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Lipoxin synthase activity of human platelet 12-lipoxygenase.

ABSTRACT: Human platelets and megacaryocytes generate lipoxins from exogenous leukotriene A4 (LTA4). We examined the role of human 12-lipoxygenase (12-LO) in lipoxin generation with recombinant histidine-tagged human platelet enzyme (6His-12-LO), partially purified 12-LO from human platelets (HPL 12-LO) and, for the purposes of direct comparison, permeabilized platelets. Recombinant and HPL 12-LO catalysed the conversion of intact LTA4 into both lipoxin A4 (LXA4) and lipoxin B4 (LXB4). In contrast, only negligible quantities of LXA4 were generated when recombinant 12-LO was incubated with the non-enzymic hydrolysis products of LTA4.6His-12-LO also converted a non-allylic epoxide, 5(6)-epoxy-(8Z,11Z,14Z)-eicosatrienoic acid. The apparent Km and Vmax. for lipoxin synthase activity of 6His-12-LO were estimated to be 7.9 +/- 0.8 microM and 24.5 +/- 2.5 nmol/min per mg respectively, and the LXB4 synthase activity of this enzyme was selectively regulated by suicide inactivation. Aspirin gave a 2-fold increase in lipoxin formation by platelets but did not enhance the conversion of LTA4 by the recombinant 12-LO. These results provide direct evidence for LXA4 and LXB4 synthase activity of human platelet 12-LO. Moreover, they suggest that 12-LO is a dual-function enzyme that carries both oxygenase and lipoxin synthase activity.

SUBMITTER: Romano M

PROVIDER: S-EPMC1137664 | biostudies-other | 1993 Nov

REPOSITORIES: biostudies-other

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5 S,15 S-Dihydroperoxyeicosatetraenoic Acid (5,15-diHpETE) as a Lipoxin Intermediate: Reactivity and Kinetics with Human Leukocyte 5-Lipoxygenase, Platelet 12-Lipoxygenase, and Reticulocyte 15-Lipoxygenase-1.

Project description:The reaction of 5 S,15 S-dihydroperoxyeicosatetraenoic acid (5,15-diHpETE) with human 5-lipoxygenase (LOX), human platelet 12-LOX, and human reticulocyte 15-LOX-1 was investigated to determine the reactivity and relative rates of producing lipoxins (LXs). 5-LOX does not react with 5,15-diHpETE, although it can produce LXA4 when 15-HpETE is the substrate. In contrast, both 12-LOX and 15-LOX-1 react with 5,15-diHpETE, forming specifically LXB4. For 12-LOX and 5,15-diHpETE, the kinetic parameters are kcat = 0.17 s-1 and kcat/ KM = 0.011 μM-1 s-1 [106- and 1600-fold lower than those for 12-LOX oxygenation of arachidonic acid (AA), respectively]. On the other hand, for 15-LOX-1 the equivalent parameters are kcat = 4.6 s-1 and kcat/ KM = 0.21 μM-1 s-1 (3-fold higher and similar to those for 12-HpETE formation by 15-LOX-1 from AA, respectively). This contrasts with the complete lack of reaction of 15-LOX-2 with 5,15-diHpETE [Green, A. R., et al. (2016) Biochemistry 55, 2832-2840]. Our data indicate that 12-LOX is markedly inferior to 15-LOX-1 in catalyzing the production of LXB4 from 5,15-diHpETE. Platelet aggregation was inhibited by the addition of 5,15-diHpETE, with an IC50 of 1.3 μM; however, LXB4 did not significantly inhibit collagen-mediated platelet activation up to 10 μM. In summary, LXB4 is the primary product of 12-LOX and 15-LOX-1 catalysis, if 5,15-diHpETE is the substrate, with 15-LOX-1 being 20-fold more efficient than 12-LOX. LXA4 is the primary product with 5-LOX but only if 15-HpETE is the substrate. Approximately equal proportions of LXA4 and LXB4 are produced by 12-LOX but only if LTA4 is the substrate, as described previously [Sheppard, K. A., et al. (1992) Biochim. Biophys. Acta 1133, 223-234].

| S-EPMC7270142 | biostudies-literature

Investigations of human platelet-type 12-lipoxygenase: role of lipoxygenase products in platelet activation.

