黄嘌呤氧化酶在高尿酸血症患者血小板高反应性中的作用及机制

doi:10.3969/j.issn.1674-8115.2025.12.002

摘要/Abstract

摘要：

目的·分析高尿酸血症患者血小板的活化指标与黄嘌呤氧化酶（xanthine oxidase，XO）活性的相关性，并探索XO在血小板聚集、铺展过程中的作用及其可能的分子机制。方法·2023年1月至2025年6月纳入复旦大学附属华山医院风湿科及体检中心的高尿酸血症患者24例，以同期该院体检中心的24例健康志愿者为对照，收集所有受试者的年龄、性别、合并症及用药史资料，并采集其清晨空腹静脉血。制备洗涤血小板，采用流式细胞术检测血小板活化指标P选择素及血小板活化复合物-1（platelet activation complex-1，PAC-1）的表达水平；采用吸光度法测定血小板内XO活性，并运用Spearman相关性分析评估血尿酸水平、XO活性与血小板活化指标的相关性。检测不同浓度非布司他对胶原、腺苷二磷酸（adenosine diphosphate，ADP）、U46619及凝血酶诱导的血小板聚集的影响；检测血小板在纤维蛋白原上的铺展情况；同时检测凝血酶诱导下血小板内活性氧（reactive oxygen species，ROS）的表达。结果·与健康对照组相比，高尿酸血症组患者血小板表面P选择素、PAC-1的表达水平显著升高（均P<0.05），但二者与患者血尿酸水平无明显相关（均P>0.05）。血小板XO活性与凝血酶诱导的P选择素表达（r=0.453，P=0.001）及PAC-1表达（r=0.478，P=0.001）呈正相关。50 μmol/L及100 μmol/L非布司他可显著抑制胶原、ADP、U46619及凝血酶诱导的血小板聚集，同时显著抑制血小板在纤维蛋白原上的铺展，差异均有统计学意义（均P<0.05），且抑制作用随非布司他浓度升高而增强，呈现浓度依赖性；同时，在凝血酶刺激条件下，经非布司他处理后的血小板内ROS生成水平显著降低（P<0.05）。结论·高尿酸血症患者的血小板活化指标高于对照组，且其血小板表面P选择素、PAC-1的表达水平与血小板内XO活化水平呈正相关；XO抑制剂可通过调控血小板内ROS生成参与血小板活化的调节。

关键词: 黄嘌呤氧化酶, 血小板, 活性氧, 高尿酸血症

Abstract:

Objective ·To analyze the correlation between platelet activation indices and xanthine oxidase (XO) activity in patients and the underlying molecular mechanisms. Methods ·Between January 2023 and June 2025, 24 patients with hyperuricemia were recruited from the Department of Rheumatology and the Physical Examination Center of Huashan Hospital, Fudan University. Twenty-four healthy volunteers from the same center during the same period served as the healthy control group. Demographic data (age, gender), comorbidities, and medication history of all subjects were collected, and their fasting venous blood was drawn. Washed platelets were prepared for subsequent assays. The expression levels of P-selectin and platelet activation complex-1 (PAC-1) on the platelet surface were determined by flow cytometry, while platelet XO activity was measured via an absorbance assay. Spearman correlation analysis was performed to evaluate the relationships between uric acid levels, XO activity, and platelet activation markers. The effects of various concentrations of febuxostat on platelet aggregation induced by collagen, adenosine diphosphate (ADP), U46619, and thrombin were assessed. Platelet spreading on fibrinogen was analyzed. Furthermore, thrombin-induced intraplatelet reactive oxygen species (ROS) production was measured. Results ·Compared with the healthy control group, patients in the hyperuricemia group exhibited significantly higher platelet surface expression of P-selectin and PAC-1 (both P<0.05). However, neither marker showed a significant correlation with serum uric acid levels (both P>0.05). Platelet XO activity showed a positive correlation with thrombin-induced P-selectin expression (r=0.453, P=0.001) and PAC-1 expression (r=0.478, P=0.001). Febuxostat at concentrations of 50 μmol/L and 100 μmol/L significantly inhibited collagen, ADP, U46619, and thrombin-induced platelet aggregation and also significantly suppressed the spreading of platelets on fibrinogen. These inhibitory effects were statistically significant (all P<0.05) and intensified with increasing concentrations of febuxostat, demonstrating a concentration-dependent manner. Meanwhile, febuxostat treatment significantly reduced intraplatelet ROS generation in response to thrombin stimulation (P<0.05). Conclusion ·Platelet activation markers are higher in hyperuricemia patients than in the healthy control group. The expression levels of P-selectin and PAC-1 on the platelet surface show a positive correlation with intraplatelet XO activity. XO inhibitors can modulate platelet activation by reducing intracellular ROS production.

