Abstract

Intracellular cyclic GMP (cGMP) inhibits platelet function. Platelet cGMP levels are controlled by phosphodiesterase 5A (PDE5A)-mediated degradation. However, the exact role of PDE5A in platelet function and thrombus formation remains poorly understood. In this study, we characterized the role of PDE5A in platelet activation and function. Platelets were isolated from WT or PDE5A-/- mice to measure platelet aggregation, activation and phosphatidylserine exposure (annexin-V binding) and ROS generation, platelet spreading as well as clot retraction. Cytosolic calcium mobilization was measured using Fluo-4 AM by a microplate reader. Western blot was used to measure the phosphorylation of VASP, ERK1/2, p38, JNK and AKT. FeCl3-induced arterial thrombosis and venous thrombosis was performed to evaluate the in vivo hemostatic function and thrombus formation. Additionally, in vitro thrombus formation was assessed in a microfluidic whole-blood perfusion assay. PDE5A deficient mice presented significantly prolonged tail bleeding time, delayed arterial and venous thrombus formation. PDE5A deficiency significantly inhibited platelet aggregation, ATP release, P-selectin expression and integrin aIIbb3 activation. In addition, an impaired spreading on collagen or fibrinogen and clot retraction was observed in PDE5A-deficient platelets. Moreover, PDE5A deficiency reduced phosphatidylserine exposure, calcium mobilization, ROS production and increased intracellular cGMP level along with elevated VASP phosphorylation and reduced phosphorylation of ERK1/2, p38, JNK and AKT. In conclusion, PDE5A modulates platelet activation, function and thrombus formation, indicating that therapeutic targeting it might be beneficial for the treatment of thrombotic diseases.