Oury Cécile, Toth-Zsamboki Emese, Vermylen Jos, Hoylaerts Marc F
Center for Molecular and Vascular Biology, University of Leuven, Belgium.
Blood. 2002 Oct 1;100(7):2499-505. doi: 10.1182/blood-2002-03-0812.
Adenosine triphosphate (ATP) and its stable analog, alpha,beta-methylene ATP, activate the platelet P2X(1) ion channel, causing a rapid Ca(++) influx. Here, we show that, in washed apyrase-treated platelets, alpha,beta-methylene ATP elicits reversible extracellular signal-regulated kinase 2 (ERK2) phosphorylation through a Ca(++)- and protein kinase C-dependent pathway. In contrast, high-performance liquid chromatography-purified adenosine diphosphate (ADP) did not trigger ERK2 phosphorylation. alpha,beta-Methylene ATP also activated the ERK2 pathway in P2X(1)-transfected HEK293 cells but not in cells expressing mutated P2X(1)delL nonfunctional channels. Because ATP released from the dense granules during platelet activation contributes to platelet aggregation elicited by low doses of collagen, and because collagen causes ERK2 phosphorylation, we have investigated the role of P2X(1)-mediated ERK2 activation in these platelet responses. We found that the antagonism of P2X(1) with ADP or desensitization of this ion channel with alpha,beta-methylene ATP both resulted in impaired ERK2 phosphorylation, ATP secretion, and platelet aggregation induced by low concentrations of collagen (< or = 1 microg/mL) without affecting the minor early dense granule release. Selective MEK1/2 inhibition by U-0126 and Ca(++) chelation with EGTA (ethyleneglycoltetraacetic acid) behaved similarly, whereas the PKC inhibitor GF109203-X totally prevented collagen-induced secretion and ERK2 activation. In contrast, when elicited by high collagen concentrations (2 microg/mL), platelet aggregation and secretion no longer depended on P2X(1) or ERK2 activation, as shown by the lack of their inhibition by alpha,beta-methylene ATP or U-0126. We thus conclude that mild platelet stimulation with collagen rapidly releases ATP, which activates the P2X(1)-PKC-ERK2 pathway. This process enhances further degranulation of the collagen-primed granules allowing platelet aggregation to be completed.
三磷酸腺苷（ATP）及其稳定类似物α,β-亚甲基ATP可激活血小板P2X(1)离子通道，导致钙离子迅速内流。在此，我们表明，在经洗涤并经腺苷三磷酸双磷酸酶处理的血小板中，α,β-亚甲基ATP通过钙离子和蛋白激酶C依赖性途径引发可逆的细胞外信号调节激酶2（ERK2）磷酸化。相比之下，高效液相色谱纯化的二磷酸腺苷（ADP）不会触发ERK2磷酸化。α,β-亚甲基ATP也可在转染P2X(1)的HEK293细胞中激活ERK2途径，但在表达突变的无功能通道P2X(1)delL的细胞中则不能。由于血小板激活过程中从致密颗粒释放的ATP有助于低剂量胶原蛋白引发的血小板聚集，且由于胶原蛋白可导致ERK2磷酸化，我们研究了P2X(1)介导的ERK2激活在这些血小板反应中的作用。我们发现，用ADP拮抗P2X(1)或用α,β-亚甲基ATP使该离子通道脱敏均会导致ERK2磷酸化、ATP分泌以及低浓度胶原蛋白（≤1μg/mL）诱导的血小板聚集受损，而不影响早期少量致密颗粒的释放。U-0126对MEK1/2的选择性抑制以及用乙二醇双四乙酸（EGTA）螯合钙离子表现出类似的效果，而蛋白激酶C抑制剂GF109203-X则完全阻止了胶原蛋白诱导的分泌和ERK2激活。相比之下，当由高浓度胶原蛋白（2μg/mL）引发时，血小板聚集和分泌不再依赖于P2X(1)或ERK2激活，α,β-亚甲基ATP或U-0126对其无抑制作用即表明了这一点。因此，我们得出结论，用胶原蛋白轻度刺激血小板可迅速释放ATP，后者激活P2X(1)-蛋白激酶C-ERK2途径。这一过程增强了经胶原蛋白预处理的颗粒的进一步脱颗粒作用，从而使血小板聚集得以完成。
