Recognition and management of palatogingival groove for tooth survival: a literature review

Palatogingival groove (PGG) is an anomaly in the maxillary anterior teeth, often accompanied by the area of bony destruction adjacent to the teeth with no carious or traumatic history. The hidden trap in the tooth can harbor plaque and bacteria, resulting in periodontal destruction with or without pulpal pathologic change. Related diseases can involve periodontal destruction, combined endodontic-periodontal lesions, or separate endodontic and periodontal lesions. Disease severity and prognosis related to PGG depend on several factors, including location, range, depth, and type of the groove. Several materials have been used and recommended for cases of extensive periodontal destruction from PGG to remove and block the inflammatory source and recover the health of surrounding periodontal tissues. Even in cases of severe periodontal destruction, several studies have reported favorable treatment outcomes with proper management. With new options in diagnosis and treatment, clinicians need a detailed understanding of the characteristics, treatment, and prognosis of PGG to successfully manage the condition.

Dynamics of Radical Intermediates in Prostaglandin H Synthase-1 Cyclooxygenase Reactions is Modulated by Multiple Factors.

Prostaglandin H synthase (PGHS) catalyzes the biosynthesis of PGG2 and PGH2, the precursor of all prostanoids, from arachidonic acid (AA). PGHS exhibits two enzymatic activities following a branched-chain radical mechanism: 1) a peroxidase activity (POX) that utilizes hydroperoxide through heme redox cycles to generate the critical Tyr385 tyrosyl radical for coupling both enzyme activities; 2) the cyclooxygenase (COX) activity inserting two oxygen molecules into AA to generate endoperoxide/hydroperoxide PGG2 through a series of radical intermediates. Upon the generation of Tyr385 radical, COX catalysis is initiated, with C13 pro-S hydrogen abstraction from AA by Tyr385 radical to generate arachidonyl substrate radical. Oxygen provides a large driving force for the subsequent fast steps leading to the formation of PGG2, including radical redistributions, ring formations, and rearrangements. On the other hand, if the supply of oxygen is severed, equilibrium between arachidonyl radical and tyrosyl radical(s) biases largely towards the latter. In this study, we demonstrate that such equilibrium is shifted by many factors, including temperature, chemical structures of fatty acid substrates and limited supply of oxygen. We also, for the first time, reveal that this equilibrium is significantly affected by co-substrates of POX. The presence of efficient POX co-substrates, which reduces heme to its ferric state, apparently biases the equilibrium towards arachidonyl radical. Therefore a dynamic interplay exists between the two activities of PGHS.

Immunomodulatory Effects of Adipose Tissue-Derived Stem Cells on Elastin Scaffold Remodeling in Diabetes

Diabetes is a major risk factor for the progression of vascular disease, contributing to elevated levels of glycoxidation, chronic inflammation and calcification. Tissue engineering emerges as a potential solution for the treatment of vascular diseases however there is a considerable gap in the understanding of how scaffolds and stem cells will perform in patients with diabetes. We hypothesized that adipose tissue-derived stem cells (ASCs) by virtue of their immunosuppressive potential would moderate the diabetes-intensified inflammatory reactions and induce positive construct remodeling. To test this hypothesis, we prepared arterial elastin scaffolds seeded with autologous ASCs and implanted them subdermally in diabetic rats and compared inflammatory markers, macrophage polarization, matrix remodeling, calcification and bone protein expression to control scaffolds implanted with and without cells in nondiabetic rats. ASC-seeded scaffolds exhibited lower levels of CD8+ T-cells and CD68+ pan-macrophages and higher numbers of M2 macrophages, smooth muscle cell-like and fibroblast-like cells. Calcification and osteogenic markers were reduced in ASCseeded scaffolds implanted in non-diabetic rats but remained unchanged in diabetes, unless the scaffolds were first pre-treated with penta-galloyl glucose (PGG), a known anti-oxidative elastin-binding polyphenol. In conclusion, autologous ASC seeding in elastin scaffolds is effective in combating diabetes-related complications. To prevent calcification, the oxidative milieu needs to be reduced by elastin-binding antioxidants such as PGG.

