Interleukin-6 signaling drives fibrosis in unresolved inflammation.

Fibrosis in response to tissue damage or persistent inflammation is a pathological hallmark of many chronic degenerative diseases. By using a model of acute peritoneal inflammation, we have examined how repeated inflammatory activation promotes fibrotic tissue injury. In this context, fibrosis was strictly dependent on interleukin-6 (IL-6). Repeat inflammation induced IL-6-mediated T helper 1 (Th1) cell effector commitment and the emergence of STAT1 (signal transducer and activator of transcription-1) activity within the peritoneal membrane. Fibrosis was not observed in mice lacking interferon-γ (IFN-γ), STAT1, or RAG-1. Here, IFN-γ and STAT1 signaling disrupted the turnover of extracellular matrix by metalloproteases. Whereas IL-6-deficient mice resisted fibrosis, transfer of polarized Th1 cells or inhibition of MMP activity reversed this outcome. Thus, IL-6 causes compromised tissue repair by shifting acute inflammation into a more chronic profibrotic state through induction of Th1 cell responses as a consequence of recurrent inflammation.

Targeting the TLR co-receptor CD14 with TLR2-derived peptides modulates immune responses to pathogens.

Dysregulation of Toll-like receptor (TLR) responses to pathogens can lead to pathological inflammation or to immune hyporesponsiveness and susceptibility to infections, and may affect adaptive immune responses. TLRs are therefore attractive therapeutic targets. We assessed the potential of the TLR co-receptor CD14 as a target for therapeutics by investigating the magnitude of its influence on TLR responses. We studied the interaction of CD14 with TLR2 by conducting peptide screening and site-directed mutagenesis analysis and found TLR2 leucine-rich repeats 5, 9, 15, and 20 involved in interaction with CD14. Peptides representing these regions interacted with CD14 and enhanced TLR2- and TLR4-mediated proinflammatory responses to bacterial pathogens in vitro. Notably, the peptides' immune boosting capacity helped to rescue proinflammatory responses of immunosuppressed sepsis patients ex vivo. In vivo, peptide treatment increased phagocyte recruitment and accelerated bacterial clearance in murine models of Gram-negative and Gram-positive bacterial peritonitis. Up-modulating CD14's co-receptor activity with TLR2-derived peptides also enhanced antigen-induced dendritic cell (DC) maturation and interleukin-2 production and, most notably, differentially affected DC cytokine profile upon antigen stimulation, promoting a T helper 1-skewed adaptive immune response. Biochemical, cell imaging, and molecular docking studies showed that peptide binding to CD14 accelerates microbial ligand transfer from CD14 to TLR2, resulting in increased and sustained ligand occupancy of TLR2 and receptor clustering for signaling. These findings reveal the influence that CD14 exerts on TLR activities and describe a potential therapeutic strategy to amplify responses to different pathogens mediated by different TLRs by targeting the common TLR co-receptor, CD14.

TLR activation enhances C5a-induced pro-inflammatory responses by negatively modulating the second C5a receptor, C5L2.

TLR and complement activation ensures efficient clearance of infection. Previous studies documented synergism between TLRs and the receptor for the pro-inflammatory complement peptide C5a (C5aR/CD88), and regulation of TLR-induced pro-inflammatory responses by C5aR, suggesting crosstalk between TLRs and C5aR. However, it is unclear whether and how TLRs modulate C5a-induced pro-inflammatory responses. We demonstrate a marked positive modulatory effect of TLR activation on cell sensitivity to C5a in vitro and ex vivo and identify an underlying mechanistic target. Pre-exposure of PBMCs and whole blood to diverse TLR ligands or bacteria enhanced C5a-induced pro-inflammatory responses. This effect was not observed in TLR4 signalling-deficient mice. TLR-induced hypersensitivity to C5a did not result from C5aR upregulation or modulation of C5a-induced Ca(2+) mobilization. Rather, TLRs targeted another C5a receptor, C5L2 (acting as a negative modulator of C5aR), by reducing C5L2 activity. TLR-induced hypersensitivity to C5a was mimicked by blocking C5L2 and was not observed in C5L2KO mice. Furthermore, TLR activation inhibited C5L2 expression upon C5a stimulation. These findings identify a novel pathway of crosstalk within the innate immune system that amplifies innate host defense at the TLR-complement interface. Unravelling the mutually regulated activities of TLRs and complement may reveal new therapeutic avenues to control inflammation.

