New Eicosanoids Formed During Platelet-Neutrophil Interactions

Abstract

In physiologic and pathologic processes such as hemostasis, thrombosis and inflammation, multiple cell types are brought into close proximity - thereby increasing the possibility of metabolic interactions in the microenvironment. Activated platelets synthesize12-hydroxyeicosatetraenoic acid (12-HETE) in the presence or absence of aspirin. During a cell-cell interaction, platelet 12-HETE is metabolized by a cytochrome P-450 enzyme system in unstimulated neutrophils to 12.20-dihydroxyeicosatetraenoic acid (12,20-DiHETE). Recently, we observed time-dependent formation of a new eicosanoid following exposure of neutrophils to 12-HETE. This compound is more polar than the parent eicosanoid 12,20-DiHETE (reversed-phase HPLC). Incubation of purified 12,20-DiHETE with neutrophils resulted in a progressive decrease in the 12,20-DiHETE with increasing formation of the polar metabolite. In the absence of neutrophils, 12.20-DiHETE was quantitatively unchanged. The new metabolite of 12.20-DiHETE has been tentatively identified as 12-hydroxyeicosatetraen-l,20-dioic acid. The UV absorption maximum of the new compound is 237 nm which is identical to that of 12-HETE and 12,20-DiHETE. 20-hydroxy-LTB4 is the omega-hydroxylated derivative of the pro-inflammatory eicosanoid LTB4. When added to neutrophils 15 sec prior to 12,20-DiHETE, equimolar concentrations of 20-hydroxy-LTB4 (2.8uM) inhibited formation of the new metabolite by 28%. A concentration of 8uM 20-hydroxy-LTB4 inhibited the reaction by 49%. These results indicate that the neutrophil enzyme system responsible for conversion of 20-hydroxy-LTB4 to 20-carboxy-LTB4 may also be involved in further metabolism of-12,20-DiHETE. Neutrophil homogenization resulted in loss of the capacity to transform 12.20-DiHETE to the new metabolite despite pretreatment with DFP and addition of NADPH. Our data provide further evidence for the occurrence of transcellular metabolic events during thrombosis and the inflammatory response.

Enhancement of mononuclear procoagulant activity by platelet 12-hydroxyeicosatetraenoic acid.

“…In platelets, free arachidonic acid serves to activate the cyclooxygenase and lipoxygenase. However, lipoxygenation does not occur in leukocytes in the absence of a strong agonist, such as ionophore (23,24). In our experiments, free arachidonate alone had no effect on leukocyte TFa generation in the absence of platelets, suggesting a requirement for metabolic conversion of added arachidonate.…”

“…Lipoxygenase is not inhibited, and continues to oxygenate free arachidonate until it is no longer available (25). Thus, in the presence of aspirin, arachidonate is diverted toward hydroxy acid production (24,25). Aspirin-treatment exerted an enhancing effect on TFa generation (Fig.…”

Studies on the mechanism of omega-hydroxylation of platelet 12-hydroxyeicosatetraenoic acid (12-HETE) by unstimulated neutrophils.

“…Upon return to isotonicity with 2 ml of 3.5% NaCl, 10 ml of phosphate-buffered saline (pH 7.4) was added. The suspension was centrifuged at 280 g for 5 min (4°C), and the neutrophils were suspended in Hepes buffer (pH 7.45) containing calcium and magnesium (8). In experiments involving subcellular fractionation, the Hepes buffer was modified (no calcium, 2.5 mM magnesium).…”

“…We have previously established that neutrophils can metabolize platelet 12-HETE in vitro by two different mechanisms (8,9). Stimulated neutrophils, in which the 5-lipoxygenase has been activated, convert platelet 12-HETE to 5S, 12S-dihydroxy-6-trans,8-cis,10-trans,14-cis-eicosatetraenoic acid (5S,12S-Di-HETE) (8,10).…”

“…Stimulated neutrophils, in which the 5-lipoxygenase has been activated, convert platelet 12-HETE to 5S, 12S-dihydroxy-6-trans,8-cis,10-trans,14-cis-eicosatetraenoic acid (5S,12S-Di-HETE) (8,10). In contrast, unstimulated neutrophils metabolize platelet 12-HETE to (12S)-12,20-dihydroxy-5,8,10,14-eicosatetraenoic acid (12, ( 9, 1 1).…”