A Study on the Statistical Method for the Target Component of a Glucose-lowering Functional Material in Extracts of Evening Primerose Seeds

BACKGROUND: The use of the extracts from evening primrose seeds as a health functional food has been gradually increased. Therefore, the monitoring and screening process has been considerably required for its quality control. OBJECTIVE: This study aimed to estimate the measurement uncertainty associated with determination of penta-o-galloyl β-D-glucose (PGG) in extracts from evening primrose seeds by high-performance liquid chromatography. METHODS: The sources of measurement uncertainty was expressed in accordance with mathematical/statistical theories of GUM & EURACHEM Guide. The expanded uncertainty was calculated by using the relative standard uncertainty between analytical result and sources of uncertainty in measurement (sample weight, final volume, extraction volume, standard solution, matrix and instrument etc). RESULTS: In the results of 95% confidence interval, the uncertainty in measurement was 10,253.34 ± 1,844.50 µg/kg (k = 2.0). CONCLUSION: In this study, it showed that the value of uncertainty in measurement for determination of PGG in extracts from evening primrose seeds by HPLC has about 18.0% influence on PGG contents of the analytical results. The results would be very useful for the monitoring and screening of evening primrose seeds marketed in Korea for its quality control as dietary supplement.

COX-2-derived endocannabinoid metabolites as novel inflammatory mediators.

Cyclooxygenase-2 (COX-2) is an enzyme that plays a key role in inflammatory processes. Classically, this enzyme is upregulated in inflammatory situations and is responsible for the generation of prostaglandins (PGs) from arachidonic acid (AA). One lesser-known property of COX-2 is its ability to metabolize the endocannabinoids, N-arachidonoylethanolamine (AEA) and 2-arachidonoylglycerol (2-AG). Endocannabinoid metabolism by COX-2 is not merely a means to terminate their actions. On the contrary, it generates PG analogs, namely PG-glycerol esters (PG-G) for 2-AG and PG-ethanolamides (PG-EA or prostamides) for AEA. Although the formation of these COX-2-derived metabolites of the endocannabinoids has been known for a while, their biological effects remain to be fully elucidated. Recently, several studies have focused on the role of these PG-G or PG-EA in vivo. In this review we take a closer look at the literature concerning these novel bioactive lipids and their role in inflammation.

pH-dependent effect of pectinase secretion in Penicillium griseoroseum recombinant strains.

A number of parameters, including culture medium pH, affect growth and enzyme production by microorganisms. In the present study, the production and secretion of pectin lyase (PL) and polygalacturonase (PG) by recombinant strains of Penicillium griseoroseum cultured in mineral-buffered media (MBM; initial pH 6.8) and mineral-unbuffered medium (MUM; initial pH 6.3) were evaluated. Under these culture conditions, no change in the transcriptional levels of plg1 and pgg2 was observed. However, the levels of secreted total protein ranged from 7.80 ± 1.1 to 3.25 ± 1.50 µg ml(-1) in MBM and MUM, respectively, and were evaluated by SDS-PAGE. PL and PG enzymatic activities decreased 6.4 and 3.6 times, respectively, when P. griseoroseum was cultivated under acidic pH conditions (MUM). Furthermore, differences were observed in the hypha and mycelium morphology. These findings suggest that acidic growing conditions affect PL and PG secretion, even though the transcription and translation processes are successful. The data obtained in this study will help to establish optimal culture conditions that increase production and secretion of recombinant proteins by filamentous fungi.

