Palmitic acid-labeled lipids selectively incorporated into platelet cytoskeleton during aggregation |
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Authors: | M A Packham M A Guccione N L Bryant A Livne |
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Affiliation: | (1) Department of Biochemistry, University of Toronto, M5S 1A8 Toronto, Ontario, Canada;(2) Department of Biology, ben-Gurion University of the Negev, Beersheva, Israel |
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Abstract: | Previous experiments showed that during the early stages (20–30 seconds) of aggregation induced by adenosine diphosphate (ADP,
2 μM) or thrombin (0.1 U/mL) of rabbit or human platelets prelabeled with 3H]palmitic acid, labeled lipid became associated with the cytoskeleton isolated after lysis with 1% Triton X-100, 5 mM EGTA
ethylene glycol-bis-(β-aminoethyl ether)]-N,N,N',N'-tetra-acetic acid. The association appeared to be related to the number of sites of contact and was independent of the release
of granule contents. We have now investigated the nature of the labeled lipids by thin-layer and column chromatography and
found differences between the distribution of the label in intact platelets (both stimulated and unstimulated) and the isolated
cytoskeletons. In both species and with either ADP or thrombin as aggregating agent, 70–85% of the label in both intact platelets
and in the cytoskeletons was in phospholipids. The distribution of label among the phospholipids in the cytoskeletons was
similar to that in intact platelets except that the percentage of label in phosphatidylcholine was significantly higher in
the cytoskeletons of human platelets than in the intact platelets, and the percentage of label in phosphatidylserine/phosphatidylinositol
was significantly lower in the cytoskeletons of rabbit platelets and thrombin-aggregated human platelets than in intact platelets.
The cytoskeletons contained a lower percentage of label in triacylglycerol, diacylglycerol, and cholesterol ester than the
intact platelets. Contrary to a report in the literature, we found no evidence for the incorporation of diacylglycerol and
palmitic acid into the cytoskeleton. Although intact rabbit platelets had more label in ceramide (6.7±2.9%) than intact human
platelets (1.5±0.9%), platelets of both species exhibited a three- to four-fold enrichment of labeled ceramide in the cytoskeletons.
Thus phospholipids and ceramide that are readily labeled with palmitic acid are selectively incorporated into the cytoskeleton
during the initial stages of platelet aggregation. |
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