RAFTK/Pyk2 involvement in platelet activation is mediated by phosphoinositide 3-kinase

Platelet activation by different agonists initiates a signalling cascade in volving the phosphorylation of several protein kinases, which control key r egulatory events. Previously, we demonstrated that the related adhesion foc al tyrosine kinase (RAFTK, Pyk2) was involved in an early phase of platelet activation, independent of integrin and glycoprotein IIb-IIIa activation. In this study, we demonstrate that RAFTK is co-immunoprecipitated with phos phoinositide 3-kinase (PI3K) upon platelet activation, and that thrombin, A DP and collagen induced the phosphorylation of both PI3K and RAFTK. A low d ose of thrombin (0.015 U/ml) induced RAFTK phosphorylation and platelet agg regation in a PI3K activity-dependent manner, whereas a high dose of thromb in (0.1 U/ml) induced these events in a PI3K activity-independent manner. A DP and collagen also induced RAFTK phosphorylation and platelet aggregation in a PI3K activity-dependent manner, similar to that of the low-dose throm bin. Furthermore, protein tyrosine phosphatase activity was associated with RAFTK in response to platelet activation, and was found to be that of prot ein tyrosine phosphatase-2 (SHP-2). The association of SHP-2 with RAFTK was PI3K-dependent and was increased upon RAFTK phosphorylation. Taken togethe r, our results strongly suggest that the involvement of RAFTK in platelet a ctivation is mediated via the PI3K pathway.