The Longevity Properties of 1,2,3,4,6-Penta-O-Galloyl-beta-D-Glucose from Curcuma longa in Caenorhabditis elegans

Here in this study, we isolated 1,2,3,4,6-penta-O-galloyl-beta-D-glucose (PGG) from Curcuma longa L. and elucidated the lifespanextending effect of PGG using Caenorhabditis elegans model system. In the present study, PGG demonstrated potent lifespan extension of worms under normal culture condition. Then, we determined the protective effects of PGG on the stress conditions such as thermal and oxidative stress. In the case of heat stress, PGG-treated worms exhibited enhanced survival rate, compared to control worms. In addition, PGG-fed worms lived longer than control worms under oxidative stress induced by paraquat. To verify the possible mechanism of PGG-mediated increased lifespan and stress resistance of worms, we investigated whether PGG might alter superoxide dismutase (SOD) activities and intracellular ROS levels. Our results showed that PGG was able to elevate SOD activities of worms and reduce intracellular ROS accumulation in a dose-dependent manner.

Endovascular Therapy to Salvage Hemodialysis Access / 대한혈관외과학회지

PURPOSE: Maintaining function of dialysis access is very important to end stage renal disease patients, and it is critical for these patients' overall well-bing. This study was performed to evaluate the success rate of endovascular interventions, risk factors and patencies of interventions. METHODS: From January 2008 to June 2010, 315 interventions were performed on 189 patients with malfunctioning hemodialysis access. Angioplastic intervention (n=147), and percutaneous mechanical thrombectomies (n=168) were done to restore flow of vascular access. RESULTS: Most common cause of malfunctioning hemodialysis accesses was stenosis (71%) in autogenous fistula group (AFG) and thrombosis (73%) in prosthetic graft group (PGG). Common stenotic site was juxta-anastomotic area (48%) in AFG, and venous anastomotic area (42%) in PGG, primary patencies of interventions at 6, 12, and 18 months were 67%, 45%, and 37% and secondary patencies were 83%, 66%, and 49%, respectively. Finally, thrombotic events (P=0.005) and numbers of procedure (P=0.000) were independent predictive factors of shorter access patency after endovascular treatment. CONCLUSION: Endovascular treatment in malfunctioning hemodialysis access is highly successful procedure with acceptable primary and secondary patency results. Especially, in the cases of stenotic lesion without thrombosis, percutaneous transluminal angioplasty was successfully done in AFG and PGG.

Prostaglandin Gbetagamma signaling stimulates gastrulation movements by limiting cell adhesion through Snai1a stabilization.

Gastrulation movements form the germ layers and shape them into the vertebrate body. Gastrulation entails a variety of cell behaviors, including directed cell migration and cell delamination, which are also involved in other physiological and pathological processes, such as cancer metastasis. Decreased Prostaglandin E(2) (PGE(2)) synthesis due to interference with the Cyclooxygenase (Cox) and Prostaglandin E synthase (Ptges) enzymes halts gastrulation and limits cancer cell invasiveness, but how PGE(2) regulates cell motility remains unclear. Here we show that PGE(2)-deficient zebrafish embryos, impaired in the epiboly, internalization, convergence and extension gastrulation movements, exhibit markedly increased cell-cell adhesion, which contributes to defective cell movements in the gastrula. Our analyses reveal that PGE(2) promotes cell protrusive activity and limits cell adhesion by modulating E-cadherin transcript and protein, in part through stabilization of the Snai1a (also known as Snail1) transcriptional repressor, an evolutionarily conserved regulator of cell delamination and directed migration. We delineate a pathway whereby PGE(2) potentiates interaction between the receptor-coupled G protein betagamma subunits and Gsk3beta to inhibit proteasomal degradation of Snai1a. However, overexpression of beta-catenin cannot stabilize Snai1a in PGE(2)-deficient gastrulae. Thus, the Gsk3beta-mediated and beta-catenin-independent inhibition of cell adhesion by Prostaglandins provides an additional mechanism for the functional interactions between the PGE(2) and Wnt signaling pathways during development and disease. We propose that ubiquitously expressed PGE(2) synthesizing enzymes, by promoting the stability of Snai1a, enable the precise and rapid regulation of cell adhesion that is required for the dynamic cell behaviors that drive various gastrulation movements.