Project description:Human platelet-type 12-lipoxygenase (12-LOX) has recently been shown to play an important role in regulation of human platelet function by reacting with arachidonic acid (AA). However, a number of other fatty acids are present on the platelet surface that, when cleaved from the phospholipid, can be oxidized by 12-LOX. We sought to characterize the substrate specificity of 12-LOX against six essential fatty acids: AA, dihomo-?-linolenic acid (DGLA), eicosapentaenoic acid (EPA), ?-linolenic acid (ALA), eicosadienoic acid (EDA), and linoleic acid (LA). Three fatty acids were comparable substrates (AA, DGLA, and EPA), one was 5-fold slower (ALA), and two showed no reactivity with 12-LOX (EDA and LA). The bioactive lipid products resulting from 12-LOX oxidation of DGLA, 12-(S)-hydroperoxy-8Z,10E,14Z-eicosatrienoic acid [12(S)-HPETrE], and its reduced product, 12(S)-HETrE, resulted in significant attenuation of agonist-mediated platelet aggregation, granule secretion, ?IIb?3 activation, Rap1 activation, and clot retraction. Treatment with DGLA similarly inhibited PAR1-mediated platelet activation as well as platelet clot retraction. These observations are in surprising contrast to our recent work showing 12(S)-HETE is a prothrombotic bioactive lipid and support our hypothesis that the overall effect of 12-LOX oxidation of fatty acids in the platelet is dependent on the fatty acid substrates available at the platelet membrane.

| S-EPMC3494251 | biostudies-literature

Protein kinase C regulation of 12-lipoxygenase-mediated human platelet activation.

Project description:Platelet activation is important in the regulation of hemostasis and thrombosis. Uncontrolled activation of platelets may lead to arterial thrombosis, which is a major cause of myocardial infarction and stroke. After activation, metabolism of arachidonic acid (AA) by 12-lipoxygenase (12-LOX) may play a significant role in regulating the degree and stability of platelet activation because inhibition of 12-LOX significantly attenuates platelet aggregation in response to various agonists. Protein kinase C (PKC) activation is also known to be an important regulator of platelet activity. Using a newly developed selective inhibitor for 12-LOX and a pan-PKC inhibitor, we investigated the role of PKC in 12-LOX-mediated regulation of agonist signaling in the platelet. To determine the role of PKC within the 12-LOX pathway, a number of biochemical endpoints were measured, including platelet aggregation, calcium mobilization, and integrin activation. Inhibition of 12-LOX or PKC resulted in inhibition of dense granule secretion and attenuation of both aggregation and αIIbβ(3) activation. However, activation of PKC downstream of 12-LOX inhibition rescued agonist-induced aggregation and integrin activation. Furthermore, inhibition of 12-LOX had no effect on PKC-mediated aggregation, indicating that 12-LOX is upstream of PKC. These studies support an essential role for PKC downstream of 12-LOX activation in human platelets and suggest 12-LOX as a possible target for antiplatelet therapy.

| S-EPMC3286293 | biostudies-literature

Discovery of potent and selective inhibitors of human platelet-type 12- lipoxygenase.

Project description:We report the discovery of novel small molecule inhibitors of platelet-type 12-human lipoxygenase, which display nanomolar activity against the purified enzyme, using a quantitative high-throughput screen (qHTS) on a library of 153607 compounds. These compounds also exhibit excellent specificity, >50-fold selectivity vs the paralogues, 5-human lipoxygenase, reticulocyte 15-human lipoxygenase type-1, and epithelial 15-human lipoxygenase type-2, and >100-fold selectivity vs ovine cyclooxygenase-1 and human cyclooxygenase-2. Kinetic experiments indicate this chemotype is a noncompetitive inhibitor that does not reduce the active site iron. Moreover, chiral HPLC separation of two of the racemic lead molecules revealed a strong preference for the (-)-enantiomers (IC(50) of 0.43 ± 0.04 and 0.38 ± 0.05 ?M) compared to the (+)-enantiomers (IC(50) of >25 ?M for both), indicating a fine degree of selectivity in the active site due to chiral geometry. In addition, these compounds demonstrate efficacy in cellular models, which underscores their relevance to disease modification.

| S-EPMC3150642 | biostudies-literature

12-lipoxygenase activity plays an important role in PAR4 and GPVI-mediated platelet reactivity.

