Impaired Platelet Function and Thrombus Formation in PDE5A-Deficient Mice

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 wild type or PDE5A^−/− mice to measure platelet aggregation, activation, phosphatidylserine exposure (annexin-V binding), reactive oxygen species (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 were assessed 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 and 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 and function and thrombus formation, indicating that therapeutically targeting it might be beneficial for the treatment of thrombotic diseases.

Keywords

PDE5A - cGMP - platelets - thrombosis - hemostasis

Author Contributions

X. Gui and X. Chu performed research, analyzed data, and wrote the manuscript. S. Zhang, Y. Wang, Y. Du, Y. Ding, H. Tong, M. Xu, Y. Li, W. Ju, Z. Sun, and Z. Li performed research and analyzed data, L. Zeng, K. Xu, and J. Qiao conceived and designed the study and wrote the manuscript.

^# These authors share first authorship.

^* These authors share senior authorship.

Publication History

Received: 29 June 2022

Accepted: 11 October 2022

Accepted Manuscript online:
17 October 2022

Article published online:
30 December 2022

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