Differential effects of phosphodiesterases (PDE) on platelet function

Background: The family of the phosphodiesterase enzymes (PDEs) play a pivotal role in regulating platelet activity by modulating intracellular levels of cAMP and cGMP. The pharmacological modulation of PDE activity by suitable PDE inhibitors has been found to reduce platelet activity and thus, thrombus formation. Platelets contain three PDE isoforms (2, 3 and 5). PDE2 and PDE3 have been shown to limit hydrolysis of cAMP, whereas cGMP levels are primarily modulated through PDE2 and PDE5. Their role in platelet function, however, is incomplete understood.
Methods: We examined the effect of three distinct PDE inhibitors (sildenafil, ibudilast, IBMX) on platelet function in human platelet-rich-plasma (PRP). Sildenafil is a selective inhibitor of PDE5, ibudilast is a non-selective inhibitor of several PDEs including PDE3, IBMX is a nonspecific PDE inhibitor. The classical platelet agonists collagen-related peptide (CRP, 2 / 5 µg/ml), adenosine diphosphate (ADP, 2.5 / 5 µM) and thrombin receptor activator peptide (TRAP, 10 / 20 µM) were used to activate platelets after preincubation with various PDE inhibitors. Platelet activity was assessed using flow cytometry (α_IIbβ₃ integrin activation and P-selectin expression) and light transmission aggregometry. Furthermore, we examined the impact of PDE inhibitors on platelet adhesion on immobilized collagen in whole blood using an ex vivo flow chamber system.
Results: The unselective inhibition of all three PDEs expressed in platelets by IBMX was shown to substantially reduced platelet activation (α_IIbβ₃ integrin activation and P-selectin expression) and aggregation in response to all tested platelet agonists. In contrast, the selective inhibition of only one of the three PDEs expressed in platelets was insufficient to reliably affect platelet activity. The inhibition of PDE3 by ibudilast demonstrated a selective effect on ADP- and TRAP-stimulated platelets, with almost no effect on CRP-stimulated platelets. The inhibition of PDE5 by sildenafil showed no discernible effect in the flow cytometry and light transmission aggregometry studies. In contrast, all tested PDE inhibitors demonstrated a significant effect on platelet-dependent thrombus formation on immobilized collagen under flow (1000 s^-1 shear rate).
Conclusion: The effect of PDE inhibitors on platelet function is influenced by two primary factors: the pharmacological profile of the substance and the pathways induced during platelet activation. Platelet activation by ADP via P₂Y₁₂ and TRAP via PAR1 showed a greater response to the PDE inhibitors than platelet activation by CRP via GPVI. This indicates that the GPVI-mediated signaling pathway is less dependent on intracellular cAMP/cGMP levels than the P₂Y₁₂- and PAR1-mediated signaling pathways.