Lyn and Fyn Kinases Positively Regulate Thromboxane Generation in Platelets.

Src family Kinases (SFKs) are important tyrosine kinases in platelets. The family consists of 9 members (viz., Blk, Fgr, Fyn, Hck, Lck, Lyn, Src, Yes and Yrk) and many of the isoforms are expressed in platelets. SFKs are key kinases in glycoprotein (GPVI) mediated platelet activation and we have shown that these kinases play an important role in thromboxane generation. Until now, the role of specific SFKs downstream of G protein signaling in platelets is not well understood. In the present study we characterized functional roles of specific SFK members Fyn, Lyn, Lck and Src Kinases downstream of ADP receptors. Src, Lyn and Fyn are activated downstream of ADP receptors (P2Y) receptors in a time and concentration dependent manner. The presence of fibrinogen receptor antagonist, GR144053, did not affect the activation of Src and Fyn; however, Lyn is not activated in the presence of GR144053, suggesting that Lyn requires outside-in signaling for it’s activation. On the other hand, Lck kinase is not activated downstream of ADP receptors. ADP activates P2Y1 and P2Y12 receptors which in turn couple to Gq and Gi, respectively. In order to further delineate the Src activation pathways we used P2Y1 and P2Y12 receptor antagonists MRS 2179 and AR-C69931MX respectively. Src activation is not inhibited by either P2Y1 or P2Y12 receptor antagonists, suggesting that both receptors independently contribute to the activation of Src kinase. Platelets from mice deficient in Src, Fyn or Lyn were analyzed for thromboxane generation upon stimulation with ADP. Lyn and Fyn KO mice had reduced levels of thromboxane A2 compared to wild type littermates. However, thromboxane generation from platelets lacking Src was unaffected. Hence, we conclude Lyn and Fyn kinases, but not Src, positively regulate thromboxane generation downstream of ADP receptors. We also conclude that Lyn requires outside-in signaling for its activation.