<p>Platelet aggregation plays an important role in both the formation of a hemostatic plug and in the generation of thrombus. The mechanism of platelet aggregation has not been fully elucidated. It is conceivable that changes in the synthesis, breakdown or rate of turnover of some of the constituents of the platelet membrane may be important in the mechanisms of aggregation. Recent experimental evidence in other mammalian tissues has implicated the closed circle conversion between MPI and PA as part of the membrane receptor-effector interaction process, and a possible role permeability toward of TPI-DPI interconversion in the regulation of membrane permeability toward Na⁺ and K⁺. Platelets have been recognized to be a useful model for these studies because of its abundance in phosphoinositides and easiness of obtaining pure platelet suspensions. Previous studies showed that addition of ADP to ³²P-labeled washed platelets caused an increase in radioactivity in PA in 2 sec, DPI in 30 sec and MPI in 2-3 min. TPI-DPI interconversion was suspected but consistent changes in ³²P-radioactivity in TPI was not detected. Similar changes were observed in platelets in response to thrombin stimulation. In none of the studies in other tissues all the metabolic pathways for inositol lipids were studied simultaneously. The aims of the present experiments were to investigate TPI-DPI interconversion and MPI metabolism during platelet aggregation and the release reaction caused by ADP, ionophore A23,187 and thrombin. Since these stimuli are considered to act on platelets by different mechanisms, their effects on the platelet phosphoinositide metabolism were compared. Furthermore, experiments were carried out with unstimulated platelets labeled with ³²PO₄ to find out if there vere differences in the patterns of ³²PO₄ incorporation into the major platelet phospholipids (PC, PE, PS) under in vitro and in vivo conditions, as claimed by former investigators.</p> <p>Using improved methods for separation of phosphoinositides, it was found that the pattern of ³²P-incorporation into the major phospholipids in platelet suspension was similar to that of in vivo platelets when the incubation was carried out for many hours. Therefore, the phosphate moiety in all phospholipids turn over; but the phosphate in the major phospholipids turn over much more slowly than that in the phosphoinositides.</p> <p>During ADP-induced platelet aggregation, hydrolysis of TPI to DPI was measurable at 60 sec, with a loss of ³²PO₄, ¹⁴C-arachidonic acid and ³H-inositol from TPI. This hydrolysis was abolished by AMP, an inhibitor of ADP-induced aggregation. Resynthesis of TPI occurred during platelet deaggregation, with the ³²PO₄ radioactivity of this compound being restored to the control level. With A23,187-induced aggregation and release, hydrolysis of TPI did not occur. While there was a significant amount of MPI converted into PA, the majority of MPI appeared to be hydrolysed to fatty acid and lyso MPI. Using platelets labelled with ¹⁴C-arachidonic acid, ³H-glycerol, ³H-inositol, ³²PO₄ and phosphorus assay, it was found that thrombin caused a decrease in TPI (-5.64 ± 1.55%, P < 0.005) as early as 9 sec when platelet shape change was maximal, ³²P-content was also decreased. Resynthesis of TPI was measurable at 60 sec. Most of the MPI was metabolised via the 1,2-diacylglycerol pathway. The the amount of PA was increased. A small amount of the MPI was converted to polyphosphoinositides or lyso MPI and free fatty acid. These experiments have shown a close relationship between changes in phosphoinositide metabolism and platelet aggregation and the release reaction.</p> <p>These principal changes are: (1) increased TPI- DPI interconversion; (2) increased conversion of MPI to l,2-diacylglycerol PA and MPI; (3) with the release reaction conversion of MPI to lyso MH and free fatty acids.</p> / Doctor of Philosophy (PhD)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/7996 |
| Date | 11 1900 |
| Creators | Leung, Lin N. |
| Contributors | Mustard, J.F., Medical Sciences |
| Source Sets | McMaster University |
| Detected Language | English |
| Type | thesis |
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