An anti‐platelet agent, OPC‐29030, inhibits translocation of 12‐lipoxygenase and 12‐hydroxyeicosatetraenoic acid production in human platelets

Ozeki, Yasushi; Nagamura, Yoshie; Ito, Hideki; Unemi, Fumiko; Kimura, Yukio; Igawa, Takehiro; Kambayashi, Junichi; Takahashi, Yoshitaka; Yoshimoto, Tanihiro

doi:10.1038/sj.bjp.0702976

Cited by 27 publications

(20 citation statements)

References 25 publications

(24 reference statements)

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“…This compound was first found to inhibit translocation of 5-lipoxygenase (24) but later shown to inhibit translocation of other lipoxygenases without affecting the enzyme activity per se (25). As shown in Fig.…”

Section: Membrane Translocation Of 12/15-lipoxygenase By Ldl-12/mentioning

confidence: 90%

“…In 5-lipoxygenase, the N-terminal C2-like domain is demonstrated to be a calciumdependent membrane-targeting domain without requirement of any special docking protein (33). We have reported that 12-lipoxygenase in human platelets is activated by membrane translocation when stimulated by collagen or thrombin and that a translocation inhibitor of 5-lipoxygenase, L655238, inhibits production of 12-HETE from platelets without affecting the enzyme activity (25). The results clearly indicate that L655238 is not a 5-lipoxygenase-specific inhibitor but a general translocation inhibitor of lipoxygenases.…”

Section: Membrane Translocation Of 12/15-lipoxygenase By Ldl-12/mentioning

confidence: 98%

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Low Density Lipoprotein Receptor-related Protein-mediated Membrane Translocation of 12/15-Lipoxygenase Is Required for Oxidation of Low Density Lipoprotein by Macrophages

Zhu

Takahashi

et al. 2003

Journal of Biological Chemistry

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Oxidation of low density lipoprotein (LDL) is the key step for the development of atherosclerosis. The 12/15-lipoxygenase expressed in macrophages is capable of oxygenating linoleic acid esterified to cholesterol in the LDL particle, and thus this enzyme is presumed to initiate LDL oxidation. We recently reported that LDL receptor-related protein (LRP) was required for the enzyme-mediated LDL oxidation by macrophages and suggested the selective uptake of cholesterol ester from LDL to the plasma membrane (Xu, W., Takahashi, Y., Sakashita, T., Iwasaki, T., Hattori, H., and Yoshimoto. T. (2001) J. Biol. Chem. 276, 36454 -36459). To elucidate precise mechanisms of lipoxygenase-mediated LDL oxidation, we investigated the intracellular localization of 12/15-lipoxygenase. The 12/15-lipoxygenase was predominantly detected in cytosol of resting peritoneal macrophages and of macrophage-like J774A.1 cells permanently transfected with the cDNA for the enzyme. When the cells were treated with LDL and subjected to subcellular fractionation, the 12/15-lipoxygenase was detected in the membranes with a concomitant decrease in cytosol as shown by Western blot analysis. The levels of the enzyme associated with the membrane reached maximum in 15 min after LDL addition and then decreased. However, the enzymatic activity of 12/15-lipoxygenase in the membrane fraction was very weak even after LDL treatment. This fact supports the suicide inactivation of the enzyme by the oxygenation of cholesterol ester transferred from the LDL particle to the plasma membrane. Immunohistochemical analysis using an antibody against 12/15-lipoxygenase revealed that the plasma membrane was the major site of the enzyme translocation by the LDL treatment. LDL-dependent 12/ 15-lipoxygenase translocation was inhibited by a blocking antibody against LRP. Furthermore, an enzyme translocation inhibitor, L655238, inhibited the LDL oxidation caused by the 12/15-lipoxygenase. We propose that cholesterol ester selectively transferred from the LDL particle to the plasma membrane via LRP is oxygenated by 12/15-lipoxygenase translocated to this membrane.12/15-Lipoxygenase is a member of the lipoxygenase family, which incorporates one molecule of oxygen in regiospecific and stereospecific manners to unsaturated fatty acids such as arachidonic and linoleic acids (1-4). The enzyme consists of leukocyte-type 12-lipoxygenase found in rats, mice, cows, and pigs, and reticulocyte-type 15-lipoxygenase (15-lipoxygenase-1) expressed in humans and rabbits, oxygenating the position 12 and 15 of arachidonic acid, respectively (5). The notable feature of the 12/15-lipoxygenase is that the enzyme directly oxygenates not only free fatty acids but also complex substrates such as phospholipids, cholesterol ester, and the cholesterol ester present in the low density lipoprotein (LDL) 1 particle (1-4). Oxidation of LDL is the first key step for the development of atherosclerosis (6, 7), and the roles of the 12/15-lipoxygenase in the process of LDL oxidation and the progress of atherogene...

