Lipid mediator class switching during acute inflammation: signals in resolution.

Leukotrienes (LTs) and prostaglandins (PGs) amplify acute inflammation, whereas lipoxins (LXs) have unique anti-inflammatory actions. Temporal analyses of these eicosanoids in clinical and experimental exudates showed early coordinate appearance of LT and PG with polymorphonuclear neutrophil (PMN) recruitment. This was followed by LX biosynthesis, which was concurrent with spontaneous resolution. Human peripheral blood PMNs exposed to PGE2 (as in exudates) switched eicosanoid biosynthesis from predominantly LTB4 and 5-lipoxygenase (5-LO)-initiated pathways to LXA4, a 15-LO product that "stopped" PMN infiltration. These results indicate that first-phase eicosanoids promote a shift to anti-inflammatory lipids: functionally distinct lipid-mediator profiles switch during acute exudate formation to "reprogram" the exudate PMNs to promote resolution.

Selectivity of recombinant human leukotriene D(4), leukotriene B(4), and lipoxin A(4) receptors with aspirin-triggered 15-epi-LXA(4) and regulation of vascular and inflammatory responses.

Aspirin-triggered lipoxin A(4) (ATL, 15-epi-LXA(4)) and leukotriene D(4) (LTD(4)) possess opposing vascular actions mediated via receptors distinct from the LXA(4) receptor (ALX) that is involved in leukocyte trafficking. Here, we identified these receptors by nucleotide sequencing and demonstrate that LTD(4) receptor (CysLT(1)) is induced in human vascular endothelia by interleukin-1beta. Recombinant CysLT(1) receptor gave stereospecific binding with both [(3)H]-LTD(4) and a novel labeled mimetic of ATL ([(3)H]-ATLa) that was displaced with LTD(4) and ATLa ( approximately IC(50) 0.2 to 0.9 nmol/L), but not with a bioinactive ATL isomer. The clinically used CysLT(1) receptor antagonist, Singulair, showed a lower rank order for competition with [(3)H]-ATLa (IC(50) approximately 8.3 nmol/L). In contrast, LTD(4) was an ineffective competitive ligand for recombinant ALX receptor with [(3)H]-ATLa, and ATLa did not compete for [(3)H]-LTB(4) binding with recombinant LTB(4) receptor. Endogenous murine CysLT(1) receptors also gave specific [(3)H]-ATLa binding that was displaced with essentially equal affinity by LTD(4) or ATLa. Systemic ATLa proved to be a potent inhibitor (>50%) of CysLT(1)-mediated vascular leakage in murine skin (200 microg/kg) in addition to its ability to block polymorphonuclear leukocyte recruitment to dorsal air pouch (4 microg/kg). These results indicate that ATL and LTD(4) bind and compete with equal affinity at CysLT(1), providing a molecular basis for aspirin-triggered LXs serving as a local damper of both vascular CysLT(1) signals as well as ALX receptor-regulated polymorphonuclear leukocyte traffic.

Novel functional sets of lipid-derived mediators with antiinflammatory actions generated from omega-3 fatty acids via cyclooxygenase 2-nonsteroidal antiinflammatory drugs and transcellular processing.

Aspirin therapy inhibits prostaglandin biosynthesis without directly acting on lipoxygenases, yet via acetylation of cyclooxygenase 2 (COX-2) it leads to bioactive lipoxins (LXs) epimeric at carbon 15 (15-epi-LX, also termed aspirin-triggered LX [ATL]). Here, we report that inflammatory exudates from mice treated with omega-3 polyunsaturated fatty acid and aspirin (ASA) generate a novel array of bioactive lipid signals. Human endothelial cells with upregulated COX-2 treated with ASA converted C20:5 omega-3 to 18R-hydroxyeicosapentaenoic acid (HEPE) and 15R-HEPE. Each was used by polymorphonuclear leukocytes to generate separate classes of novel trihydroxy-containing mediators, including 5-series 15R-LX(5) and 5,12,18R-triHEPE. These new compounds proved to be potent inhibitors of human polymorphonuclear leukocyte transendothelial migration and infiltration in vivo (ATL analogue > 5,12,18R-triHEPE > 18R-HEPE). Acetaminophen and indomethacin also permitted 18R-HEPE and 15R-HEPE generation with recombinant COX-2 as well as omega-5 and omega-9 oxygenations of other fatty acids that act on hematologic cells. These findings establish new transcellular routes for producing arrays of bioactive lipid mediators via COX-2-nonsteroidal antiinflammatory drug-dependent oxygenations and cell-cell interactions that impact microinflammation. The generation of these and related compounds provides a novel mechanism(s) for the therapeutic benefits of omega-3 dietary supplementation, which may be important in inflammation, neoplasia, and vascular diseases.

