Role of ADP ribosylation factor (ARF) in platelet fanctions

磷脂酶D （PLD）在细胞表面受体激活后的刺激在许多细胞类型中都有报道。我们研究了这种酶的激活机制，特别是ADP核糖基化因子（ARF）依赖的PLD，由ADP或胶原在人血小板。对激动剂诱导的聚集机制尚不清楚。我们用免疫印迹法检测小细胞中ADP核糖化因子（ARF）的表达，用RP-PCR法检测ARF依赖型PLD （PLD1）的表达。然后，我们发现ADP和胶原蛋白在小细胞膜上诱导ARF1激活（[^<35>S]GTPgS结合）高达10倍。通过添加GTPgammaS和myrisoylated重组ARF1，血小板膜上PLD的活性被刺激了4- 5倍。ADP和胶原蛋白均能诱导ARF依赖性PLD活性约150%。Brefeldine A （BFA）对ARF和PLD的抑制作用分别为40%和50%，而ADP对血小板聚集的抑制作用不明显（仅为25%）。然而，BFA抑制50%的血小板由胶原最大聚集。考虑到ADP诱导血小板初级聚集，胶原诱导血小板次级聚集，BFA可能具有抑制血小板颗粒释放等作用。BFA抑制血小板中60%的β - tg释放。综上所述，ARF-PLD信号似乎在GPIa/IIa或GPIb/V/IX下作为胶原受体，并作为颗粒释放和血小板聚集的信号转导。

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.