Impaired Platelet Function and Thrombus Formation in PDE5A-Deficient Mice
Impaired Platelet Function and Thrombus Formation in PDE5A-Deficient Mice
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DOI：
10.1055/a-1962-1613
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发表时间：
2022-12-30
影响因子：
6.7
通讯作者：
Qiao, Jianlin
中科院分区：
文献类型：
--
作者：
Gui, Xiang;Chu, Xiang;Qiao, Jianlin
细胞内环GMP （cGMP）抑制血小板功能。血小板cGMP水平由磷酸二酯酶5A （PDE5A）介导的降解控制。然而，PDE5A在血小板功能和血栓形成中的确切作用尚不清楚。在本研究中，我们描述了PDE5A在血小板活化和功能中的作用。从野生型或PDE5A（-/-）小鼠中分离血小板，测定血小板聚集、活化、磷脂酰丝氨酸暴露（膜联蛋白- v结合）、活性氧（ROS）产生、血小板扩散和凝块缩回。细胞内钙动员使用Fluo-4 AM通过微孔板读取仪进行测定。Western blot检测VASP、ERK1/2、p38、JNK、AKT的磷酸化水平。评估fecl3诱导的动脉血栓形成和静脉血栓形成，评估体内止血功能和血栓形成情况。此外，用微流体全血灌注法评估体外血栓形成。pde5a缺陷小鼠尾出血时间明显延长，动脉和静脉血栓形成延迟。PDE5A缺乏显著抑制血小板聚集、ATP释放、p -选择素表达和整合素aIIbb3激活。此外，在缺乏pde5a的血小板中观察到胶原蛋白或纤维蛋白原的扩散受损和凝块收缩。此外，PDE5A缺乏减少了磷脂酰丝氨酸暴露、钙动员、ROS产生，增加了细胞内cGMP水平，同时VASP磷酸化升高，ERK1/2、p38、JNK和AKT磷酸化降低。综上所述，PDE5A调节血小板活化和功能以及血栓形成，表明靶向治疗PDE5A可能有益于血栓性疾病的治疗。
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.