Effect of cholesterol and cholesterol analogues on platelet function
McLeod, Andrew J.
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This thesis reports studies carried out to investigate further the effect of alteration of the platelet cholesterol content on platelet function. Recent research has shown that cholesterol - enriched human platelets were capable of synthesising greater quantities of pro- aggregating compounds compared to control and cholesterol- depleted platelets. Phospholipase A2 activity in the platelet membrane may be an important rate limiting step in the metabolism of arachidonic acid to pro- aggregating compounds for example thromboxane A2. Thus in the present project, cholesterol - enriched platelets have been investigated with particular reference to phospholipase A2 activity.Rat platelets were enriched with and depleted of cholesterol by in vitro incubation with cholesterol -rich and cholesterol -poor phospholipid liposome suspensions respectively. Aggregation studies shoved that cholesterol- enriched rat platelets (in platelet -liposome mixtures) were more sensitive to collagen and ADP induced aggregation. Phospholipase A2 assays were carried out by analysing the conversion of phosphatidylcholine containing a radiolabelled sn -2 fatty acid. Rat platelets were resuspended once, and no difference in phospholipase A2 activity was detected between cholesterol- enriched and cholesterol normal platelets.Rabbits were fed a cholesterol -rich diet for 4 weeks. Platelets isolated from cholesterol fed rabbits had a significantly higher cholesterol :phospholipid molar ratio than platelets from rabbits fed a normal diet. The once resuspended cholesterol- enriched rabbit platelets showed significantly higher phospholipase A2 activity compared to control platelets treated in the same way. Also, the cholesterol- enriched rabbit platelets were observed to have a more active arachidonic acid metabolic pathway, as assayed by measuring arachidonic acid induced thromboxane A2 and MDA production.Platelets were also examined from hypercholesterolaemic human subjects and compared with platelets from normal human subjects. In these experiments, no differences were detected in cholesterol content or phospholipase A2 activities of crude platelet membrane fractions prepared from these platelets. This may have been due to this assay not being suitable for the very labile human platelet phospholipase A2 activity.Crude platelet membrane fractions were also prepared from rat platelets with altered cholesterol content. Phospholipase A2 assays consistently showed that the activity was significantly higher in cholesterol -enriched rat platelet membrane fractions than in control platelet membrane fractions. These results and those from the rabbit platelet experiments suggested that platelet hyper sensitivity induced by cholesterol -enrichment may be mediated partly through hyperactivity of the membrane bound phospholipase A2.Rat platelets were also enriched with cholesterol analogues which were different from cholesterol only in that they had side chains of reduced length. The analogues studied were pregn -5 -en- 30-ol, chol- 5- en- 3ß -ol, and 27 norcholest- 5- en- 3/ -ol. These analogues were readily taken up by the platelets on incubation with analogue loaded liposome suspensions. Aggregation tests and scanning electron microscopy indicated that major changes occurred in the physiology of platelets enriched with analogues which had a side chain 3 or more carbon atoms shorter than cholesterol. Enrichment of rat platelets with the analogue which had a side chain shorter by one carbon atom had the same effects as cholesterol- enrichment. These platelets became hypersensitive to collagen induced aggregation.The results presented in this thesis suggest that the activity of phospholipase A2 may be influenced by the membrane cholesterol content, and clearly indicate a requirement by the platelet membrane of a sterol of precise dimensions to maintain normal platelet function.