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

Abstract

Stimulated platelets, in the presence or absence of aspirin, synthesize significant quantities of 12-hydroxyeicosatetraenoic acid (12-HETE), which is chemotactic and chemokinetic, and enhances mononuclear cell procoagulant activity. During a cellcell interaction between stimulated platelets and unstimulated neutrophils, platelet 12-HETE is metabolized to 12,20-dihydroxyeicosatetraenoic acid (12,20-DiHETE) by neutrophils. Characteristics ofthe enzyme system in unstimulated neutrophils responsible for this -hydroxylation were investigated. A broad range of cytochrome P-450 inhibitors, as well as leukotriene B4, blocked formation of 12,20-DiHETE. Owing largely to released proteases, neutrophil homogenization abolished activity. Pretreatment with diisopropylfluorophosphate preserved activity in neutrophil homogenates. w-Hydroxylation of 12-HETE was confined solely to the microsomal fraction. Specific activity increased 6.6-fold compared with neutrophil sonicates. The electron donor NADPH was a required cofactor. These results indicate that the enzyme in unstimulated human neutrophils, which metabolizes 12-HETE from stimulated platelets to 12,20-DiHETE in this cell-cell interaction, is a cytochrome P-450 monooxygenase.

Thrombin‐activated platelets promote leukotriene B4 synthesis in polymorphonuclear leucocytes stimulated by physiological agonists

“…The product of the co-oxidation of the 5S,12S-DiHETE in the PMNL, the 5S,12S,20-TriHETE (Lindgren et al, 1981), was always formed in substantial amounts, while the 12-HETE metabolite, 12,20-DiHETE (Marcus et al, 1984) was also present but in smaller amounts. The mechanism of formation of these three products of platelet-PMNL interaction have been discussed previously (Lindgren et al, 1981;Marcus et al, 1987). The h.p.l.c.…”

The endothelial cell ecto-ADPase responsible for inhibition of platelet function is CD39.

“…Washed HUVEC monolayers were harvested with collagenase-EDTA, washed twice in HBS and treated with 2 mM diisopropylfluorophosphate in HBS (5 min, 4 Њ C), followed by two more washes (14,15). Cells were sonicated (10 s, 3-5 times), centrifuged (150 g , 10 min), and supernatants differentially centrifuged (10,000 g , 15 min, followed by 100,000 g , 1 h) (14,15). The 10,000 g and 100,000 g pellets were resuspended in Tris-buffered saline (TSG) buffer (15 mM Tris, 134 mM NaCl, 5 mM glucose, 0.5 mM DTT, pH 7.4) (3).…”

Platelet–neutrophil interactions as drivers of inflammatory and thrombotic disease

“…Furthermore, platelets stimulate neutrophil transmigration over the endothelium, likely by a combination of assisting neutrophil adhesion to the endothelium and enhancing endothelial cell permeability (Gros et al 2015a ). Finally, platelets stimulate the formation of various leukotrienes by neutrophils in an intriguing mechanism involving transfer of the arachidonic acid metabolite 12-HETE from platelet to neutrophils, which further process this molecule to bioactive leukotrienes (Marcus et al 1984 , 1987 , 1988 ). This transcellular arachidonic acid pathway presumably requires platelet–neutrophil contact given the key role of P-selectin in this process (Maugeri et al 1994 ).…”