Spectroelectrochemical characterization of pain biomarkers.

This article reports the first electrochemical characterization of pain biomarkers that include arachidonic acid (AA), prostaglandin G(2) (PGG(2)), and cyclooxygenase 2 (COX-2). These biomarkers are mediators of pathophysiology of pain, inflammation, and cell proliferation in cancer. The article also reports the development of an electrochemical immunosensor for monitoring these pain biomarkers. The results revealed that direct electron transfer between AA metabolites and the electrode could be easily monitored and that an enzyme-modified electrode dramatically enhanced bioelectrocatalytic activity toward AA. Cyclic voltammetric analysis of AA revealed a concentration-dependent anodic current with a slope of 2.37 and a limit of detection (LOD) of 0.25nM. This unique AA/gold electrode electron transfer provides a good electrochemical sensing platform for prostaglandin H(2) (PGH(2)) as the basis for quantitation of pain. An amperometric signal intensity of a COX-2 antibody-modified gold electrode was linear with COX-2 concentration in the range of 0.1-0.5microg/ml and an LOD of 0.095microg/ml. The results also revealed a linear correlation of the concentration of PGG(2) with an LOD of 0.227microM.

Co-oxidation by cyclooxygenases.

Cyclooxygenases (COXs; prostaglandin H(2) synthases) catalyze the bis-dioxygenation of arachidonic acid (AA) to generate prostaglandin (PG) G(2) followed by the peroxidative cleavage of PGG(2) to yield PGH(2), the precursor to all of the vasoactive PGs. These enzymes utilize a Fe-protoporhyrin IX (heme) co-factor to catalyze peroxide bond cleavage, which puts the Fe at a higher oxidation state (Fe(3+) → Fe(5+)). The heme Fe requires two electrons (e(-)) to return to its resting state (Fe(3+)) for the next round of catalysis. Peroxide bond cleavage thus occurs via compound I and compound II, observed for horseradish peroxidase. To return to Fe(3+), electrons come from "co-reductants" and their subsequent oxidation by the enzyme is known as "co-oxidation". The protocols in this unit are aimed at characterizing this side reaction of COXs.

Effects of nimesulide, a cyclooxygenase-2 selective inhibitor, on colitis induced tumors.

Cyclooxygenase-2 (COX-2) is known to suppress sporadic colorectal cancer, but effect of selective COX-2 inhibitor in UC-associated neoplasia is still unknown. This study investigated effect of a selective COX-2 inhibitor on colorectal carcinogenesis in experimental murine UC. Chronic colitis was induced in mice by four cycles of administration of dextran sulfate sodium (DSS) (i. e., 5 % DSS for 7 days and distilled water for the following 14 days), and the mice were sacrificed 120 days after the end of the fourth cycle. The mice were divided into the following five groups: Group A, served as a disease control; Group B, received a diet mixed with 400 ppm of nimesulide (NIM), a selective COX-2 inhibitor, during the whole period; Group C, received NIM during the four cycles of DSS administration; Group D, received NIM for 120 days from the end of the fourth cycle; Group E, served as a normal control. In Group D, NIM significantly suppressed the occurrence of dysplasia and/or cancer. The results show that NIM inhibited both dysplasia and cancer in DSS-treated mice, thus showing that NIM has preventive effects on the remission phase of carcinogenesis.

Prostacyclin among prostanoids.

