Abstract
Physiological agonists induce a rapid increase of cytosolic free Ca2+ concentration ([Ca2+]i) in platelets, as well as shape change, aggregation and1 secretion. They activate a phosphodiesterase which specifically splits polyphosphoinositides, generating the second messengers diacylglycerol, a protein kinase C ativator, and inositol 1,4,5 triphosphate, which releases Ca2+ from the intracellular stores - [Ca2+]i rise and protein kinase C activation are responsible for aggregation and exocytosis.We have now studied the effects of the synthetic antioxidants: butyl-hydroxytoluene (BHT), butyl- hydroxyanisole (BHA), nordihydroguaiaretic acid (NDGA) and the one electron donor 1-1' dimethylferrocene plus ascorbate (FC) on the increase of [Ca2+]i, aggregation and ATP secretion. Human platelets were loaded1 with 20μM quin-2-acetoxymethylester and incubated with aspirin (100μM). Changes in the [Ca2+]. were measured with a spectrofluorimeter. Platelet aggregation and ATP secretion (luciferin/luciferase system) were evaluated in parallel. In the presence of 1mM external Ca2+,0.15 u/ml thrombin induced a [Ca2+]. increase to about 1μM, that was inhibited by 50% and abolished by 25μM and 100μM BHT respectively; [Ca2+]. increase induced by 75 nM PAF, 1μM vasopressin, 10 μM ADP was also inhibited by BHA, NDGA and FC in a range of 30-150 μM. Shape change, aggregation and ATP secretion were also inhibited. In the absence of external Ca2+ (1mM ECTA), 80μM BHT inhibits the [Ca2+]i increase originating only from the intracellular stores. Tumor-promoting phorbol esters induce aggregation and secretion without raising [Ca2+]i; 80 μM BHT induced 80-90% inhibition of aggregation and ATP secretion by 0.12nM TPA. Our results suggest that some free radical dependent reactions are involved both in the processes of platelet activation leading to the increase of [Ca2+]i and in those leading to aggregation and exocytosis.
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