Esterified eicosanoids are acutely generated by 5-lipoxygenase in primary human neutrophils and in human and murine infection.

5-Lipoxygenase (5-LOX) plays key roles in infection and allergic responses. Herein, four 5-LOX-derived lipids comprising 5-hydroxyeicosatetraenoic acid (HETE) attached to phospholipids (PLs), either phosphatidylethanolamine (PE) or phosphatidylcholine (18:0p/5-HETE-PE, 18:1p/5-HETE-PE, 16:0p/5-HETE-PE, and 16:0a/5-HETE-PC), were identified in primary human neutrophils. They formed within 2 minutes in response to serum-opsonized Staphylococcus epidermidis or f-methionine-leucine-phenylalanine, with priming by lipopolysaccharide, granulocyte macrophage colony-stimulating factor, or cytochalasin D. Levels generated were similar to free 5-HETE (0.37 ± 0.14 ng vs 0.55 ± 0.18 ng/10(6) cells, esterified vs free 5-HETE, respectively). They remained cell associated, localizing to nuclear and extranuclear membrane, and were formed by fast esterification of newly synthesized free 5-HETE. Generation also required Ca(2+), phospholipase C, cytosolic and secretory phospholipase A(2), 5-LOX activating protein, and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1. 5-HETE-PLs were detected in murine S epidermidis peritonitis, paralleling neutrophil influx, and in effluent from Gram-positive human bacterial peritonitis. Formation of neutrophil extracellular traps was significantly enhanced by 5-LOX inhibition but attenuated by HETE-PE, whereas 5-HETE-PE enhanced superoxide and interleukin-8 generation. Thus, new molecular species of oxidized PL formed by human neutrophils during bacterial infection are identified and characterized.

Phosphatidylethanolamine-esterified eicosanoids in the mouse: tissue localization and inflammation-dependent formation in Th-2 disease.

In this study, murine peritoneal macrophages from naïve lavage were found to generate four phospholipids that contain 12-hydroxyeicosatetraenoic acid (12-HETE). They comprise three plasmalogen and one diacyl phosphatidylethanolamines (PEs) (16:0p, 18:1p, 18:0p, and 18:0a at sn-1) and are absent in macrophages from 12/15-lipoxygenase (12/15-LOX)-deficient mice. They are generated acutely in response to calcium mobilization, are primarily cell-associated, and are detected on the outside of the plasma membrane. Levels of 12-HETE-PEs in naïve lavage are in a similar range to those of free 12-HETE (5.5 +/- 0.2 ng or 18.5 +/- 1.03 ng/lavage for esterified versus free, respectively). In healthy mice, 12/15-LOX-derived 12-HETE-PEs are found in the peritoneal cavity, peritoneal membrane, lymph node, and intestine, with a similar distribution to 12/15-LOX-derived 12-HETE. In vivo generation of 12-HETE-PEs occurs in a Th2-dependent model of murine lung inflammation associated with interleukin-4/interleukin-13 expression. In contrast, in Toll receptor-dependent peritonitis mediated either by live bacteria or bacterial products, 12-HETE-PEs are rapidly cleared during the acute phase then reappear during resolution. The human homolog, 18:0a/15-HETE-PE inhibited human monocyte generation of cytokines in response to lipopolysaccharide. In summary, a new family of lipid mediators generated by murine macrophages during Th2 inflammation are identified and structurally characterized. The studies suggest a new paradigm for lipids generated by 12/15-LOX in inflammation involving formation of esterified eicosanoids.

Soluble TLR2 reduces inflammation without compromising bacterial clearance by disrupting TLR2 triggering.

TLR overactivation may lead to end organ damage and serious acute and chronic inflammatory conditions. TLR responses must therefore be tightly regulated to control disease outcomes. We show in this study the ability of the soluble form of TLR2 (sTLR2) to regulate proinflammatory responses, and demonstrate the mechanisms underlying sTLR2 regulatory capacity. Cells overexpressing sTLR2, or stimulated in the presence of the sTLR2 protein, are hyporesponsive to TLR2 ligands. Regulation was TLR2 specific, and affected NF-kappaB activation, phagocytosis, and superoxide production. Natural sTLR2-depleted serum rendered leukocytes hypersensitive to TLR2-mediated stimulation. Mice administered sTLR2 together with Gram-positive bacteria-derived components showed lower peritoneal levels of the neutrophil (PMN) chemoattractant, keratinocyte-derived chemokine; lower PMN numbers; and a reduction in late apoptotic PMN. Mononuclear cell recruitment remained unaffected, and endogenous peritoneal sTLR2 levels increased. Notably, the capacity of sTLR2 to modulate acute inflammatory parameters did not compromise the ability of mice to clear live Gram-positive bacteria-induced infection. Mechanistically, sTLR2 interfered with TLR2 mobilization to lipid rafts for signaling, acted as a decoy microbial receptor, and disrupted the interaction of TLR2 with its coreceptor, CD14, by associating with CD14. These findings establish sTLR2 as a regulator of TLR2-mediated inflammatory responses, capable of blunting immune responses without abrogating microbial recognition and may inform the design of novel therapeutics against acute and chronic inflammatory conditions.

