Regulation and functional consequences of ADP receptor-mediated ERK2 activation in platelets

We have previously shown that ADP-induced thromboxane generation in platelets requires signalling events from the G(q)-coupled P2Y(1) receptor (platelet ADP receptor coupled to stimulation of phospholipase C) and the Gi-coupled P2Y(12) receptor (platelet ADP receptor coupled to inhibition of adenylate cyclase) in addition to outside-in signalling. While it is also known that extracellular calcium negatively regulates ADP-induced thromboxane A(2) generation, the underlying mechanism remains unclear. In the present study we sought to elucidate the signalling mechanisms and regulation by extracellular calcium of ADP-induced thromboxane A2 generation in platelets. ERK (extracllular-signal-regulated kinase) 2 activation occurred when outside-in signalling was blocked, indicating that it is a downstream event from the P2Y receptors. However, blockade of either P2Y(1) or the P2Y(12) receptors with corresponding antagonists completely abolished ERK phosphorylation, indicating that both P2Y receptors are required for ADP-induced ERK activation. Inhibitors of Src family kinases or the ERK upstream kinase MEK [MAPK (mitogen-activated protein kinase)/ERK kinase] abrogated ADP-induced ERK phosphorylation and thromboxane A(2) generation. Finally ADP- or G(i) + G(z)-induced ERK phosphorylation was blocked in the presence of extracellular calcium. The present studies show that ERK2 is activated downstream of P2Y receptors through a complex mechanism involving Src kinases and this plays an important role in ADP-induced thromboxane A, generation. We also conclude that extracellular calcium blocks ADP-induced thromboxane A2 generation through the inhibition of ERK activation.