Aspirin-tolerant asthmatics generate more lipoxins than aspirin-intolerant asthmatics.

Asthma is characterized by chronic airway inflammation resulting from overproduction of pro-inflammatory mediators, such as leukotrienes (LT). The authors questioned the biosynthetic capacity of asthmatic patients for lipoxins (LX) and 15-epimer lipoxins (15-epi-LX), endogenous regulators of inflammatory responses that inhibit pro-inflammatory events. Levels of LXA4, 15-epi-LXA4 and LTC4 were determined in 14 clinically characterized aspirin-intolerant asthmatics (AIA), 11 aspirin-tolerant asthmatics (ATA) and eight healthy volunteers using a stimulated whole blood protocol. Both LXA4 and 15-epi-LXA4 were generated in whole blood activated by the divalent cation ionophore, A23187. Higher levels of LXA4 were produced in ATA than either AIA or healthy volunteers. Exposure of AIA whole blood to interleukin-3 prior to A23187 did not elevate their reduced capacity to generate LXA4. Generation of a bronchoconstrictor, LTC4, was similar in both AIA and ATA. Consequently, the ratio of LXA4:LTC4 quantitatively favoured the bronchoconstrictor for AIA and differed from both ATA and healthy subjects. In addition, the capacity for 15-epi-LXA4 generation was also diminished in AIA, since whole blood stimulated in the presence of aspirin gave increased levels only in samples from ATA. The present results indicate that asthmatics possess the capacity to generate both lipoxins and 15-epimer-lipoxins, but aspirin-intolerant asthmatics display a lower biosynthetic capacity than aspirin-tolerant asthmatics for these potentially protective lipid mediators. This previously unappreciated, diminished capacity for lipoxin formation by aspirin-intolerant asthmatic patients may contribute to their more severe clinical phenotype, and represents a novel paradigm for the development of chronic inflammatory disorders.

Lipoxins, aspirin-triggered 15-epi-lipoxin stable analogs and their receptors in anti-inflammation: a window for therapeutic opportunity.

LXs and 15-epimer LXs are generated during cell-cell interactions that occur during multicellular host response to inflammation, tissue injury or host defense. Results indicate that they are present in vivo during human illness and carry predominantly counter-regulatory biological actions opposing the action of well-characterized mediators of inflammation that appear to lead to resolution of the inflammatory response or promotion of repair and wound healing. The first selective receptor of LXA4 was identified by direct ligand binding and was cloned and characterized. Its signaling involves a novel polyisoprenyl-phosphate pathway that directly regulates PLD (Levy et al. 1999a). LX- and 15-epimer-LX-stable analogs that resist metabolic inactivation were designed, synthesized and shown to be potent LX mimetics and novel topically active anti-inflammatory agents in animal models. These new investigational tools enable structure-function studies of LX signal transduction, further elucidation of the role of LX and 15-epimer LX in host responses and exploitation of their potent bioactions in the design of novel pharmacologic agents.

Activation of lipoxin A(4) receptors by aspirin-triggered lipoxins and select peptides evokes ligand-specific responses in inflammation.

Lipoxin (LX) A(4) and aspirin-triggered LX (ATL) are endogenous lipids that regulate leukocyte trafficking via specific LXA(4) receptors (ALXRs) and mediate antiinflammation and resolution. ATL analogues dramatically inhibited human neutrophil (polymorphonuclear leukocyte [PMN]) responses evoked by a potent necrotactic peptide derived from mitochondria as well as a rogue synthetic chemotactic peptide. These bioactive lipid analogues and small peptides each selectively competed for specific (3)H-LXA(4) binding with recombinant human ALXR, and its N-glycosylation proved essential for peptide but not LXA(4) recognition. Chimeric receptors constructed from receptors with opposing functions, namely ALXR and leukotriene B(4) receptors (BLTs), revealed that the seventh transmembrane segment and adjacent regions of ALXR are essential for LXA(4) recognition, and additional regions of ALXR are required for high affinity binding of the peptide ligands. Together, these findings are the first to indicate that a single seven-transmembrane receptor can switch recognition as well as function with certain chemotactic peptides to inhibitory with ATL and LX (lipid ligands). Moreover, they suggest that ALXR activation by LX or ATL can protect the host from potentially deleterious PMN responses associated with innate immunity as well as direct effector responses in tissue injury by recognition of peptide fragments.