Circulating neutrophils maintain physiological blood pressure by suppressing bacteria and IFNgamma-dependent iNOS expression in the vasculature of healthy mice.

Whether leukocytes exert an influence on vascular function in vivo is not known. Here, genetic and pharmacologic approaches show that the absence of neutrophils leads to acute blood pressure dysregulation. Following neutrophil depletion, systolic blood pressure falls significantly over 3 days (88.0 +/- 3.5 vs 104.0 +/- 2.8 mm Hg, day 3 vs day 0, mean +/- SEM, P < .001), and aortic rings from neutropenic mice do not constrict properly. The constriction defect is corrected using l-nitroarginine-methyl ester (L-NAME) or the specific inducible nitric oxide synthase (iNOS) inhibitor 1400W, while acetylcholine relaxation is normal. iNOS- or IFNgamma-deficient mice are protected from neutropenia-induced hypotension, indicating that iNOS-derived nitric oxide (NO) is responsible and that its induction involves IFNgamma. Oral enrofloxacin partially inhibited hypotension, implicating bacterial products. Roles for cyclooxygenase, complement C5, or endotoxin were excluded, although urinary prostacyclin metabolites were elevated. Neutrophil depletion required complement opsinization, with no evidence for intravascular degranulation. In summary, circulating neutrophils contribute to maintaining physiological tone in the vasculature, at least in part through suppressing early proinflammatory effects of infection. The speed with which hypotension developed provides insight into early changes that occur in the absence of neutrophils and illustrates the importance of constant surveillance of mucosal sites by granulocytes in healthy mice.

Classic interleukin-6 receptor signaling and interleukin-6 trans-signaling differentially control angiotensin II-dependent hypertension, cardiac signal transducer and activator of transcription-3 activation, and vascular hypertrophy in vivo.

Interleukin (IL)-6 acts via a receptor complex consisting of the cognate IL-6 receptor (IL-6R) or the soluble IL-6 receptor (sIL-6R) and glycoprotein 130 (gp130). Here, we investigated the role of these IL-6R components in hypertension and vascular hypertrophy in mice. Angiotensin (Ang) II (1.1 mg/kg/day) caused hypertension and cardiac/aortic hypertrophy in wild-type, but not IL-6(-/-), mice throughout 7 days. A recombinant dimeric soluble gp130 (sgp130Fc; 50 to 100 microg, i.p.) blocked Ang II hypertension but not hypertrophy in wild-type mice. Cognate IL-6R was detected in aortic smooth muscle, but its levels and those of plasma sIL-6R were approximately 50% decreased in IL-6(-/-) mice. Ang II infusion activated signal transducer and activator of transcription-3 in heart of WT and decreased Ang II receptor 1 (ATR1) expression in aorta. Both responses were unaffected by sgp130Fc and absent in IL-6(-/-) mice. In summary, we show that IL-6 trans-signaling is required for Ang II-dependent hypertension, but that hypertrophy, down-regulation of AT1R, and cardiac signal transducer and activator of transcription-3 activation are mediated via cognate IL-6R. These data show that IL-6 responses in a single disease context are governed by both modes of IL-6 signaling, with each pathway eliciting different outcomes. Inhibition of IL-6 signaling is suggested as a potential therapy for hypertension and cardiac hypertrophy.

Activated platelets and monocytes generate four hydroxyphosphatidylethanolamines via lipoxygenase.

