Lipoxin generation by permeabilized human platelets.
Human platelets convert leukocyte-derived leukotriene (LT) A4 to lipoxins during transcellular lipoxin biosynthesis. Here, we examined lipoxin generation in intact human platelets and compared it with that elicited from permeabilized platelets. Conversion of LTA4 to lipoxins by permeabilized cells exceeded (10-15 times) that to peptidoleukotrienes, while intact cells exposed to thrombin generated similar amounts of these two series (LT/LX). Permeabilized platelets also generated 3-5 times more lipoxins than intact cells. Lipoxin A4 (LXA4), lipoxin B4 (LXB4), and their respective all-trans isomers were identified by physical methods including HPLC and GC-MS. Chiral analysis of platelet-derived all-trans-containing LXs revealed that greater than 69.5 +/- 0.5% carried alcohol groups in the R configuration at carbons 6 and 14 (e.g., 11-trans-LXA4 and 8-trans-LXB4), respectively. More than 50% of these all-trans LX were formed by isomerization of native LXA4 and LXB4 during isolation. Lipoxin formation with permeabilized platelets gave an apparent Km of 8.9 microM and Vmax of 83.3 ng/(min-10(9) platelets) with maximal conversion in pH range 7-9. In addition, permeabilized platelets converted 14,15-LTA4 and LTA5, but not LTA3, to lipoxins. Consecutive exposure to LTA4 did not alter LXA4 generation but inhibited LXB4 by 40-50%, suggesting that LXB4 formation can be regulated by suicide inactivation. Unlike platelets, human endothelial cells did not convert LTA4 to lipoxins. These results indicate that lipoxin formation is a major route of LTA4 metabolism in thrombin-activated platelets and those that have undergone a loss of membrane barriers.(ABSTRACT TRUNCATED AT 250 WORDS)
Romano
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Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115.
Serhan
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CN
D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D002463:Cell Membrane Permeability; D015777:Eicosanoids / Q000097:blood* / Q000302:isolation & purification; D006801:Humans; D006893:Hydroxyeicosatetraenoic Acids / Q000097:blood*; D016285:Iloprost / Q000494:pharmacology; D066298:In Vitro Techniques; D007536:Isomerism; D007700:Kinetics; D017572:Leukotriene A4; D015289:Leukotrienes / Q000097:blood*; D044045:Lipoxins
D015777:Eicosanoids; D006893:Hydroxyeicosatetraenoic Acids; D017572:Leukotriene A4; D015289:Leukotrienes; D044045:Lipoxins; C040527:lipoxin A4; C042651:lipoxin B4; D016285:Iloprost