The platelet P2Y12 receptor contributes to granule secretion through Ephrin A4 receptor

The main responses of P2Y(1) ligation are platelet shape change and transient aggregation while P2Y(12) ligation amplifies P2Y(1)-induced aggregation and accelerates aggregation, secretion and thromboxane A(2) production induced by other agonist-receptor complexes. We searched for new targets of P2Y signalling using micro-arrays with 144 peptides representing known phosphosites of protein tyrosine kinases. ADP induced phosphorylation of peptides representing surface receptors, second messenger enzymes and cytoskeletal proteins. Strong phosphorylation was found in peptides representing Ephrin-receptor family members. Blockade of P2Y(1/12) inhibited phosphorylation of EphA4- and EphB1-peptides on micro-arrays. The EphA2/4 inhibitor 2,5-dimethylpyrrolyl benzoic acid derivative interfered with P2Y(1/12)-induced EphA4 phosphorylation, left P2Y(1)-induced aggregation unchanged but inhibited with P2Y(12)-induced secretion, second phase aggregation and thrombus formation on collagen at 1600 s(-1). These results show that platelet EphA4 is an important intermediate in P2Y(12)-induced granule secretion.