Prostanoids are cyclic lipid mediators which arise from enzymic cyclooxygenation of linear polyunsaturated fatty acids, e.g. arachidonic acid (20:4 n 6, AA). Biologically active prostanoids deriving from AA include stable prostaglandins (PGs), e.g. PGE(2), PGF(2alpha), PGD(2), PGJ(2) as well as labile prostanoids, i.e. PG endoperoxides (PGG(2), PGH(2)), thromboxane A(2) (TXA(2)) and prostacyclin (PGI(2)). A "Rabbit aorta Contracting Substance" (RCS) played important role in discovering of labile PGs. RCS was discovered in the Vane's Cascade as a labile product released along with PGs from the activated lung or spleen. RCS was identified as a mixture of PG endoperoxides and thromboxane A(2). Stable PGs regulate the cell cycle, smooth muscle tone and various secretory functions; they also modulate inflammatory and immune reactions. PG endoperoxides are intermediates in biosynthesis of all prostanoids. Thromboxane A(2) (TXA(2)) is the most labile prostanoid (with a half life of 30 s at 37 degrees C). It is generated mainly by blood platelets. TXA(2) is endowed with powerful vasoconstrictor, cytotoxic and thrombogenic properties. Again the Vane's Cascade was behind the discovery of prostacyclin (PGI(2)) with a half life of 4 min at 37 degrees C. It is produced by the vascular wall (predominantly by the endothelium) and it acts as a physiological antagonist of TXA(2). Moreover, prostacyclin per se is a powerful cytoprotective agent that exerts its action through activation of adenylate cyclase, followed by an intracellular accumulation of cyclic-AMP in various types of cells. In that respect PGI(2) collaborates with the system consisting of NO synthase (eNOS)/nitric oxide free radical (NO)/guanylate cyclase/cyclic-GMP. Both cyclic nucleotides (c-AMP and c-GMP) act in synergy as two energetic fists which defend the cellular machinery from being destroyed by endogenous or exogenous aggressors. Recently, a new partner has been recognized in this endogenous defensive squadron, i.e. a system consisting of heme oxygenase (HO-1)/carbon monoxide (CO)/biliverdin/biliverdin reductase/bilirubin. The expanding knowledge on the pharmacological steering of this enzymic triad (PGI(2)-S/eNOS/HO-1) is likely to contribute to the rational therapy of many systemic diseases such as atherosclerosis, diabetes mellitus, arterial hypertension or Alzheimer diseases. The discovery of prostacyclin broadened our pathophysiological horizon, and by itself opened new therapeutic possibilities. Prostacyclin sodium salt and its synthetic stable analogues (iloprost, beraprost, treprostinil, epoprostenol, cicaprost) are useful drugs for the treatment of the advanced critical limb ischemia, e.g. in the course of Buerger's disease, and also for the treatment of pulmonary artery hypertension (PAH). In this last case a synergism between prostacyclin analogues and sildenafil (a selective phosphodiesterase 5 inhibitor) or bosentan (an endothelin ET-1 receptor antagonist) points our to complex mechanisms controlling pulmonary circulation. At the Jagiellonian University we have demonstrated that several well recognised cardiovascular drugs, e.g. ACE inhibitors (ACE-I), statins, some of beta-adrenergic receptor antagonists, e.g. carvedilol or nebivolol, anti-platelet thienopyridines (ticlopidine, clopidogrel) and a metabolite of vitamin PP--N(1)-methyl-nicotinamide--all of them are endowed with the in vivo PGI(2)-releasing properties. In this way, the foundations for the Endothelial Pharmacology were laid.

Molecular oxygen (a substrate of the cyclooxygenase reaction) in the kinetic mechanism of the bifunctional enzyme prostaglandin-H-synthase.

Prostaglandin-H-synthase is a bifunctional enzyme catalyzing conversion of arachidonic acid into prostaglandin H2 as a result of cyclooxygenase and peroxidase reactions. The dependence of the rate of the cyclooxygenase reaction on oxygen concentration in the absence and in the presence of electron donor was determined. A two-dimensional kinetic scheme accounting for independent proceeding and mutual influence of the cyclooxygenase and peroxidase reactions and also for hierarchy of the rates of these reactions was used as a model. In the context of this model, it was shown that there are irreversible stages in the mechanism of the cyclooxygenase reaction between points of substrate donation (between donation of arachidonic acid and the first oxygen molecule and also between donation of two oxygen molecules).