Anti-microinflammatory lipid signals generated from dietary N-3 fatty acids via cyclooxygenase-2 and transcellular processing: a novel mechanism for NSAID and N-3 PUFA therapeutic actions.

Aspirin therapy inhibits prostaglandin biosynthesis; yet via acetylation of cyclooxygenase 2 (COX-2) it leads to bioactive lipoxins epimeric at carbon 15 (15-epi-LX, also termed aspirin-triggered lipoxin or ATL). Here, we review our findings indicating that inflammatory exudates from mice treated with omega-3 PUFA and aspirin (ASA) generate a novel array of bioactive lipid signals. Also, human endothelial cells, both HUVEC and microvascular, with upregulated COX-2 and treated with ASA converted C20:5 omega-3 to 18R-hydroxyeicosapentaenoic acid (HEPE) and 15R-HEPE. Human PMN activated with serum treated zymosan (STZ) utilized each of these R-HEPEs to generate novel classes of trihydroxy-containing mediators including 5-series 15R-LX and 5,12,18R-triHEPE. The novel products were potent inhibitors of human PMN transendothelial migration and infiltration of PMN in dorsal air pouches in vivo. In addition to ASA, both acetaminophen and indomethacin also permitted 18R-HEPE and 15R-HEPE generation with recombinant human COX-2 as well as omega-5 and omega-9 oxygenations of other fatty acids that act on leukocytes, platelets and endothelial cells. These findings establish new transcellular routes for producing arrays of lipid mediators via COX-2-NSAIDs and cell-cell interactions that impact microinflammation. Moreover, they provide novel mechanism(s) that could underlie the many reported therapeutic benefits of omega-3 dietary supplementation of interest in inflammation, cancer, and vascular disorders.

Leukotriene B4 receptor transgenic mice reveal novel protective roles for lipoxins and aspirin-triggered lipoxins in reperfusion.

Polymorphonuclear neutrophil (PMN) activation is pivotal in acute inflammation and injury from reperfusion. To elucidate components controlling PMNs in vivo, we prepared novel transgenic mice with the human leukotriene (LT) B4 receptor (BLTR) for functional characterization. Overexpression of BLTR in leukocytes dramatically increased PMN trafficking to skin microabscesses and lungs after ischemia-reperfusion, whereas mice deficient in 5-lipoxygenase (5-LO) showed diminished PMN accumulation in reperfused lungs. Hence, both BLTR expression and LT biosynthesis are critical for PMN infiltration in reperfusion-initiated second-organ injury. Also, in BLTR transgenic mice, 5-LO expression and product formation were selectively increased in exudates, demonstrating that receptor overexpression amplifies proinflammatory circuits. Endogenous lipoxin (LX) A4 was produced in ischemic lungs and elevated by reperfusion. Because LXA4 and aspirin-triggered 15-epimeric LXA4 (ATL) selectively regulate leukocyte responses, they were tested in BLTR transgenic mice. Despite excessive PMN recruitment in BLTR transgenic mice, intravenous injection of ATL sharply diminished reperfusion-initiated PMN trafficking to remote organs, and topical application of LX was protective in acute dermal inflammation. These results demonstrate a direct role for BLTR with positive feedback, involving BLTR and 5-LO signaling in controlling PMNs. Moreover, LXA4 and ATL counter BLTR-amplified networks, revealing a novel protective role for LX and ATL in stress responses that has applications in perioperative medicine.

Local and systemic delivery of a stable aspirin-triggered lipoxin prevents neutrophil recruitment in vivo.

