Effect of phosphodiesterase inhibitors on platelet function

Abstract

Phosphodiesterase enzymes (PDEs) play a pivotal role in regulating platelet activity by modulating intracellular levels of cAMP and cGMP. Modulation of PDE-2, -3 and -5 activity by suitable inhibitors has been found to reduce platelet activity, and thus thrombus formation. Our aim was to study Ibudilast effects on platelet activation, degranulation and aggregation. Therefore, we used the nonspecific PDE inhibitors IBMX as well as the PDE-5 inhibitor Sildenafil as controls. Platelet agonists collagen-related peptide (CRP-A), adenosine diphosphate (ADP) and thrombin receptor activator peptide (TRAP6) were used to induce distinct activation pathways. PDE inhibition was quantified by western blot analysis. Platelet activity was assessed using flow cytometry, light transmission aggregometry and in vitro thrombus formation. Inhibition of all platelet PDEs by IBMX substantially reduced platelet activation and aggregation in response to all tested platelet agonists. Ibudilast preferentially inhibits PDE-3 in platelets. Ibudilast decreased platelet activation and aggregation induced by ADP and TRAP, but not CRP-A. Sildenafil alone induced no reduction in PDE activity, platelet activation or aggregation. However, the combination of Sildenafil and Ibudilast had an additive effect on platelet activation. Interestingly, all tested PDE inhibitors demonstrated a significant effect on platelet-dependent thrombus formation. In conclusion, the effect of PDE inhibitors on platelet function is influenced by two primary factors: the pharmacological target of the inhibitor and the cAMP/cGMP interaction with the activation pathways induced. Platelet activation by ADP via P₂Y₁₂ and TRAP via PAR1 showed a greater response to PDE inhibitors than platelet activation by CRP via GPVI.

Keywords: PDE inhibitors; Phosphodiesterase; Platelets.

Graphical abstract

Fig. 1

Effect of PDE inhibitors on platelet fibrinogen receptor activation and $\alpha$-degranulation. A-L Statistical analysis of flow cytometry measurements of activated integrin IIb/IIIa (antibody clone PAC-1) surface expression. Human PRP was pre-treated with A-C IBMX, D-F Ibudilast, G-I Sildenafil or J-L combination of Ibudilast and Sildenafil and then stimulated with CRP-A, ADP or TRAP6 as indicated. vc vehicle control. Plotted: Mean ± SD, n ≥ 5, ordinary one-way ANOVA; ns not significant, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. M-X Statistical analysis of flow cytometry measurements of CD62P surface expression. Human PRP was pretreated with M-O IBMX, P–R Ibudilast, S–U Sildenafil or V-X combination of Ibudilast and Sildenafil and then stimulated with CRP-A, ADP or TRAP6 as indicated. vc vehicle control. Plotted: Mean ± SD, n ≥ 4, ordinary one-way ANOVA; ns not significant, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Fig. 2

Effect of PDE inhibitors on platelet aggregation. A-L Representative curves and statistical analysis of light transmission aggregometry measurements. CRP-A-, ADP- or TRAP6-induced aggregation at 37 °C in human PRP after pre-treatment with A-C IBMX, D-F Ibudilast, G-I Sildenafil or J-L combination of Ibudilast und Sildenafil as indicated. vc vehicle control. Plotted: Mean ± SD; n ≥ 4, ordinary one-way ANOVA; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗∗p < 0.0001.

Fig. 3

Effect of PDE inhibitors on thrombus formation on immobilized collagen under flow. A Representative fluorescence microscopy images of thrombi stained with DiOC₆ (scale: 100 μM) after human whole blood was perfused over a collagen-coated surface at a shear rate of 1.000 s⁻¹. B-D Statistical analysis of thrombus area (area fraction) after pretreatment of whole blood with B IBMX, C Ibudilast or D Sildenafil as indicated. vc vehicle control. Plotted: Mean ± SD, n = 5, Repeated measures one-way ANOVA; ns not significant, ∗p < 0.05, ∗∗p < 0.01.

Fig. 4

A Representative western blots images of IBMX, Ibudilast and Sildelnafil treated platelet samples, first panel pVASP S157, second panel pVASP S239, third panel VASP and fourth panel GAPDH loading control. B/C Densitometric analysis of the 50 kDa signal of pVASP S157 and pVASP S329 under stimulation B pVASP S157 and C pVASP S329. vc vehicle control. Plotted: Mean ± SD; n = 5, repeated measure one-way ANOVA; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.