15-Hydroxyprostaglandin dehydrogenase is an in vivo suppressor of colon tumorigenesis.

15-Hydroxyprostaglandin dehydrogenase (15-PGDH) is a prostaglandin-degrading enzyme that is highly expressed in normal colon mucosa but is ubiquitously lost in human colon cancers. Herein, we demonstrate that 15-PGDH is active in vivo as a highly potent suppressor of colon neoplasia development and acts in the colon as a required physiologic antagonist of the prostaglandin-synthesizing activity of the cyclooxygenase 2 (COX-2) oncogene. We first show that 15-PGDH gene knockout induces a marked 7.6-fold increase in colon tumors arising in the Min (multiple intestinal neoplasia) mouse model. Furthermore, 15-PGDH gene knockout abrogates the normal resistance of C57BL/6J mice to colon tumor induction by the carcinogen azoxymethane (AOM), conferring susceptibility to AOM-induced adenomas and carcinomas in situ. Susceptibility to AOM-induced tumorigenesis is mediated by a marked induction of dysplasia, proliferation, and cyclin D1 expression throughout microscopic aberrant crypt foci arising in 15-PGDH null colons and is concomitant with a doubling of prostaglandin E(2) in 15-PGDH null colonic mucosa. A parallel role for 15-PGDH loss in promoting the earliest steps of colon neoplasia in humans is supported by our finding of a universal loss of 15-PGDH expression in microscopic colon adenomas recovered from patients with familial adenomatous polyposis, including adenomas as small as a single crypt. These models thus delineate the in vivo significance of 15-PGDH-mediated negative regulation of the COX-2 pathway and moreover reveal the particular importance of 15-PGDH in opposing the neoplastic progression of colonic aberrant crypt foci.

The productive conformation of prostaglandin G2 at the peroxidase site of prostaglandin endoperoxide H synthase: docking, molecular dynamics, and site-directed mutagenesis studies.

We present a plausible productive conformation obtained by docking calculations for the binding of prostaglandin G2 (PGG2) to the peroxidase site of prostaglandin endoperoxide H synthase-1 (PGHS-1, COX-1). The enzyme-substrate complex stability was verified by molecular dynamics. Structural analysis reveals the requirements for enzyme-substrate recognition and binding: the PGG2 15-hydroperoxide group is in the proximity of the heme iron and participates in a hydrogen bond network with the conserved His207 and Gln203 and a water molecule, whereas the carboxylate group forms salt bridges with the remote Lys215 and Lys222. Site-directed mutagenesis showed that a single mutation of Lys215 or Lys222 does not affect enzyme activity, whereas dual mutation of these residues, to either alanine or glutamate, significantly decreases turnover. This indicates that the conserved cationic pocket is involved in enzyme-substrate binding.

The effect of PGG-beta-glucan on neutrophil chemotaxis in vivo.

The beta-glucans are long-chain polymers of glucose in beta-(1,3)(1,6) linkages, which comprise the fungal cell wall and stimulate cells of the innate immune system. Previous in vitro studies have shown the ability of beta-glucan to increase the chemotactic capacity of human neutrophils. The current study examined an in vivo correlate of that observation by testing the hypothesis that systemic beta-glucan treatment would result in enhanced migration of neutrophils into a site of inflammation and improve antimicrobial function. A model of acute inflammation was used in which polyvinyl alcohol sponges were implanted subcutaneously into the dorsum of rats. Animals treated with beta-glucan showed a 66 +/- 6% and 186 +/- 42% increase in wound cell number recovered 6 and 18 h postwounding, respectively. Increased migration did not correlate with increased chemoattractant content of wound fluid, alterations in neutrophil-induced loss of endothelial barrier function, or changes in neutrophil adhesion to endothelial cells. Systemic administration of SB203580 abrogated the enhanced migration by beta-glucan without altering normal cellular entry into the wound. Studies also showed a priming effect for chemotaxis and respiratory burst in circulating neutrophils isolated from beta-glucan-treated animals. Heightened neutrophil function took place without cytokine elicitation. Furthermore, beta-glucan treatment resulted in a 169 +/- 28% increase in neutrophil number and a 60 +/- 9% decrease in bacterial load in the bronchoalveolar lavage fluid of Escherichia coli pneumonic animals. Taken together, these findings demonstrate that beta-glucan directly affects the chemotactic capacity of circulating neutrophils through a p38 mitogen-activated protein kinase-dependent mechanism and potentiates antimicrobial host defense.

