vapiprost and lysophosphatidylethanolamine

Two TxA2/PGH2 receptor binding sites linked to different effector systems have recently been identified. Since plasmenylethanolamine represents the major phospholipid reservoir of arachidonic acid (AA) in resting human platelets, we assessed the differential role of these binding sites on plasmenylethanolamine hydrolysis by phospholipase A2 activity upon platelet activation by determining the generation of the corresponding [3H]lysoplasmenylethanolamine. Ethanolamine-containing phospholipids in platelets were pre-labelled with [3H]ethanolamine prior to platelet stimulation with U46619 (1 microM), a TxA2 mimetic, in the presence or absence of S-145, an antagonist of the low affinity TxA2/PGH2 receptor. Labelled platelets were also treated with the TxA2/PGH2 receptor antagonist, GR32191B, prior to washing (which blocks the low affinity site of the receptor) and subsequent stimulation. The above conditions provided for blockage of platelet aggregation but not shape change with U46619. The rise in [3H]lysoplasmenylethanolamine accumulation (170% of unstimulated controls) with U46619 as the agonist was inhibited in platelets pre-treated with S-145 and in platelets washed from GR32191B. Similar findings were also obtained for [3H]lysophosphatidylethanolamine accumulation. The present results indicate that the TxA2-dependent activation of plasmenylethanolamine cleavage by phospholipase A2 in intact human platelets is predominantly linked to the low affinity site of the TxA2/PGH2 receptor and may be important for platelet aggregation but not shape change.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Binding Sites; Biphenyl Compounds; Blood Platelets; Bridged Bicyclo Compounds; Fatty Acids, Monounsaturated; Heptanoic Acids; Humans; Lysophospholipids; Phospholipases A; Phospholipases A2; Plasmalogens; Platelet Aggregation; Prostaglandin Endoperoxides, Synthetic; Receptors, Prostaglandin; Receptors, Thromboxane; Receptors, Thromboxane A2, Prostaglandin H2; Thromboxane A2

Collagen-induced human platelet stimulation is dependent on the release of arachidonic acid (AA) from membrane phospholipid and the formation of thromboxane A2 (TxA2) for TxA2-induced platelet activation. Since plasmenylethanolamine represents the single major phospholipid reservoir of AA in resting human platelets, we assessed its hydrolysis via phospholipase A2 upon platelet stimulation with low levels of collagen by determining the generation of [3H]lysoplasmenylethanolamine via eicosanoid/TxA2-independent and -dependent processes. Ethanolamine phospholipids in platelets were prelabelled with [3H]ethanolamine before stimulation with either collagen or the TxA2 mimetic U46619, in the presence or absence of BW755C, a dual inhibitor of the cyclooxygenase/lipoxygenase activities, or GR32191B, a TxA2-receptor antagonist. Collagen stimulation promoted a marked generation of [3H]lysoplasmenylethanolamine, which was only moderately decreased when TxA2 synthesis or TxA2 receptors were blocked by BW755C or GR32191B respectively. The moderate rise in [3H]lysoplasmenylethanolamine formation with U46619 as the agonist was only slightly affected by BW755C and blocked by GR32191B. Evidence for eicosanoid/TxA2-independent and -dependent generation of [3H]lysophosphatidylethanolamine was also obtained. A significant quantitative loss of AA from plasmenylethanolamine was also demonstrated in collagen-stimulated platelets. The present findings indicate the activation of plasmenylethanolamine cleavage via phospholipase A2 in collagen-stimulated human platelets, which, to a considerable extent, does not depend on eicosanoid/TxA2 synthesis. This may represent an important source of releasable AA for TxA2 generation and the promotion of further liberation of AA and phospholipid-mediated signalling pathways.

Topics: 4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyrazol-3-amine; Adult; Biphenyl Compounds; Collagen; Eicosanoids; Heptanoic Acids; Humans; Lysophospholipids; Male; Phospholipases A; Phospholipases A2; Platelet Activation; Prostaglandin Endoperoxides, Synthetic; Thromboxane A2