Abstract
Exogenous unlabeled arachidonic acid (AA) added to human platelets prelabeled with [3H]AA induces breakdown of [3H]phosphatidylinositol and the rapid and transient formation of [3H]1,2-diacylglycerol and [3H]phosphatidic acid (PA), indicating activation of phosphatidylinositol-specific phospholipase C. Formation of [3H]1,2-diacylglycerol and [3H]PA is inhibited by pretreatment of platelets with aspirin, which suggests that endoperoxides or thromboxane A2 are responsible for AA-induced stimulation of phospholipase C. Exogenous unlabeled AA also induces the formation of [32P]PA or [14C]PA in platelets that have been prelabeled with 32Pi or [14C]AA, respectively. Increased radioactivity in PA reflects increased content of PA as measured by the fatty acid composition of PA. The relation of PA production, which reflects stimulation of phospholipase C, to specific platelet responses was further investigated. Low concentrations of AA (0.05-0.2 microM) induces platelet shape change in parallel to formation of 50-100% PA and phosphorylation of a 40,000 molecular weight protein. Higher concentrations of AA (0.5-50 microM) stimulate the formation of a further amount of PA (200-250%), and phosphorylation of 40,000 molecular weight protein, platelet aggregation, and serotonin release. Indomethacin inhibits all these observed changes by inhibiting the conversion of AA by platelet cyclooxygenase. In contrast, prostacyclin blocks these responses without affecting conversion of AA by platelet cyclooxygenase and thromboxane synthetase. We conclude that formation of endoperoxides and thromboxane A2 is necessary but not sufficient for platelet activation by AA. Only if PA is formed are platelets activated. The results indicate a central role for the phospholipase C pathway in the process of platelet activation.
Highlights
Exogenous unlabeled arachidonic acid(AA) added to vation of phospholipases C and AS [1,2,3,4,5,6]
We suggest that actiparallel to formationof 50-100% P A and phosphoryl- vation of phospholipase C may be acommon pathway for ation of a 40,000 molecular weight protein
Arachidonic Acid Stimulates Phospholipase C in Human extraction [1,6], and PwAas separated by thin layer chromatography Platelets-Addition of exogenousunlabeled AA ( 5 KM) to as described previously [1, 6]. [32P]phosphatidic acid (PA) was localized by autoradiog- washed human platelets prelabeledwith[3H]AA stimulates raphy and the radioactivity measurbeyd liquid scintillation counting. the rapid and transient formationof [3H]1,2-DGand [3H]PA
Summary
At various times as indicated, 0.1-ml aliquots were transferred into tubes containing 3.75 volumes of chloroform/methanol [12]for lipid. Arachidonic Acid Stimulates Phospholipase C in Human extraction [1,6], and PwAas separated by thin layer chromatography Platelets-Addition of exogenousunlabeled AA ( 5 KM) to as described previously [1, 6]. Incubations were stopped by AA (0.2 WM) induces the formation of [I4C]PA during transferring the samples into chloroform/methan(o1l 9)as described platelet shape change 1.2-DIACYLGLYCEROL assays were mixed before the separation of PA by one-dimensional thin layer chromatography [1].PA was localized by a very brief period of autoradiography and thespecific areas of silica gel containingPAisolated.Heptadecanoicacid (Supelco,Belleforte, PA) was added as internal standard in an appropriate amount to each of the samples.
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