Identification of two cyclooxygenase active site residues, Leucine 384 and Glycine 526, that control carbon ring cyclization in prostaglandin biosynthesis.

The cyclooxygenase (COX) reaction of prostaglandin (PG) biosynthesis begins with the highly specific oxygenation of arachidonic acid in the 11R configuration and ends with a 15S oxygenation to form PGG2. To obtain new insights into the mechanisms of stereocontrol of oxygenation, we mutated active site residues of human COX-2 that have potential contacts with C-11 of the reacting substrate. Although the 11R oxygenation was not perturbed, changing Leu-384 (into Phe, Trp), Trp-387 (Phe, Tyr), Phe-518 (Ile, Trp, Tyr), and Gly-526 (Ala, Ser, Thr, Val) impaired or abrogated PGG2 synthesis, and typically 11R-HETE was the main product formed. The Gly-526 and Leu-384 mutants formed, in addition, three novel products identified by LC-MS, NMR, and circular dichroism as 8,9-11,12-diepoxy-13R-(or 15R)-hydro(pero)xy derivatives of arachidonic acid. Mechanistically, we propose these arise from a free radical intermediate in which a C-8 carbon radical displaces the 9,11-endoperoxide O-O bond to yield an 8,9-11,12-diepoxide that is finally oxygenated stereospecifically in the 13R or 15R configuration. Formation of these novel products signals an arrest in the normal course of prostaglandin synthesis just prior to closing of the 5-membered carbon ring, and points to a crucial role for Leu-384 and Gly-526 in the correct positioning of the reacting fatty acid intermediate. Some of the Gly-526 and Leu-384 mutants catalyzed both formation of PGG2 (with the normal 15S configuration) and the 13R- or 15R-oxygenated diepoxides. This result suggests that oxygenation specificity can be determined by the orientation of the reacting fatty acid radical and is not a predetermined outcome based solely on the structure of the cyclooxygenase active site.

Levuglandinyl adducts of proteins are formed via a prostaglandin H2 synthase-dependent pathway after platelet activation.

The product of oxygenation of arachidonic acid by the prostaglandin H synthases (PGHS), prostaglandin H(2) (PGH(2)), undergoes rearrangement to the highly reactive gamma-ketoaldehydes, levuglandin (LG) E(2), and LGD(2). We have demonstrated previously that LGE(2) reacts with the epsilon-amine of lysine to form both the levuglandinyl-lysine Schiff base and the pyrrole-derived levuglandinyl-lysine lactam adducts. We also have reported that these levuglandinyl-lysine adducts are formed on purified PGHSs following the oxygenation of arachidonic acid. We now present evidence that the levuglandinyl-lysine lactam adduct is formed in human platelets upon activation with exogenous arachidonic acid or thrombin. After proteolytic digestion of the platelet proteins, and isolation of the adducted amino acid residues, this adduct was identified by liquid chromatography-tandem mass spectrometry. We also demonstrate that formation of these adducts is inhibited by indomethacin, a PGHS inhibitor, and is enhanced by an inhibitor of thromboxane synthase. These data establish that levuglandinyl-lysine adducts are formed via a PGHS-dependent pathway in whole cells, even in the presence of an enzyme that metabolizes PGH(2). They also demonstrate that a physiological stimulus is sufficient to lead to the lipid modification of proteins through the levuglandin pathway in human platelets.