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A.S. Macwan et al.
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Figure 4. Mechanistic characterization of gradient-dependent inhibition. (A) Western blotting was performed on resting platelets (RST: treated with a 320 s infusion of saline), activated platelets (ACT: treated with a 2 s infusion of 30 μM PAR1-AP) or platelets showing gradient-dependent inhibition (GDI: treated with a 320 s infusion of 30 μM PAR1-AP) with staining for VASP phosphorylation at Ser157 or total VASP. Experiments were performed with or without pre-treatment with a PKA inhibitor (H89) or PGI2. (B) Quantitation of the signal intensity, with the PGI2-mediated phosphorylation signal set at 100%, n=3. (C) Effects of the PKA inhibitor H89 on platelet aggregation induced by 30 μM PAR1-AP added at a 2 s or 320 s infusion time. (D) Levels of total and phosphorylated AKT (Ser 473) in resting, activated or GDI platelets determined by western blotting. (E) Effect of pre-incubation with PGI2 (0.01, 0.1 and 1 nM) on aggregation induced by 30 μM PAR1-AP added with different infusion times. (F) Effect of the phosphodiesterase-3 inhibitor milrinone (Mil; 3 μM) on maximal platelet aggregation induced by Cagg (30 μM PAR1-AP, 300 μM PAR4-AP, 5 μM ADP, 2 μM U46619, 0.16 μg/mL CRP-XL) added with different infusion times compared to control (Ctr). (G,H) Effects of epinephrine (Epi; 0.1, 1 and 10 μM) on GDI for the agonists PAR1-AP (G) and thrombin (H) at different infusion times presented as a log scale on the x-axis (GDI calculated as % inhibition of maximal aggregation compared to that with the 2 s infusion time). For all the experiments, data represent mean ± standard deviation, n≥3. *P<0.05, **P<0.01 and ***P<0.001.
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haematologica | 2019; 104(7)