Key words: xanthine oxidase, platelet, reactive oxygen species, hyperuricemia

中图分类号:

R543.3

高稳, 潘杰松, 赵奕凯, 罗心平, 李剑. 黄嘌呤氧化酶在高尿酸血症患者血小板高反应性中的作用及机制[J]. 上海交通大学学报（医学版）, 2025, 45(12): 1568-1577.

GAO Wen, PAN Jiesong, ZHAO Yikai, LUO Xinping, LI Jian. Role and mechanism of xanthine oxidase in platelet hyperreactivity of hyperuricemia patients[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2025, 45(12): 1568-1577.

图/表 9

表1 高尿酸血症患者组与对照组的基线特征

Tab 1 Baseline characteristics of the hyperuricemia group and the healthy control group

Item	Hyperuricemia group (n=24)	Healthy control group (n=24)	P value
Age/year	62.13±15.92	70.17±7.52	0.079
Gender (male)/n(%)	17 (70.83)	14 (58.33)	0.365
Hypertension/n(%)	3 (12.5)	0 (0)	0.234
Platelet (×10¹²)/L	206.75±57.25	214.92±61.27	0.636
Red blood cell/(g·L^-1)	4.58±0.52	4.46±0.65	0.485
White blood cell (×10⁹)/L	6.81±1.74	7.34±2.08	0.342
Anti-hyperuricemic drugs/n(%)	6 (25.00)	0 (0)	0.022
Febuxostat/n(%)	5 (20.83)	0 (0)	0.050
Alopurinol/n(%)	1 (4.17)	0 (0)	1.000

图1 高尿酸血症患者组和健康对照组血小板表面P-选择素和PAC-1的表达Note： A. P-selectin expression. B. PAC-1 expression. Thrombin: 0.05 U·mL-1. HU—hyperuricemia; MFI—mean fluorescence intensity.

Fig 1 Expression of P-selectin and PAC-1 on platelet surface in the hyperuricemia patient group and the healthy control group

图2 血小板活化指标P-选择素及PAC-1与血尿酸水平及血小板XO活性的相关性Note： A. Correlation between serum uric acid level and P-selectin expression. B. Correlation between serum uric acid level and PAC-1 expression. C. Correlation between XO activity and P-selectin expression. D. Correlation between XO activity and PAC-1 expression. MFI—mean fluorescence intensity.

Fig 2 Correlation of platelet activation indicators P-selectin and PAC-1 with serum uric acid levels and platelet XO activity

图3 非布司他对不同刺激剂诱导的血小板聚集的影响Note： Representative platelet aggregation tracings induced by different agonists and summary data from 3 experiments. A. 2 μg·mL-1 collagen. B. 10 μmol·L-1 ADP. C. 0.1 μg·mL-1 U46619. Febu—febuxostat.

Fig 3 Effect of febuxostat on platelet aggregation induced by different agonists

图4 非布司他对不同浓度凝血酶诱导的血小板聚集的影响Note： Representative platelet aggregation tracings induced by different concentrations of thrombin and summary data from 3 experiments. A. 0.05 U·mL-1 thrombin. B. 0.1 U·mL-1 thrombin. Febu—febuxostat.

Fig 4 Effect of febuxostat on platelet aggregation induced by different concentrations of thrombin

图5 别嘌呤醇对胶原和凝血酶诱导的血小板聚集的影响Note： Representative platelet aggregation and summary data from 3 experiments. A. 2 μg·mL-1 collagen. B. 0.1 U·L-1 thrombin. Allo—allopurinol.

Fig 5 Effect of allopurinol on platelet aggregation induced by collagen and thrombin

图6 非布司他对血小板铺展的影响Note： A. Representative fluorescence images of platelet spreading on immobilized fibrinogen. B. Summary data from 3 experiments are presented. Febu—febuxostat.

Fig 6 Effect of febuxostat on platelet spreading

图7 非布司他对血小板ROS生成的影响Note： A. Representative histogram obtained by flow cytometry for DCF (dichlorodihydro-fluorescein) fluorescence. B. Summary data from 3 experiments are presented. Febu—febuxostat.

Fig 7 Effect of febuxostat on the production of ROS in platelets

图8 非布司他联合H2O2 或NACA对血小板聚集的影响Note： Effect of febuxostat combined with H2O2 (A) or NACA (B) on platelet aggregation and summary data from 3 experiments. Febu—febuxostat.

Fig 8 Effect of febuxostat combined with H2O2 or NACA on platelet aggregation

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