12/15-Lipoxygenase (LOX) mediates immune-regulatory activities not accounted for by its known free acid eicosanoids, suggesting that additional lipids may be generated by activated cells. To characterize novel LOX-derived lipids, a lipidomic approach was utilized. Ionophore-activated interleukin-4-treated human peripheral monocytes generated up to 10-fold more esterified 15-hydroxyeicosatetraenoic acid (15-HETE) than free in a phosphatidylinositol 3-kinase- and protein kinase C-sensitive manner. Precursor scanning electrospray ionization/tandem spectroscopy for m/z 319 (HETE, [M-H](-)) showed 4 ions at m/z 738, 764, 766, and 782 that were identified using tandem spectroscopy and MS3 as specific diacyl and plasmalogen 15-HETE phosphatidylethanolamines. Using H (18)(2)O water, the compounds were shown to form by direct oxidation of endogenous phosphatidylethanolamine (PE) by 15-LOX, with PE being the preferred phospholipid pool containing 15-HETE. Similarly, human platelets generated 4 analogous PE lipids that contained 12-HETE and increased significantly in response to ionophore, collagen, or convulxin. These products were retained in the cells, in contrast to free acids, which are primarily secreted. Precursor scanning of platelet extracts for the major platelet-derived prostanoid, thromboxane B2 (m/z 369.2), did not reveal PE esters, indicating that this modification is restricted to the LOX pathway. In summary, we show formation of PE-esterified HETEs in immune cells that may contribute to LOX signaling in inflammation.

CD59 or C3 are not requred for angiotensin II-dependent hypertension or hypertrophy in mice.

Complement is a major pro-inflammatory innate immune system whose serum activity correlates with systolic blood pressure in humans. To date, no studies using in vivo models have directly examined the role of individual complement components in regulating vessel function, hypertension and cardiac hypertrophy. Herein, in vivo responses to angiotensin (ang) II were characterized in mice deficient in CD59a or C3. CD59a(-/-) mice had slightly but significantly elevated systolic blood pressure (107.2 +/- 1.7 mmHg versus 113.8 +/- 1.31 mmHg, P < 0.01, for wild-type and CD59a(-/-), respectively). Aortic rings from CD59a(-/-) mice showed significantly less platelet endothelial cell adhesion molecule-1 (PECAM-1) expression, with elevated deposition of membrane attack complex. However, acetylcholine- and sodium nitroprusside-dependent dilatation, plasma nitrate/nitrite and aortic cyclic guanosine monophosphate levels were unchanged from wild-type. Also, in vivo infusion with either ang II or noradrenaline caused similar hypertension and vascular hypertrophy to wild-type. Mice deficient in C3 had similar basal blood pressure to wild type and showed no differences in hypertension or hypertrophy responses to in vivo infusion with ang II. These data indicate that CD59a deficiency is associated with some vascular alterations that may represent early damage occurring as a result of increased complement attack. However, a direct role for CD59a or C3 in modulating development of ang II-dependent hypertension or hypertrophy in vivo is excluded and we suggest caution in development of complement intervention strategies for hypertension and heart failure.

Nitric oxide deficiency promotes vascular side effects of cyclooxygenase inhibitors.

The cardiovascular safety of COX-2 selective and nonselective nonsteroidal anti-inflammatory drugs (NSAIDs) has recently been called into question. The factors that predispose to adverse events by NSAIDs are unknown. Because patients with arthritis have decreased nitric oxide (NO) bioavailability, the in vivo effects of NSAIDs on murine vascular tone and platelet activity in the presence or absence of NO were examined. Here, we show that acute hypertensive and prothrombotic activities of the COX-2-selective inhibitor celecoxib are revealed only after in vivo inhibition of NO generation. The nonselective NSAID indomethacin was hypertensive but antithrombotic when NO was absent. In vitro myography of aortic rings confirmed that vasoconstriction required inhibition of both NOS and COX-2 and was abolished by supplementation with exogenous NO. These data indicate that NO suppresses vascular side effects of NSAIDs, suggesting that risk will be greatest in patients with impaired vascular function associated with decreased NO bioavailability.

In vivo aspirin supplementation inhibits nitric oxide consumption by human platelets.

