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. 2006 Nov 15;400(1):127-34.
doi: 10.1042/BJ20061015.

Rapid stimulation of tyrosine phosphorylation signals downstream of G-protein-coupled receptors for thromboxane A2 in human platelets

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Rapid stimulation of tyrosine phosphorylation signals downstream of G-protein-coupled receptors for thromboxane A2 in human platelets

Pietro Minuz et al. Biochem J. .

Abstract

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.

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Figures

Figure 1
Figure 1. Low doses of U46619 trigger tyrosine phosphorylation signals
ASA-treated platelets were pre-incubated with 10 units/ml apyrase and 10 μg/ml eptifibatide and then stimulated with 0.05 μmol/l U46619. At the indicated times, the reaction was stopped by the addition of 4×sample buffer, the proteins were subjected to electrophoresis and transferred on to nitrocellulose. (A) Blots were probed with anti-phosphotyrosine antibodies. A representative experiment of three performed is shown. Molecular masses (in kDa) are indicated on the left. (B) Blots were probed with phospho-specific antibodies recognizing the indicated tyrosine residues [FAK Tyr397 (αFAKpY397), Src Tyr416 (αSrcpY416) and Syk Tyr352 (αSykpY352)], followed by stripping and re-probing with antibodies recognizing total protein. (C) Densitometric analysis of blots probed with phospho-specific antibodies. Results are means±S.D. of three independent experiments.
Figure 2
Figure 2. Low doses of U46619 are unable to trigger Akt phosphorylation, and their capability to trigger protein tyrosine phosphorylation is not inhibited by ADP receptor antagonists
(A) ASA-treated platelets were stimulated with the indicated concentrations of U46619 in the presence (+) or absence (−) of apyrase (10 units/ml). After 5 min of incubation, the reaction was stopped by the addition of 4× sample buffer, and equal amounts of protein (15 μg) were analysed by Western blot analysis with a phospho-specific anti-Akt antibody (αAktpSer 473), followed by stripping and re-probing with an antibody recognizing total Akt (αAkt). A representative experiment of three performed is shown. (B) ASA-treated platelets, pre-incubated with 10 units/ml apyrase and 10 μg/ml eptifibatide, were incubated with 0.5 μmol/l AR-C69931MX and 0.1 mmol/l MRS2179 prior to stimulation with 0.05 μmol/l U46619. At the times indicated, the reaction was stopped by the addition of 4× sample buffer, proteins were subjected to electrophoresis and transferred on to nitrocellulose. Blots were probed with an anti-phosphotyrosine antibody. A representative experiment of two performed is shown. Molecular masses (in kDa) are indicated on the left.
Figure 3
Figure 3. Tyrosine phosphorylation signals triggered by low doses of U46619 are independent of the Rho/Rho-kinase pathway
ASA-treated platelets, pre-incubated with 10 units/ml apyrase and 10 μg/ml eptifibatide, were incubated with 30 μmol/l Y-27632 prior to stimulation with 0.05 μmol/l U46619. At the times indicated, the reaction was stopped by the addition of 4× sample buffer, proteins were subjected to electrophoresis and transferred on to nitrocellulose. (A) Blots were probed with an anti-phosphotyrosine antibody. Molecular masses (in kDa) are indicated on the left. (B) Blots were probed with phospho-specific antibodies recognizing the indicated threonine/serine residues of MLC [MLC Thr18/Ser19 (αMLCpThr18/Ser19)], followed by stripping and reprobing with an antibody recognizing total MLC (αMLC). A representative experiment of three performed is shown.
Figure 4
Figure 4. Platelet aggregation induced by simultaneous stimulation of G12/13 and Gz pathways requires both tyrosine phosphorylation and Rho-kinase-mediated signals
(AC) ASA-treated platelets were stimulated in the presence or absence of 10 units/ml apyrase with the indicated doses of U46619 and adrenaline alone or in combination. (D) ASA-treated platelets were stimulated with the indicated doses of U46619 and adrenaline in combination following pre-incubation with 10 μmol/l PP2, 30 μmol/l Y-27632 or 10 μmol/l PP2+30 μmol/l Y-27632.
Figure 5
Figure 5. PP2 inhibits tyrosine phosphorylation signals triggered by the combination of U46619 and adrenaline
ASA-treated platelets were stimulated in the presence of 10 units/ml apyrase with 0.05 μmol/l U46619 and 10 μmol/l adrenaline, alone or in combination, following pre-incubation with 10 μmol/l PP2, 30 μmol/l Y-27632 or 10 μmol/l PP2+30 μmol/l Y-27632. After 40 s, the reaction was stopped by the addition of 4× sample buffer, then proteins were subjected to electrophoresis and were transferred on to nitrocellulose. (A) Blots were probed with anti-phosphotyrosine antibodies. Molecular masses (in kDa) are indicated on the left. (B) Blots were probed with phospho-specific antibodies recognizing the indicated threonine/serine residues of MLC [MLC Thr18/Ser19 (αMLCpThr18/Ser19)], followed by stripping and reprobing with an antibody recognizing total MLC (αMLC). A representative experiment of three performed is shown.

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