Aspirin (ASA) triggers a switch in the biosynthesis of lipid mediators, inhibiting prostanoid production and initiating 15-epi-lipoxin generation through the acetylation of cyclooxygenase II. These aspirin-triggered lipoxins (ATL) may mediate some of ASA's beneficial actions and therefore are of interest in the search for novel antiinflammatories that could manifest fewer unwanted side effects. Here, we report that design modifications to native ATL structure prolong its biostability in vivo. In mouse whole blood, ATL analogs protected at carbon 15 [15(R/S)-methyl-lipoxin A4 (ATLa1)] and the omega end [15-epi-16-(para-fluoro)-phenoxy-LXA4 (ATLa2)] were recoverable to approximately 90 and 100% at 3 hr, respectively, compared with a approximately 40% loss of native lipoxin A4. ATLa2 retains bioactivity and, at levels as low as approximately 24 nmol/mouse, potently inhibited tumor necrosis factor-alpha-induced leukocyte recruitment into the dorsal air pouch. Inhibition was evident by either local intra-air pouch delivery (approximately 77% inhibition) or systemic delivery by intravenous injection (approximately 85% inhibition) and proved more potent than local delivery of ASA. Rank order for inhibiting polymorphonuclear leukocyte infiltration was: ATLa2 (10 micrograms, i.v.) approximately ATLa2 (10 micrograms, local) approximately dexamethasone (10 micrograms, local) >ASA (1.0 mg, local). Applied topically to mouse ear skin, ATLa2 also inhibited polymorphonuclear leukocyte infiltration induced by leukotriene B4 (approximately 78% inhibition) or phorbol ester (approximately 49% inhibition), which initiates endogenous chemokine production. These results indicate that this fluorinated analog of natural aspirin-triggered lipoxin A4 is bioavailable by either local or systemic delivery routes and is a more potent and precise inhibitor of neutrophil accumulation than is ASA.

Lipoxin and aspirin-triggered 15-epi-lipoxin cellular interactions anti-inflammatory lipid mediators.

Eicosanoids are known to play important roles in inflammation. Recent findings have given rise to several new concepts regulating the generation of eicosanoids, illustrated in Figure 1. Lipoxins (LX) are trihydroxytetraene-containing eicosanoids that are generated within vascular lumen by platelet-leukocyte interactions and at mucosal surfaces by leukocyte-epithelial cell interactions. During these cell-cell interactions, transcellular biosynthetic pathways are used as major routes, and thus, in humans, LX are formed in vivo during multicellular responses such as inflammation, atherosclerosis, and thrombosis. This branch of the eicosanoid cascade generates specific tetraene-containing products that appear to function as stop signals, since they inhibit key steps in leukocyte-mediated inflammation. Of special interest, it appears that aspirin also functions in part via production of novel epimers of lipoxins or 15-epi-lipoxins (Figure 1). Here, we review recent developments on the cellular interactions of these novel anti-inflammatory mediators.

Transcellular regulation of eicosanoid biosynthesis.

Models for in vivo scenarios of transcellular biosynthesis provide invaluable information about the regulation of eicosanoid biosynthesis that is likely to occur during multicellular events in vivo. The experimental approach of studying eicosanoid generation during cell-cell interactions and receptor-mediated cell activation represents a significant advancement beyond initial observations of eicosanoid formation and bioaction in isolated cell types that were activated under less physiologically relevant conditions. The experimental models reviewed in this chapter should be viewed as specific examples or as approaches to the study of cell-cell interactions. These examples may serve as guidelines to investigate novel cell-cell scenarios (see Fig. 3) and advance the emerging area of transcellular biosynthesis of bioactive lipid mediators.

Pathogen-induced chemokine secretion from model intestinal epithelium is inhibited by lipoxin A4 analogs.

Enteric pathogens induce intestinal epithelium to secrete chemokines that direct movement of polymorphonuclear leukocytes. Mechanisms that might downregulate secretion of these proinflammatory chemokines and thus contain intestinal inflammation have not yet been elucidated. The antiinflammatory activities exhibited by the arachidonate metabolite lipoxin A4 (LXA4) suggests that this eicosanoid, which is biosynthesized in vivo at sites of inflammation, might play such a role. We investigated whether chemokine secretion could be regulated by stable analogs of LXA4. Monolayers of T84 intestinal epithelial cells were infected with Salmonella typhimurium, which elicits secretion of distinct apical (pathogen-elicited epithelial chemoattractant) and basolateral (IL-8) chemokines. Stable analogs of LXA4 inhibited S. typhimurium-induced (but not phorbol ester-induced) secretion of both IL-8 and pathogen-elicited epithelial chemoattractant. LXA4 stable analogs did not alter bacterial adherence to nor internalization by epithelia, indicating that LXA4 stable analogs did not block all signals that Salmonella typhimurium activates in intestinal epithelia, but likely led to attenuation of signals that mediate chemokine secretion. Inhibition of S. typhimurium-induced IL-8 secretion by LXA4 analogs was concentration- (IC50 approximately 1 nM) and time-dependent (maximal inhibition approximately 1 h). As a result of these effects, LXA4 stable analogs inhibited the ability of bacteria-infected epithelia to direct polymorphonuclear leukocyte movement. These data suggest that LXA4 and its stable analogs may be useful in downregulating active inflammation at mucosal surfaces.