Antiplatelet therapies improve endothelial function in atherosclerosis, suggesting that platelets regulate vascular nitric oxide (NO) bioactivity in vivo. Herein, washed platelets consumed NO on activation in an aspirin-sensitive manner, and aspirin enhanced platelet NO responses in vitro. To examine whether in vivo aspirin can inhibit platelet NO consumption, a double-blind placebo-controlled study was conducted. After a 2-week nonsteroidal anti-inflammatory drug (NSAID)-free period, healthy men were randomly assigned and administered aspirin (75 mg/d orally) or identical placebo for 14 days, then crossed over to the opposite arm. Following in vivo aspirin, NO consumption by platelets was inhibited 91%. Rate of onset and recovery following aspirin withdrawal was consistent with cyclooxygenase 1 (COX-1) inhibition. In a small substudy, NO consumption by platelets from postmenopausal women was faster in hypercholesterolemics and less sensitive to aspirin (ie, 39% versus 76% inhibition for hypercholesterolemics or normocholesterolemics, respectively). However, 150 mg aspirin/day increased inhibition of NO consumption by platelets of hypercholesterolemics to 80%. Comparisons of platelet COX-1 or -2 expression and urinary 11-dehydro-thromboxane B2 excretion suggested that aspirin was less able to block platelet activation in vivo in hypercholesterolemia. In conclusion, aspirin inhibits NO consumption by platelets from healthy subjects, but its beneficial effects on NO bioactivity may be compromised in some hypercholesterolemic patients.

Elevated endothelial nitric oxide bioactivity and resistance to angiotensin-dependent hypertension in 12/15-lipoxygenase knockout mice.

12/15-Lipoxygenase (12/15-LOX) plays a pathogenic role in atherosclerosis. To characterize whether 12/15-LOX also contributes to endothelial dysfunction and hypertension, regulation of vessel tone and angiotensin II (ang II) responses were characterized in mice deficient in 12/15-LOX. There was a twofold increase in the magnitude of l-nitroarginine-methyl ester-inhibitable, acetylcholine-dependent relaxation or phenylephrine-dependent constriction in aortic rings isolated from 12/15-LOX(-/-) mice. Plasma NO metabolites and aortic endothelial NO synthase (eNOS) expression were also elevated twofold. Angiotensin II failed to vasoconstrict 12/15-LOX(-/-) aortic rings in the absence of L-nitroarginine-methyl ester, and ang II impaired acetylcholine-induced relaxation in wild-type, but not 12/15-LOX(-/-) rings. In vivo, 12/15-LOX(-/-) mice had similar basal systolic blood pressure measurements to wild type, however, blood pressure elevations in response to ang II infusion (1.1 mg/kg/day) were significantly attenuated (maximal pressure, 143.4 +/- 4 mmHg versus 122.1 +/- 5.3 mmHg for wild type and 12/15-LOX(-/-), respectively). In contrast, vascular hypertrophic responses to ang II, and ang II type 1 receptor (AT1-R) expression were similar in both strains. This study shows that 12/15-LOX(-/-) mice have increased NO biosynthesis and impaired ang II-dependent vascular responses in vitro and in vivo, suggesting that 12/15-LOX signaling contributes to impaired NO bioactivity in vascular disease in vivo.

Interactions of 12-lipoxygenase with phospholipase A2 isoforms following platelet activation through the glycoprotein VI collagen receptor.

Recent studies implicate the collagen receptor, glycoprotein VI (GPVI) in activation of platelet 12-lipoxygenase (p12-LOX). Herein, we show that GPVI-stimulated 12-hydro(peroxy)eicosatetraenoic acid (H(P)ETE) synthesis is inhibited by palmityl trifluromethyl ketone or oleyloxyethylphosphocholine , but not bromoenol lactone, implicating secretory and cytosolic, but not calcium-independent phospholipase A2 (PLA2) isoforms. Also, following GPVI activation, 12-LOX co-immunoprecipitates with both cytosolic and secretory PLA2 (sPLA2). Finally, venoms containing sPLA2 acutely activate p12-LOX in a dose-dependent manner. This study shows that platelet 12-H(P)ETE generation utilizes arachidonate substrate from both c- and sPLA2 and that 12-LOX functionally associates with both PLA2 isoforms.

Platelet 12-lipoxygenase activation via glycoprotein VI: involvement of multiple signaling pathways in agonist control of H(P)ETE synthesis.

