Endogenous ADP prevents PGE1-induced tyrosine dephosphorylation of focal adhesion kinase in thrombin-activated platelets.
Prostaglandin E1(PGE1) inhibits tyrosine phosphorylation induced by low thrombin concentration (0.05 U/ml), but this is overcome by a high thrombin (2.0 U/ml) concentration. Thromboxane A2 and ADP are endogenous platelet agonists released during platelet activation which potentiate platelet responses. We investigated how these endogenous agonists influenced the effects of PGE1 on thrombin (2.0 U/ml)-induced tyrosine phosphorylation by removing released ADP with apyrase (2.0 U/ml) and by inhibiting thromboxane A2 synthesis with indomethacin (1 microM). Adding PGE1 (1 microM) before thrombin in apyrase/indomethacin(A/I)-treated platelets selectively prevented thrombin-induced tyrosine phosphorylation of a 117 kDa protein while other substrates were not affected. This selective effect was evident only in the presence of apyrase and was not dependent on indomethacin. Addition of PGE1 to A/I-treated platelets after thrombin also caused selective tyrosine dephosphorylation of the 117 kDa protein. Conditions which prevented thrombin-induced 117 kDa protein tyrosine phosphorylation also decreased fibrinogen binding to platelets. The 117 kDa protein was identified as the focal adhesion kinase (FAK) by immunoprecipitation with a monoclonal antibody to FAK and by absence of its tyrosine phosphorylation in the presence of RGDS peptide which inhibits fibrinogen binding and platelet aggregation. Thus, released endogenous ADP selectively prevents PGE1-mediated tyrosine dephosphorylation of platelet FAK most likely by stabilizing fibrinogen binding to platelets.
Vostal
J G
JG
Laboratory of Cellular Hematology, CBER-FDA, Bethesda, MD, USA. vostal@al.cber.fda.gov
Shafer
B
B
Mondoro
T H
TH
Fratantoni
J C
JC
D000244:Adenosine Diphosphate / Q000502:physiology*; D000527:Alprostadil / Q000502:physiology*; D001792:Blood Platelets / Q000187:drug effects* / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D000242:Cyclic AMP / Q000502:physiology; D003600:Cytosol / Q000378:metabolism; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D006801:Humans; D010766:Phosphorylation; D015539:Platelet Activation / Q000187:drug effects; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D013917:Thrombin / Q000494:pharmacology*; D013928:Thromboxane A2 / Q000502:physiology*; D014443:Tyrosine / Q000378:metabolism*
D015815:Cell Adhesion Molecules; D014443:Tyrosine; D013928:Thromboxane A2; D000244:Adenosine Diphosphate; D000242:Cyclic AMP; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; D013917:Thrombin; D000527:Alprostadil; D002118:Calcium