Identification of a human enterocyte lipoxin A4 receptor that is regulated by interleukin (IL)-13 and interferon gamma and inhibits tumor necrosis factor alpha-induced IL-8 release.

Epithelial cells of the alimentary tract play a central role in mucosal immunophysiology. Pathogens and/or agonists that interact with mucosal surfaces often elicit epithelial responses that upregulate inflammation. Therefore, it was of interest to explore potential epithelial targeted antiinflammatory signals. Here we identified and sequenced a human enterocyte lipoxin (LX) A4 [5(S), 6(R),15(S)-trihydroxy-7,9,13-trans-11-cis eicosatetraenoic acid] receptor, and demonstrate that transcription of this receptor was controlled by cytokines, of which lymphocyte-derived interleukin (IL)-13 and interferon gamma were the most potent. When lipoxins and LXA4 stable analogs were evaluated for enterocyte functional as well as immune responses, lipoxins sharply inhibited TNF-alpha-induced IL-8 release but did not alter either barrier function or agonist-stimulated chloride secretion. 15R/S-methyl-LXA4 and 16-phenoxy-LXA4 each attenuated (IC50 approximately 10 nM) IL-8 release. Cyclooxygenase (COX) II is emerging as an important component in wound healing and proliferation in intestinal epithelia and when acetylated by acetylsalicylic acid (aspirin) initiates the biosynthesis of a LXA4 receptor ligand. We therefore determined whether colonic cell lines (HT-29 Cl.19A, Caco-2, or T84) express the COX II isozyme. Results for RT-PCR and Western blot analysis showed that COX I as well as an IL-1beta- and TNF-alpha-inducible COX II are expressed in HT-29 Cl.19A. In addition, aspirin-treated enterocytes generated 15R-HETE, a precursor of 15-epi-LXA4 biosynthesis, whose potent bioactions were mimicked by the stable analog 15R/S-methyl-LXA4. Taken together, these results identify an endogenous pathway for downregulating mucosal inflammatory events and suggest a potential therapeutic benefit for LXA4 stable analogs.

Characterization of human neutrophil and endothelial cell ligand-operated extracellular acidification rate by microphysiometry: impact of reoxygenation.

Neutrophil (PMN) activation and recruitment are coordinated by ligand-operated surface receptors. These responses are involved in the tissue injury that follows hypoxia/reoxygenation. Here, we report that inflammatory mediators each evoke distinct and characteristic extracellular acidification rates (EAR) in both PMN and endothelial cells (EC) as measured by a Cytosensor microphysiometer. Leukotriene B4 (LTB4) and the peptide N-formylmethionyl-leucyl-phenylalanine were the most potent activators of EAR, whereas other potent stimuli including interleukin-8 and platelet-activating factor only weakly stimulated EAR in PMN. In contrast, other lipid-derived PMN mediators such as prostaglandin E2 and lipoxin A4 (LXA4) did not evoke EAR. Ligand-operated EAR exhibited desensitization as well as ligand specificity and sensitivity to pertussis toxin. Human endothelial cell agonists including histamine, prostacyclin stable analog and LXA4 each gave sharply different EAR responses, with only histamine evoking an EAR in these cells. Hypoxia/reoxygenation did not alter ligand-operated EAR from PMN, and similarly LTB4-stimulated PMN transendothelial migration, a functional response, was not influenced by either PMN or EC exposure to intervals of hypoxia/reoxygenation. LXA4 stable analogs inhibited PMN transendothelial migration (1 nM-1 microM), and this PMN-EC responsiveness to inhibition by a lipoxin stable analog (e.g., 16-phenoxy-LXA4) was enhanced approximately 2 log orders of magnitude after hypoxia/reoxygenation. Results demonstrate that ligand-receptor interactions evoke characteristic profiles of EAR and that some well-characterized ligand-receptor pairs (including interleukin-8, platelet-activating factor, prostaglandin E2 or LXA4) on these cell types either weakly activate the EAR pathway or are silent. Furthermore, hypoxia/reoxygenation did not alter LTB4 PMN responses but did heighten responsiveness to 16-phenoxy-LXA4, which suggests a potential protective role in leukocyte-mediated injury.