Project description:Following initial platelet activation, arachidonic acid is metabolised by cyclooxygenase-1 and 12-lipoxygenase (12-LOX). While the role of 12-LOX in the platelet is not well defined, recent evidence suggests that it may be important for regulation of platelet activity and is agonist-specific in the manner in which it regulates platelet function. Using small molecule inhibitors selective for 12-LOX and 12-LOX-deficient mice, the role of 12-LOX in regulation of human platelet activation and thrombosis was investigated. Pharmacologically inhibiting 12-LOX resulted in attenuation of platelet aggregation, selective inhibition of dense versus alpha granule secretion, and inhibition of platelet adhesion under flow for PAR4 and collagen. Additionally, 12-LOX-deficient mice showed attenuated integrin activity to PAR4-AP and convulxin compared to wild-type mice. Finally, platelet activation by PARs was shown to be differentially dependent on COX-1 and 12-LOX with PAR1 relying on COX-1 oxidation of arachidonic acid while PAR4 being more dependent on 12-LOX for normal platelet function. These studies demonstrate an important role for 12-LOX in regulating platelet activation and thrombosis. Furthermore, the data presented here provide a basis for potentially targeting 12-LOX as a means to attenuate unwanted platelet activation and clot formation.

| S-EPMC3960303 | biostudies-literature

Increased platelet sensitivity to ADP in mice lacking platelet-type 12-lipoxygenase.

Project description:Arachidonic acid metabolism is one of several mechanisms culminating in the production of an agonist for platelet activation and recruitment. Although the proaggregatory role of thromboxane A2, a product of the aspirin-inhibitable cyclooxygenase, is well established, relatively little is known regarding the biological importance of arachidonic acid metabolism via the 12-lipoxygenase (P-12LO) pathway to 12-hydro(pero)xyeicosatetraenoic acid. We observed that platelets obtained from mice in which the P-12LO gene has been disrupted by gene targeting (P-12LO-/-) exhibit a selective hypersensitivity to ADP, manifested as a marked increase in slope and percent aggregation in ex vivo assays and increased mortality in an ADP-induced mouse model of thromboembolism. The hyperresponsiveness to ADP is independent of dense granule release, cyclooxygenase-derived eicosanoid synthesis, and protein kinase C activity. The addition of 12-hydroxyeicosatetraenoic acid to P-12LO-/- platelet-rich plasma rescues the hyperresponsive phenotype resulting in a diminished ADP-induced aggregation profile. The enhanced ADP sensitivity of P-12LO-/- mice appears to reveal a mechanism by which a product of the P-12LO pathway suppresses platelet activation by ADP.

| S-EPMC19701 | biostudies-literature

Probing the Electrostatic and Steric Requirements for Substrate Binding in Human Platelet-Type 12-Lipoxygenase.

Project description:Human platelet ALOX12 (hALOX12 or h12-LOX) has been implicated in a variety of human diseases. The present study investigates the active site of hALOX12 to more thoroughly understand how it positions the substrate and achieves nearly perfect regio- and stereospecificities (i.e., 100 ± 5% of the 12(S)-hydroperoxide product), utilizing site-directed mutagenesis. Specifically, we have determined that Arg402 is not as important in substrate binding as previously seen for hALOX15 but that His596 may play a role in anchoring the carboxy terminal of the arachidonic acid during catalysis. In addition, Phe414 creates a π-stacking interaction with a double bond of arachidonic acid (Δ11), and Ala417/Val418 define the bottom of the cavity. However, the influence of Ala417/Val418 on the profile is markedly less for hALOX12 than that seen in hALOX15. Mutating these two residues to larger amino acids (Ala417Ile/Val418Met) only increased the generation of 15-HpETE by 24 ± 2%, but conversely, smaller residues at these positions converted hALOX15 to almost 100% hALOX12 reactivity [Gan et al. (1996) J. Biol. Chem. 271, 25412-25418]. However, we were able to increase 15-HpETE to 46 ± 3% by restricting the width of the active site with the Ala417Ile/Val418Met/Ser594Thr mutation, indicating both depth and width of the active site are important. Finally, residue Leu407 is shown to play a critical role in positioning the substrate correctly, as seen by the increase of 15-HpETE to 21 ± 1% for the single Leu407Gly mutant. These results outline critical differences between the active site requirements of hALOX12 relative to hALOX15 and explain both their product specificity and inhibitory differences.

| S-EPMC7270147 | biostudies-literature

Platelet-type 12-lipoxygenase deletion provokes a compensatory 12/15-lipoxygenase increase that exacerbates oxidative stress in mouse islet β cells.

