Rapid stimulation of tyrosine phosphorylation signals downstream of G-protein-coupled receptors for thromboxane A2 in human platelets.
Signals ensuing from trimeric G-protein-coupled receptors synergize to induce platelet activation. At low doses, the thromboxane A2 analogue U46619 does not activate integrin alphaIIbbeta3 or trigger platelet aggregation, but it induces shape changes. In the present study, we addressed whether low doses of U46619 trigger tyrosine phosphorylation independently of integrin alphaIIbbeta3 activation and ADP secretion, and synergize with adrenaline (epinephrine) to induce aggregation in acetylsalicylic acid (aspirin)-treated platelets. Low doses of U46619 triggered tyrosine phosphorylation of different proteins, including FAK (focal adhesion kinase), Src and Syk, independently of signals ensuing from integrin alphaIIbbeta3 or ADP receptors engaged by secreted ADP. The G(12/13)-mediated Rho/Rho-kinase pathway was also increased by low doses of U46619; however, this pathway was not upstream of tyrosine phosphorylation, because this occurred in the presence of the Rho-kinase inhibitor Y-27632. Although low doses of U46619 or adrenaline alone were unable to trigger platelet aggregation and integrin alphaIIbbeta3 activation, the combination of the two stimuli effectively induced these responses. PP2, a tyrosine kinase inhibitor, and Y-27632 inhibited platelet activation induced by low doses of U46619 plus adrenaline and, when used in combination, totally suppressed this platelet response. In addition, the two inhibitors selectively blocked tyrosine kinases and the Rho/Rho-kinase pathway respectively. These findings suggest that both tyrosine phosphorylation and the Rho/Rho-kinase pathway are required to activate platelet aggregation via G(12/13) plus G(z) signalling.
Minuz
Pietro
P
Department of Biomedical and Surgical Sciences, University of Verona, Verona, Italy.
Fumagalli
Laura
L
Gaino
Stefania
S
Tommasoli
Rosa M
RM
Degan
Maurizio
M
Cavallini
Chiara
C
Lecchi
Anna
A
Cattaneo
Marco
M
Lechi Santonastaso
Clara
C
Berton
Giorgio
G
D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid / Q000494:pharmacology*; D000328:Adult; D000577:Amides / Q000494:pharmacology; D001081:Apyrase / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D004837:Epinephrine / Q000494:pharmacology; D006801:Humans; D039421:Integrin alpha2 / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D010974:Platelet Aggregation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011720:Pyrazoles / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D043562:Receptors, G-Protein-Coupled / Q000502:physiology*; D018048:Receptors, Purinergic P2 / Q000502:physiology; D044244:Receptors, Thromboxane A2, Prostaglandin H2 / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects*; D014443:Tyrosine / Q000378:metabolism*; D054460:rho-Associated Kinases
C509621:4-amino-7-phenylpyrazol(3,4-d)pyrimidine; C412373:AG 1879; D000577:Amides; C056564:ITGA2B protein, human; D039421:Integrin alpha2; D047908:Intracellular Signaling Peptides and Proteins; D011720:Pyrazoles; D011725:Pyridines; D011743:Pyrimidines; D043562:Receptors, G-Protein-Coupled; D018048:Receptors, Purinergic P2; D044244:Receptors, Thromboxane A2, Prostaglandin H2; C108830:Y 27632; D014443:Tyrosine; D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D001081:Apyrase; D004837:Epinephrine