mutants, but had no effect on Cys17Ser/Cys270Ser P2Y(12) receptor-mediated inhibition of adenylyl cyclase. These results indicate that, unlike the P2Y(1) receptor, which has 2 essential disulfide bridges linking its extracellular domains, the P2Y(12) receptor has 2 free cysteines in its extracellular domains (Cys17 and Cys270), both of which are targets of thiol reagents. We speculate that the active metabolites of clopidogrel and CS-747 form disulfide bridges with both Cys17 and Cys270 in the P2Y(12) receptor, and thereby inactivate the receptor.

The cochaperone GrpE functions as a nucleotide exchange factor to promote dissociation of adenosine 5'-diphosphate (ADP) from the nucleotide-binding cleft of DnaK. GrpE and the DnaJ cochaperone act in concert to control the flux of unfolded polypeptides into and out of the substrate-binding domain of DnaK by regulating the nucleotide-bound state of DnaK. DnaJ stimulates nucleotide hydrolysis, and GrpE promotes the exchange of ADP for adenosine triphosphate (ATP) and also augments peptide release from the DnaK substrate-binding domain in an ATP-independent manner. The eukaryotic cytosol does not contain GrpE per se because GrpE-like function is provided by the BAG1 protein, which acts as a nucleotide exchange factor for cytosolic Hsp70s. GrpE, which plays a prominent role in mitochondria, chloroplasts, and bacterial cytoplasms, is a fascinating molecule with an unusual quaternary structure. The long alpha-helices of GrpE have been hypothesized to act as a thermosensor and to be involved in the decrease in GrpE-dependent nucleotide exchange that is observed in vitro at temperatures relevant to heat shock. This review describes the molecular biology of GrpE and focuses on the structural and kinetic aspects of nucleotide exchange, peptide release, and the thermosensor hypothesis.