ENDOGENOUS ADP PREVENTS PGE(1)-INDUCED TYROSINE DEPHOSPHORYLATION OF FOCAL ADHESION KINASE IN THROMBIN-ACTIVATED PLATELETS

Prostaglandin E(1)(PCE(1)) inhibits tyrosine phosphorylation induced b y low thrombin concentration (0.05 U/ml), but this is overcome by a hi gh thrombin (2.0 U/ml) concentration. Thromboxane A(2) and ADP are end ogenous platelet agonists released during platelet activation which po tentiate platelet responses, We investigated how these endogenous agon ists influenced the effects of PGE(1) on thrombin (2.0 U/ml)-induced t yrosine phosphorylation by removing released ADP with apyrase (2.0 U/m l) and by inhibiting thromboxane A(2) synthesis with indomethacin(1 mu M). Adding PGE(1) (1 mu M) before thrombin in apyrase/indomethacin(A/ I)-treated platelets selectively prevented thrombin-induced tyrosine p hosphorylation of a 117 kDa protein while other substrates were not af fected. This selective effect was evident only in the presence of apyr ase and was not dependent on indomethacin. Addition of PGE(1) to A/I-t reated platelets after thrombin also caused selective tyrosine dephosp horylation of the 117 kDa protein. Conditions which prevented thrombin -induced 117 kDa protein tyrosine phosphorylation also decreased fibri nogen binding to platelets. The 117 kDa protein was identified as the focal adhesion kinase (FAK) by immunoprecipitation with a monoclonal a ntibody to FAK and by absence of its tyrosine phosphorylation in the p resence of RGDS peptide which inhibits fibrinogen binding and platelet aggregation. Thus, released endogenous ADP selectively prevents PGE(1 )-mediated tyrosine dephosphorylation of platelet FAK most likely by s tabilizing fibrinogen binding to platelets.