Stimulated cholesterol-enriched platelets display increased cytosolic Ca2+ and phospholipase A activity independent of changes in inositol trisphosphates and agonist/receptor binding.
To investigate the mechanism of enhanced responsiveness of cholesterol-enriched human platelets, we compared stimulation by surface-membrane-receptor (thrombin) and post-receptor (AlF4-) G-protein-directed pathways. Platelets were labelled with [32P]Pi and [methyl-3H] choline chloride, incubated with sonicated lipid dispersions of various ratios of cholesterol and phospholipid, and loaded with the fluorescent Ca2+ indicator fura-2. We report the following. (1) Cholesterol enrichment enhances cytosolic Ca2+ accumulation and phospholipase A activation in response to both receptor-directed and post-receptor-directed agonists. No enhancement by cholesterol of phospholipase A activity at fixed Ca2+ concentrations is observed in lysed platelets, implying that no perturbation by cholesterol of phospholipase A/substrate interaction occurs in our preparations. (2) In both normal and cholesterol-enriched platelets, Ca2+ mobilization is promoted by a factor(s) apart from InsP3 that appear(s) to be modulated by cholesterol. A disproportionate increase in cytosolic Ca2+ relative to [32P]InsP3 is observed with increasing doses of thrombin in normal, and to a larger extent in cholesterol-enriched, platelets. When AlF4- is the agonist, there is no cholesterol-associated enhancement in [32P]InsP3 to account for the heightened Ca2+ rise seen with cholesterol enrichment. (3) Enhanced phospholipase A activation is not necessarily proportional to cytosolic Ca2+ increase. The magnitude of the increase in phospholipase A activity for a given rise in cytosolic Ca2+ is greater in cholesterol-enriched platelets that are stimulated by AlF4- than in those stimulated by thrombin. We conclude that increased membrane microviscosity associated with cholesterol enrichment may promote G-protein/phospholipase A interaction as well as the Ca2(+)-release mechanism, without significantly altering G-protein/phospholipase C interaction.
Sorisky
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A
Metabolic Unit, University of Vermont College of Medicine, Burlington 05405.
Kucera
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Rittenhouse
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D001792:Blood Platelets / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002784:Cholesterol / Q000494:pharmacology*; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D008055:Lipids / Q000032:analysis; D010740:Phospholipases / Q000378:metabolism*; D010741:Phospholipases A / Q000378:metabolism*; D010974:Platelet Aggregation; D015398:Signal Transduction / Q000187:drug effects; D013917:Thrombin / Q000378:metabolism
D008055:Lipids; D015544:Inositol 1,4,5-Trisphosphate; D002784:Cholesterol; D010740:Phospholipases; D010741:Phospholipases A; D013917:Thrombin; D002118:Calcium