Project description:In type 1 diabetes, an autoimmune event increases oxidative stress in islet β cells, giving rise to cellular dysfunction and apoptosis. Lipoxygenases are enzymes that catalyze the oxygenation of polyunsaturated fatty acids that can form lipid metabolites involved in several biological functions, including oxidative stress. 12-Lipoxygenase and 12/15-lipoxygenase are related but distinct enzymes that are expressed in pancreatic islets, but their relative contributions to oxidative stress in these regions are still being elucidated. In this study, we used mice with global genetic deletion of the genes encoding 12-lipoxygenase (arachidonate 12-lipoxygenase, 12S type [Alox12]) or 12/15-lipoxygenase (Alox15) to compare the influence of each gene deletion on β cell function and survival in response to the β cell toxin streptozotocin. Alox12 -/- mice exhibited greater impairment in glucose tolerance following streptozotocin exposure than WT mice, whereas Alox15 -/- mice were protected against dysglycemia. These changes were accompanied by evidence of islet oxidative stress in Alox12 -/- mice and reduced oxidative stress in Alox15 -/- mice, consistent with alterations in the expression of the antioxidant response enzymes in islets from these mice. Additionally, islets from Alox12 -/- mice displayed a compensatory increase in Alox15 gene expression, and treatment of these mice with the 12/15-lipoxygenase inhibitor ML-351 rescued the dysglycemic phenotype. Collectively, these results indicate that Alox12 loss activates a compensatory increase in Alox15 that sensitizes mouse β cells to oxidative stress.

| S-EPMC6484126 | biostudies-literature

Platelet microparticles are internalized in neutrophils via the concerted activity of 12-lipoxygenase and secreted phospholipase A2-IIA.

Project description:Platelets are anucleated blood elements highly potent at generating extracellular vesicles (EVs) called microparticles (MPs). Whereas EVs are accepted as an important means of intercellular communication, the mechanisms underlying platelet MP internalization in recipient cells are poorly understood. Our lipidomic analyses identified 12(S)-hydroxyeicosatetranoic acid [12(S)-HETE] as the predominant eicosanoid generated by MPs. Mechanistically, 12(S)-HETE is produced through the concerted activity of secreted phospholipase A2 IIA (sPLA2-IIA), present in inflammatory fluids, and platelet-type 12-lipoxygenase (12-LO), expressed by platelet MPs. Platelet MPs convey an elaborate set of transcription factors and nucleic acids, and contain mitochondria. We observed that MPs and their cargo are internalized by activated neutrophils in the endomembrane system via 12(S)-HETE. Platelet MPs are found inside neutrophils isolated from the joints of arthritic patients, and are found in neutrophils only in the presence of sPLA2-IIA and 12-LO in an in vivo model of autoimmune inflammatory arthritis. Using a combination of genetically modified mice, we show that the coordinated action of sPLA2-IIA and 12-LO promotes inflammatory arthritis. These findings identify 12(S)-HETE as a trigger of platelet MP internalization by neutrophils, a mechanism highly relevant to inflammatory processes. Because sPLA2-IIA is induced during inflammation, and 12-LO expression is restricted mainly to platelets, these observations demonstrate that platelet MPs promote their internalization in recipient cells through highly regulated mechanisms.

| S-EPMC4500271 | biostudies-literature

Molecular cloning, primary structure, and expression of the human platelet/erythroleukemia cell 12-lipoxygenase.

Project description:The major pathway of arachidonic acid metabolism in human platelets proceeds via a 12-lipoxygenase enzyme; however, the biological role of the product of this reaction, 12-hydro(pero)xyeicosatetraenoic acid [12-H(P)ETE], is unknown. Using a combination of the polymerase chain reaction and conventional screening procedures, we have isolated cDNA clones encoding the human platelet/human erythroleukemia (HEL) cell 12-lipoxygenase. From the deduced primary structure, human platelet/HEL 12-lipoxygenase would encode a Mr 75,000 protein consisting of 663 amino acids. The cDNA encoding the full-length protein (pCDNA-121x) under the control of the cytomegalovirus promoter was expressed in simian COS-M6 cells. Intact cells and lysed-cell supernatants were able to synthesize 12-H(P)ETE from arachidonic acid, whereas no 12-H(P)ETE synthesis was detected in mock-transfected cells. A single 2.4-kilobase mRNA was detected in erythroleukemia cells but not in several other tissues and cell lines evaluated by Northern blot analysis. Comparison of the human platelet/HEL 12-lipoxygenase sequence with that of porcine leukocyte 12-lipoxygenase and human reticulocyte 15-lipoxygenase revealed 65% amino acid identity to both enzymes. By contrast, the leukocyte 12-lipoxygenase is 86% identical to human reticulocyte 15-lipoxygenase. Sequence data and previously demonstrated immunochemical and biochemical evidence support the existence of distinct 12-lipoxygenase isoforms. The availability of cDNA probes for human platelet/HEL cell 12-lipoxygenase should facilitate elucidation of the biological role of this pathway.

| S-EPMC54382 | biostudies-other

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