Studies on the formation of leukotriene C4 and lipoxins from leukotriene A4 in human platelets

Sammanfattning: Human platelets lack 5-lipoxygenase activity, but possess enzymatic capacity to transformthe unstable epoxide leukotriene (LT)A4 to the potent inflammatory agent LTC4 and lipoxins, agroup of trihydroxylated tetraene-containing eicosanoids, which recently have been suggestedto have an anti-inflammatory role. A novel pathway for the formation of lipoxin (LX)A4 via platelet lipoxygenation of SS,6R-diHETE and 5S,6S-diHETE, enzymatic/non-enzymatic hydrolysis products of LTA4, wasdiscovered in human platelets. In contrast, human platelets did not transform cysteinylleukotrienes to the corresponding cysteinyl lipoxins. The results indicated that platelet-dependent lipoxin formation from LTA4 can proceed both via a direct lipoxygenation, yielding15-OH-LTA4, and through initial hydrolysis to 5,6-diHETEs. The hydroxylation of LTA4 atC-15 was more efficiently catalysed by human platelet and porcine leukocyte 12-lipoxygenasesthan by rabbit reticulocyte and soybean 15-lipoxygenases. In kinetic studies, human platelet12-lipoxygenase was found to have a high affinity for LTA4, with a Km value comparable tothose earlier reported for arachidonic acid. Platelet activation resulted in elevated lipoxin synthesis. This activation was due to stimula-tion of 12-lipoxygenase by endogenous 12-HPETE, formed after calcium-dependentphospholipase activation and liberation of arachidonic acid. In contrast to the increased lipoxin formation, platelet activation attenuated the conversion ofLTA4 to LTC4. This inhibition of platelet LTC4 synthase activity was observed both afterreceptor-mediated activation of human platelets as well as after direct stimulation of PKC withPMA. These effects were blocked by pretreatment with the protein kinase inhibitor stauro-sporine, supporting the possible regulation of LTC4 synthase through phosphorylationpathways. This was further suggested by kinetic studies demonstrating non-competitiveinhibition of the enzyme after incubation of intact platelets with thrombin or PMA. However,receptor-mediated attenuation of LTC4 formation could not be reversed by specific PKCinhibitors, even though these drugs efficiently prevented the PMA-induced effect. Theseresults indicate that the LTC4 synthase activity in platelets is phosphoregulated, both via PKC-dependent and via receptor-mediated, PKC-independent mechanisms. Involvement of proteintyrosine phosphorylations in the latter process was suggested by the finding that a tyrosinephosphatase inhibitor induced dose-dependent inhibition of LTC4 production in plateletsonicates. In agreement with the findings in platelets, PKC-induced down-regulation of the LTC4synthase activity was also demonstrated in human granulocytes. Aspirin treatment in vivo was found to block attenuation of platelet LTC4 synthase activityinduced by arachidonic acid or collagen ex vivo. The results suggest that aspirin treatment canuncouple mechanisms that normally restrict the production of cysteinyl leukotrienes, which areimportant bronchoconstrictors and asthma mediators. These findings may therefore be relevantfor our understanding of the pathophysiology of aspirin-induced asthma. The effects of the anti-inflammatory drug sulfasalazine on the arachidonic acid cascade inhuman platelets and leukocytes were investigated. The drug was found to inhibit the produc-tion of leukotrienes and tromboxanes but allowed formation of prostaglandin E2. Thispharmacological profile may contribute to the clinical mechanism of action of this drug. In conclusion: Platelet 12-lipoxygenase is well suited to initiate the conversion of LTA4 tolipoxins, indicating a physiological role for this enzyme in lipoxin formation. Also, plateletactivation may lead to elevated formation of putative anti-inflammatory lipoxins paralleled bysuppressed formation of the pro-inflammatory LTC4. According to the present findings, regu-lation of LTC4 synthase activity can be exerted via receptor-mediated, phosphoregulatorymechanisms and may be of importance in aspirin-induced asthma. ISBN 91-628-2109-1