Demonstration of leukotriene‐C₄ synthase in platelets and species distribution of the enzyme activity

Abstract

Human platelets have been demonstrated to possess leukotriene (LT)-C 4 synthase activity and may thus be involved in transcellular 5(S)-hydroxy-6(R)-S-glutathionyl-7,9-trans-11,14-cis-eicosatetraenoic acid (LTC 4 ) synthesis. In this study, platelets from seven different species were screened for LTC 4 synthase activity. Very high enzyme activity was observed in suspensions of bovine platelets, with approximately 70% conversion of 5(S)-trans-5,6-oxido-7,9-trans-11,14-cis-eicosatetraenoic acid (LTA 4 ) to LTC 4 . The capacity of equine platelets to produce LTC 4 was similar to that of human platelets. In addition, ovine, rabbit, and rat platelets also produced LTC 4 after incubation with LTA 4 . The results demonstrate that LTC 4 synthase activity is a common feature among platelets from various species. In contrast, porcine platelets failed to transform LTA 4 to LTC 4 . Instead, these cells produced 5(S),12(R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid (LTB 4), indicating the presence of LTA4 hydrolase in porcine platelets.A protein with a molecular mass of approximately 18 kDa and LTC 4 synthase activity was solubilised from lyophilised bovine platelet concentrates and purified to near homogeneity by affinity chromatography and gel filtration. The N-terminal amino acid sequence of this protein was analysed and found to be almost identical to the corresponding sequence of human LTC 4 synthase (17 of 18 amino acid residues identical). Kinetic analysis of partially purified bovine platelet LTC 4 synthase revealed K m (for LTA 4 ) and V max values of 3.3 µM and 521 nmol · mg protein Ϫ1 · min Ϫ1 , respectively. In addition, the presence of a mRNA transcript encoding LTC 4 synthase was demonstrated in equine platelets by reverse transcription (RT) PCR using primers derived from the human LTC 4 synthase cDNA sequence. Cloning and sequencing of the PCR fragment corresponding to a region near the N-terminus demonstrated very high identity between equine and human leukotriene-C 4 synthase in this region.In summary, the present study establishes that platelets contain LTC 4 synthase and indicates that this enzyme is widely distributed among platelets from various species.

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“…(3) The possibility of LTB 4 and LTC 4 production by transcellular biosynthesis is also assumed [31][32][33]. That is, LTA 4 is released into the extracellular space along with LTB 4 , and LTA 4 is converted to LTC 4 by cells that express LTC 4 synthase, such as mast cells, eosinophils, platelets [34][35][36] and endothelial cells [37][38][39]. Nasser et al [40] reported that large numbers of eosinophils and mast cells are present in tissues obtained from AIA patients by bronchial biopsy compared with those in tissues obtained from ATA patients.…”

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“…Interestingly this enzyme is also expressed in platelets, which do not contain 5-LO. (96,97) However, platelets can import LTA 4 generated in 5-LO containing cells to synthesize LTC 4 through a process termed transcellular metabolism (98). The actions of LTB 4 are mediated via at least two distinct G protein-coupled receptors, referred to as BLT 1 and BLT 2 .…”

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“…Treatment of human platelets with helenalin (86) has been shown to cause irreversible inhibition of LTC 4 synthase in a concentration-and time-dependent manner, with an IC 50 of 12µM after a 60-min preincubation (Fig. 26) [39]. This effect was not related to decreased GSH levels or cytotoxicity.…”