Neutrophil-mediated changes in vascular permeability are inhibited by topical application of aspirin-triggered 15-epi-lipoxin A4 and novel lipoxin B4 stable analogues.

Neutrophil (PMN) activation is critical in inflammation and reperfusion injury, suggesting that PMN-directed therapies may be of clinical use. Here, leukotriene B4 (LTB4)-induced PMN influx in ear skin was equivalent between 5-lipoxygenase knockout and wild-type mice. To explore actions of lipoxin (LX) in PMN-mediated tissue injury, we prepared several novel LX stable analogues, including analogues of LXA4 and aspirin-triggered 15-epi-LXA4 as well as LXB4, and examined their impact in PMN infiltration and vascular permeability. Each applied topically to mouse ears inhibited dramatically PMN-mediated increases in vascular permeability (IC50 range of 13-26 nmol) with a rank order of 15(R/S)-methyl-LXA4 > 16-para-fluoro-phenoxy-LXA4 approximately 5(S)-methyl-LXB4 >/= 16-phenoxy-LXA4 > 5(R)-methyl-LXB4. These LX mimetics were as potent as an LTB4 receptor antagonist, yet results from microphysiometry with mouse leukocytes indicated that they do not act as LTB4 receptor level antagonists. In addition, within 24 h of delivery, > 90% were cleared from ear biopsies. Neither IL-8, FMLP, C5a, LTD4, nor platelet-activating factor act topically to promote PMN influx. When applied with LTB4, PGE2 enhanced sharply both infiltration and vascular permeability, which were inhibited by a fluorinated stable analogue of aspirin-triggered LX. These results indicate that mimetics of LXs and aspirin-triggered 15-epi-LXA4 are topically active in this model and are potent inhibitors of both PMN infiltration and PMN-mediated vascular injury.

Biosynthesis of thromboxane by snake (Elaphe obsoleta) erythrocytes and the requirement of eicosanoid production for blood clotting.

Lower vertebrates provide important insights into the evolution of eicosanoid synthesis and function. Whole snake blood, purified nucleated erythrocytes, and isolated leukocytes activated by clotting or A23187 produced thromboxane, PGE2, and 5-lipoxygenase products. Indomethacin's complete inhibition of clotting suggests eicosanoids produced by these cells are important in snake blood hemostasis.

Thrombocytes are the predominant source of endogenous sulfidopeptide leukotrienes in the American bullfrog (Rana catesbeiana).

Nucleated bullfrog erythrocytes have 5-lipoxygenase (LO) and are the first non-mammalian cell to exhibit endogenous sulfidopeptide leukotriene (LT) synthesis. Non-nucleated mammalian platelets lack 5-LO, but contribute significantly to LTC4 production by transcellular synthesis. However, nucleated bullfrog thrombocytes have not been examined for 5-LO activity. Endogenous leukotriene synthesis by bullfrog thrombocytes and mixed leukocytes was analyzed by reverse-phase high-performance liquid chromatography (RP-HPLC). Calcium ionophore activated (A23187) leukocytes demonstrated 5-LO, 12-LO, and 15-LO activity. Spectral analysis demonstrated synthesis of LTB4, LTB4 isomers, 15(S)-monohydroxyicosatetraenoic acid (HETE), 5(S),12(S)-diHETE, 5(S),15(S)-di-HETE, lipoxin A4 (LXA4) and LXB4. Thrombocytes synthesized large quantities of sulfidopeptide leukotrienes but no lipoxins. Sulfidopeptide leukotriene and LTB4 radioimmunoassay analysis and the radiological RP-HPLC profile of [3H]AA metabolism further confirmed synthesis. Incubations with [3H]LTC4 demonstrated slow and incomplete conversion to [3H]LTD4. Thrombocyte leukotriene profile changed over time revealing a significant shift from the LTC4 synthase to LTA4 hydrolase pathway, corresponding with release of large amounts of LTA4. Thrombocytes potentially play a pivotal role in inflammatory and cardiovascular responses. 5-LO activity in amphibian homologs to mammalian platelets and erythrocytes compared with the lack of activity in the mammalian counterparts may correspond to the loss of the nucleus in the evolution of these cells.