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Section: Membrane Translocation Of 12/15-lipoxygenase By Ldl-12/mentioning

confidence: 90%

Section: Membrane Translocation Of 12/15-lipoxygenase By Ldl-12/mentioning

confidence: 98%

Low Density Lipoprotein Receptor-related Protein-mediated Membrane Translocation of 12/15-Lipoxygenase Is Required for Oxidation of Low Density Lipoprotein by Macrophages

Zhu

Takahashi

et al. 2003

Journal of Biological Chemistry

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“…50,51 In agreement, a translocation inhibitor, L-655238, substantially inhibited GPVI-dependent 12-H(P)ETE synthesis similar to previous reports using collagen ( Figure 2D). 15 However, Ca 2ϩ mobilization is not in itself sufficient for p12-LOX activation, because a concentration of thrombin that did not cause 12-H(P)ETE synthesis caused significantly greater Ca 2ϩ mobilization than collagen ( Figure 2C). GPVI stimulation of 12-H(P)ETE generation was inhibited by PP2, staurosporine, and wortmannin, implicating srctyrosine kinases and PI3-kinase in p12-LOX activation (Figure 3).…”

Section: Discussionmentioning

confidence: 99%

Platelet 12-Lipoxygenase Activation via Glycoprotein VI

Coffey

Jarvis

Gibbins

et al. 2004

Circulation Research

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Abstract-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 g/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 FcR␥ chain. Conversely, thrombin only activated at high concentrations (Ͼ 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, Ca 2ϩ 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. Key Words: platelets Ⅲ lipoxygenase Ⅲ collagen Ⅲ collagen receptor glycoprotein VI L ipoxygenases (LOX) catalyze lipid hydroperoxide formation from unsaturated fatty acids. 1 Many LOX isoforms are found in the vasculature: some constitutive (eg, p12-LOX in platelets, 5-LOX in neutrophils), others cytokine-inducible (eg, 15-LOX by interleukin-4 (IL-4) in monocytes). Compelling evidence exists for critical involvement of LOXs in vascular and inflammatory diseases including atherosclerosis, cancer, hypertension, and diabetes. [2][3][4][5][6][7][8][9][10][11][12][13] Cellular LOXs require direct activation before product generation occurs. Typically, this requires (1) oxidation of the ferrous iron to an intermediate that abstracts hydrogen from unsaturated lipid and (2) translocation of the cytosolic enzyme to nuclear or plasma membrane. This activation is separate from simple regulation of LOX by fatty acid supply, and its requirement for H(P)ETE synthesis is supported by the following evidence: (1) Ca 2ϩ stimulates membrane binding and arachidonate oxidation by purified 15-LOX in vitro; (2) LOX product generation (in response to Ca 2ϩ ionophore) is inhibited by membrane translocation inhibitors; and (3) 15-LOX in human monocytes oxidizes membrane-bound arachidonate after Ca 2ϩ mobilization, independently of PLA 2 . 14 -16 Currently, little is known regarding control of LOX in cells by acute mechanisms. Most studies activate LOX turnover by addition of arachidonate substrate w...

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“…The intracellular distribution of 12-LO activity varies between different cells, ranging from a predominant cytosolic 20 localization to a preferential localization in membranes. 21 In human platelets, most 22 but not all 23 investigators have observed a predominant (65%) localization of 12-LO activity and protein in the cytosolic fraction. 12(S)-HETE has also been shown to participate in the regulation of platelet function such as aggregation and P-selectin expression.…”

Section: Discussionmentioning

confidence: 99%

Increased Levels of 12(S)-HETE in Patients With Essential Hypertension

et al. 2001

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Abstract-The platelet-type 12-lipoxygenase (12-LO) catalyzes the transformation of arachidonic acid into 12-hydroperoxyeicosatetraenoic acid [12-(S)HPETE], which is reduced to 12-hydroxyeicosatetraenoic acid [12-(S)HETE]. These metabolites exhibit a variety of biological activities such as mediation of angiotensin II-induced intracellular calcium transients in cultured rat vascular smooth muscle cells. It has recently been reported that platelet 12(S)-HETE production is enhanced in the spontaneously hypertensive rat. The pronounced hypotensive effect of LO inhibition in SHR suggests that LO activity may play a role in this form of hypertension. The aim of this study was to determine the basal and thrombin-induced platelet 12(S)-HETE production and the urinary 12(S)-HETE excretion in essential hypertension. We studied 19 patients with this disease (57Ϯ2 years of age) and 9 normotensive control subjects (48Ϯ5 years of age) (Pϭ0.074). 12(S)-HETE was measured in Sep-Pack-extracted samples with specific ELISA and high-performance liquid chromatography. The platelet basal level of 12(S)-HETE was significantly higher in patients than in control subjects (3.56Ϯ1.22 versus 0.64Ϯ0.13 ng/10 6 platelets, PϽ0.025). In contrast, there were no differences in thrombin-stimulated (1 U/mL) 12(S)-HETE generation: 7.66Ϯ2.14 in patients versus 4.87Ϯ1.46 in control subjects (Pϭ0.61). Platelet 12-LO protein levels, measured by Western blotting with a polyclonal antibody, were higher in the patients than in the control subjects. The urinary excretion of 12(S)-HETE was higher in patients than in control subjects: 36.8Ϯ7.24 versus 17.1Ϯ3.14 ng/mg creatinine (PϽ0.01). These results indicate that 12(S)-HETE levels and 12-LO protein are increased in patients with essential hypertension, suggesting a role for this metabolite in human hypertension. (Hypertension. 2001;37:334-338.)

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An anti‐platelet agent, OPC‐29030, inhibits translocation of 12‐lipoxygenase and 12‐hydroxyeicosatetraenoic acid production in human platelets

Cited by 27 publications

References 25 publications

Low Density Lipoprotein Receptor-related Protein-mediated Membrane Translocation of 12/15-Lipoxygenase Is Required for Oxidation of Low Density Lipoprotein by Macrophages

Low Density Lipoprotein Receptor-related Protein-mediated Membrane Translocation of 12/15-Lipoxygenase Is Required for Oxidation of Low Density Lipoprotein by Macrophages

Platelet 12-Lipoxygenase Activation via Glycoprotein VI

Increased Levels of 12(S)-HETE in Patients With Essential Hypertension

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