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

Abstract

Human platelets lack 5-lipoxygenase activity, but possess enzymatic capacity to transform the unstable epoxide leukotriene (LT)A4 to the potent inflammatory agent LTC4 and lipoxins, a group of trihydroxylated tetraene-containing eicosanoids, which recently have been suggested to 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, was discovered in human platelets. In contrast, human platelets did not transform cysteinyl leukotrienes to the corresponding cysteinyl lipoxins. The results indicated that platelet-dependent lipoxin formation from LTA4 can proceed both via a direct lipoxygenation, yielding 15-OH-LTA4, and through initial hydrolysis to 5,6-diHETEs. The hydroxylation of LTA4 at C-15 was more efficiently catalysed by human platelet and porcine leukocyte 12-lipoxygenasesthan by rabbit reticulocyte and soybean 15-lipoxygenases.

In kinetic studies, human platelet 12-lipoxygenase was found to have a high affinity for LTA4, with a Km value comparable to those earlier reported for arachidonic acid. Platelet activation resulted in elevated lipoxin synthesis. This activation was due to stimulation of 12-lipoxygenase by endogenous 12-HPETE, formed after calcium-dependent phospholipase activation and liberation of arachidonic acid.

In contrast to the increased lipoxin formation, platelet activation attenuated the conversion of LTA4 to LTC4. This inhibition of platelet LTC4 synthase activity was observed both after receptor-mediated activation of human platelets as well as after direct stimulation of PKC with PMA. These effects were blocked by pretreatment with the protein kinase inhibitor stauro-sporine, supporting the possible regulation of LTC4 synthase through phosphorylation pathways. This was further suggested by kinetic studies demonstrating non-competitive inhibition 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 PKC inhibitors, even though these drugs efficiently prevented the PMA-induced effect. These results indicate that the LTC4 synthase activity in platelets is phosphoregulated, both via PKC-dependent and via receptor-mediated, PKC-independent mechanisms. Involvement of protein tyrosine phosphorylations in the latter process was suggested by the finding that a tyrosine phosphatase inhibitor induced dose-dependent inhibition of LTC4 production in platelet sonicates. 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 activity induced by arachidonic acid or collagen ex vivo. The results suggest that aspirin treatment can uncouple mechanisms that normally restrict the production of cysteinyl leukotrienes, which are important bronchoconstrictors and asthma mediators. These findings may therefore be relevant for 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 production of leukotrienes and tromboxanes but allowed formation of prostaglandin E2. This pharmacological 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, platelet activation may lead to elevated formation of putative anti-inflammatory lipoxins paralleled by suppressed formation of the pro-inflammatory LTC4. According to the present findings, regulation of LTC4 synthase activity can be exerted via receptor-mediated, phosphoregulatory mechanisms and may be of importance in aspirin-induced asthma.