Tyrosine phosphorylation of cortactin associated with Syk accompanies thromboxane analogue-induced platelet shape change

Thromboxane A(2) (TxA(2)) is a potent vasoconstrictor and platelet agonist. Pharmacological studies have defined two classes of thromboxane receptors (TPs) in human platelets; sites that bind the agonist 1S-(1,2(5Z),3-(1E,3S) ,4)-7- 3-(3-hydroxy-4-(4'-iodophenoxy)-1-butenyl)- 7-oxabicyclo-2.2.1-hepta n-2-yl-5-heptenoic acid (I-BOP) with high affinity support platelet shape c hange, whereas low affinity sites that bind irreversibly the antagonist GR 32191 transduce platelet aggregation. As the mechanisms of signal transduct ion involved in platelet aggregation bean to be elucidated, few results con cern those involved in platelet shape change, which is independent of the e ngagement of GPIIb/IIIa, To elucidate the respective role of the two classe s of pharmacological binding sites of TPs in shape change, platelets were i ncubated with I-BOP at low concentrations or stimulated by I-BOP at high co ncentrations after pretreatment with GR 32191 or activated with low concent rations of 8-epi-prostaglandin F(2)alpha. Under these three conditions, the re is a rapid stimulation of protein tyrosine phosphorylation of the 80/85- kDa doublet identified as the cytoskeletal protein cortactin, Tyrosine phos phorylation of cortactin is kinetically correlated with the occurrence of s hape change, These biochemical and morphological events are both inhibited by SQ 29548, a TP antagonist, indicating the specificity of the signal. Since tyrosine kinase Syk was activated early during platelet activation, w e examined the possibility that cortactin is a potential substrate of Syk i n TxA(2)-induced platelet shape change. p72 Syk phosphorylation and kinase activity took place during the period when platelets were changing shape up on low concentrations of I-BOP stimulation, Furthermore, cortactin was asso ciated with Syk, and this association increases along with the level of pho sphorylation. These data suggest a novel pathway for a Gr protein coupled T xA(2) high affinity receptor to the protein-tyrosine kinase Syk, which is a ssociated with cortactin in the very early steps of platelet activation.