Calcium-Dependent Src Phosphorylation and Reactive Oxygen Species Generation Are Implicated in the Activation of Human Platelet Induced by Thromboxane A2 Analogs

Figures

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  FIGURE 1. Ex vivo effect of dasatinib on Src phosphorylation. PRP was isolated in the presence of ASA 100 μM and apyrase VII 10 U mL−1 from health volunteer (n = 7) or from patient (n = 5) 3 and 24 h post treatment with dasatinib. PRP was stimulated by U466191 μM, U46619 50 nM, epinephrine (epi) 10 μM, U46619 50 nM plus epinephrine 10 μM. Two-way ANOVA followed by Bonferroni’s test was applied to compare platelet response to each agonist with resting conditions in the presence or absence of dasatinib. Data are expressed as Mean ± SE, ∗P < 0.05, ∗∗P < 0.01.

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  FIGURE 2. Maximal release of intracellular calcium in washed and non-aggregating platelets (in presence of ASA 100 μM, apyrase VII 10 U mL−1, eptifibatide 10 μg mL−1) stimulated by U46619 1 μM, U46619 50 nM, U46619 50 nM + epinephrine (epi) 10 μM, epi alone 10 μM, 8-iso PGF2α 10 μM, 8-iso PGF2α 10 μM + epi 10 μM (Ca2+ nM). Repeated-measures Two-Way ANOVA was applied to compare the effects of platelets agonists at different time points. Significant differences (P < 0.0001) were observed comparing U46619 1 mmol/L with all the other agonists, except U46619 50 nM + epi 10 μM. Data are expressed as Mean ± SE.

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  FIGURE 3. Intracellular Ca2+ release in stimulated washed platelets in presence/absence of extracellular calcium. Washed platelets in non-aggregating condition (eptifibatide 10 μg mL−1, ASA 100 μM, apyrase VII 10 U mL−1) were stimulated by U46619 1 μM, U46619 50 nM, U46619 50nM + epinephrine 10 μM, and epinephrine 10 μM. Fluorescent emission at 510 nm was measured induced by double excitation wavelength of 340 and 380 nm. The ratio of the two fluorescence is proportional to the concentration of the released intracellular calcium. Repeated-measures Two-Way ANOVA was applied to compare the effects of platelets agonists at different time points. Significant differences (P < 0.0001) were observed comparing intracellular Ca2+ release induced by U46619, but not epinephrine (P = n.s.), with and without EGTA. Data are expressed as Mean ± SE.

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  FIGURE 4. Western Blotting analysis of tyrosine (A) and Src (B,C) phosphorylation. Washed platelets were isolated in non-aggregating condition (eptifibatide 10 μg mL−1, ASA 100 μM, apyrase VII 10 U mL−1) and stimulated with agonists. The reaction was stopped by adding sample buffer 4× at 100°C. (B) Typical pattern of Src phosphorylation after stimulation of 10, 20, 40, and 60 s with U46619 50 nM + epi 10 μM in condition of intracellular calcium-presence or absence. Graphic is representative of four experiment. (C) Typical pattern of Src phosphorylation after stimulation of 40 s with U46619 1 μM, U46619 50 nM, U46619 50 nM + epi 10 μM, and epi 10 μM in presence or absence of BAPTA 20 μM. Graphic shows five experiments. Repeated-measures Two-Way ANOVA was applied to compare the effects of platelets agonists on Src phosphorylation at different time points in the presence of different agonists (B,C). Mean ± SE, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

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  FIGURE 5. Platelet aggregation induced by U46619 1 μM and U46619 50 nM + epi 10 μM. Washed platelets in presence or absence of apyrase VII were pre-treated with the NADPH oxidase inhibitor apocynin 300 μM, the reactive oxygen species (ROS) scavenger EUK-134 250 μM or the intracellular calcium chelator BAPTA-AM 20 μM, respectively. Data are expressed as Mean ± SE.

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  FIGURE 6. Reactive oxygen species generation (measured as Fluorescence Arbitrary Units-FUA) induced by agonists in washed platelets in presence of apyrase VII 10 U mL−1, ASA 100 μM and eptifibatide 10 μg mL−1 (A,B). Platelets were pre-treated with the NADPH oxidase inhibitor apocynin 300 μM, the intracellular calcium chelator BAPTA-AM 20 μM (A), or the tyrosine kinase inhibitor dasatinib 150 nM (B). Repeated-measures One-way ANOVA, with the Newman–Keuls test for post hoc analysis were applied to compare independently each platelet agonist in the presence or absence of inhibitors with resting conditions. Data are expressed as Mean ± SE. Indicates the signficance level: ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.0001.

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  FIGURE 7. Graphs summarize the relationships between TP receptor activation, signaling pathways and functional responses in platelets stimulated with different amounts of the TXA2 analog U46619 and the cooperative effects of epinephrine. +, ++, +++ Represent differences in the implication of the signaling pathways.