Lipoxygenases (LOX) contribute to vascular disease and inflammation through generation of bioactive lipids, including 12-hydro(pero)xyeicosatetraenoic acid (12-H(P)ETE). The physiological mechanisms that acutely control LOX product generation in mammalian cells are uncharacterized. Human platelets that contain a 12-LOX isoform (p12-LOX) were used to define pathways that activate H(P)ETE synthesis in the vasculature. Collagen and collagen-related peptide (CRP) (1 to 10 microg/mL) acutely induced platelet 12-H(P)ETE synthesis. This implicated the collagen receptor glycoprotein VI (GPVI), which signals via the immunoreceptor-based activatory motif (ITAM)-containing FcRgamma chain. Conversely, thrombin only activated at high concentrations (> 0.2 U/mL), whereas U46619 and ADP alone were ineffective. Collagen or CRP-stimulated 12-H(P)ETE generation was inhibited by staurosporine, PP2, wortmannin, BAPTA/AM, EGTA, and L-655238, implicating src-tyrosine kinases, PI3-kinase, Ca2+ mobilization, and p12-LOX translocation. In contrast, protein kinase C (PKC) inhibition potentiated 12-H(P)ETE generation. Finally, activation of the immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing platelet endothelial cell adhesion molecule (PECAM-1) inhibited p12-LOX product generation. This study characterizes a receptor-dependent pathway for 12-H(P)ETE synthesis via the collagen receptor GPVI, which is negatively regulated by PECAM-1 and PKC, and demonstrates a novel link between immune receptor signaling and lipid mediator generation in the vasculature.

Nitrolinoleate inhibits superoxide generation, degranulation, and integrin expression by human neutrophils: novel antiinflammatory properties of nitric oxide-derived reactive species in vascular cells.

Nitration of unsaturated fatty acids such as linoleate by NO-derived reactive species forms novel derivatives (including nitrolinoleate [LNO2]) that can stimulate smooth muscle relaxation and block platelet activation by either NO/cGMP or cAMP-dependent mechanisms. Here, LNO2 was observed to inhibit human neutrophil function. LNO2, but not linoleic acid or the nitrated amino acid 3-nitrotyrosine, dose-dependently (0.2 to 1 micromol/L) inhibited superoxide (O2*-) generation, Ca2+ influx, elastase release, and CD11b expression in response to either phorbol 12-myristate 13-acetate or N-formyl-Met-Leu-Phe. LNO2 did not elevate cGMP, and inhibition of guanylate cyclase by 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one did not restore neutrophil responses, ruling out a role for NO. In contrast, LNO2 caused elevations in intracellular cAMP in the presence and absence of phosphodiesterase inhibition, suggesting activation of adenylate cyclase. Compared with phorbol 12-myristate 13-acetate-activated neutrophils, N-formyl-Met-Leu-Phe-activated neutrophils were more susceptible to the inhibitory effects of LNO2, indicating that LNO2 may inhibit signaling both upstream and downstream of protein kinase C. These data suggest novel signaling actions for LNO2 in mediating its potent inhibitory actions. Thus, nitration of lipids by NO-derived reactive species yields products with antiinflammatory properties, revealing a novel mechanism by which NO-derived nitrated biomolecules can influence the progression of vascular disease.

Nitrolinoleate inhibits platelet activation by attenuating calcium mobilization and inducing phosphorylation of vasodilator-stimulated phosphoprotein through elevation of cAMP.

Reactive species formed from nitric oxide (NO) nitrate unsaturated fatty acids such as linoleate (LA) to nitrated derivatives including nitrolinoleate (LNO(2)). The effect of LNO(2) on human platelets was examined to define how nitrated lipids might behave in vivo. LNO(2), but not LA or 3-nitrotyrosine, dose dependently (0.5-10 microm) inhibited thrombin-mediated aggregation of washed human platelets, with concomitant attenuation of P-selectin expression and selective phosphorylation of VASP at the cAMP-dependent protein kinase selective site, serine 157. LNO(2) caused slight mobilization of calcium (Ca(2+)) from intracellular stores but significantly inhibited subsequent thrombin-stimulated Ca(2+) elevations. LNO(2) did not elevate platelet cGMP, and its effects were not blocked with inhibitors of NO signaling (oxyhemoglobin, 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one. 2-fold elevations in cAMP were found following LNO(2) treatment of platelets, and the adenylyl cyclase inhibitors 2',5'-dideoxyadenosine and SQ22536 partially restored thrombin-stimulated aggregation. Finally, LNO(2) significantly inhibited cAMP hydrolysis to AMP by platelet lysates. These data implicate cAMP in the anti-aggregatory action of LNO(2). The platelet inhibitory actions of LNO(2) indicate that nitration reactions that occur following NO generation in an oxidizing environment can alter the activity of lipids and lend insight into mechanisms by which NO-derived species may modulate the progression of vascular injury.