Role of ADP ribosylation factor (ARF) in platelet fanctions

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Stimulation of phospholipase D (PLD) after activation of cell surface receptors has been reported in many cell types. We have investigated the mechanisms of activation of this enzyme, especially ADP ribosylation factor (ARF) dependent PLD,by ADP or collagen in human pletelet. aggregation is still unclearinduced with agonists has been studied.We examined the expression of ADP ribosylation factor (ARF) in pletelets by immuboblotting and ARF dependent type PLD (PLD1) by RP-PCR.Then, we revealed that both ADP and collagen indused ARF1 activation ([^<35>S]GTPgS binding) on pletelets membrane upto 10-fold. PLD activity on platelet membrane was stimulated 4- to 5- fold by the addition of GTPgammaS and myrisoylated recombinant ARF1. And both ADP and collagen also induced ARF dependent PLD activity about 150%.Brefeldine A (BFA) inhibits activation of ARF and PLD about 40% and 50% respectively, aggregation of platelets by ADP,however, was not inibited so much (only 25% inhibition). However, BFA inhibited 50% of maxmal aggreation of platelets by collagen. Considering ADP induces primary aggregation and collagen induces secondary aggregation of platelete, BFA might have inhibited granule relese and so on. BFA inhibited 60% of beta-TG release from platelet.In conclution, ARF-PLD signaling seems to be under GPIa/IIa or GPIb/V/IX as collagen receptor and work as a signal transduction for granule release and platelet aggregation.