目的·探讨促凝血小板在H1N1流行性感冒（流感）病毒感染的小鼠体内血栓形成中的作用。方法·利用鼻滴注H1N1流感病毒构建小鼠病毒感染模型，持续监测病毒感染后小鼠的体质量，通过眼眶后静脉丛采集全血测定血小板数量。腹主动脉抽血后制备小鼠血浆样本，采用酶联免疫吸附试验（enzyme linked immunosorbent assay，ELISA）测定小鼠血浆凝血酶时间（thrombin time，TT），及纤维蛋白原、D-二聚体、白细胞介素-6（interleukin-6，IL-6）、肿瘤坏死因子α（tumor necrosis factor-α，TNF-α）水平。制备感染小鼠的肺和肝组织切片，进行苏木精-伊红（hematoxylin and eosin，HE）染色，观察小鼠肺和肝组织内血栓形成情况；利用酪酰胺信号放大（tyramide signal amplification，TSA）技术对肺和肝组织进行多重荧光免疫组织化学染色，分析血栓的组成成分。利用流式细胞术监测病毒感染后小鼠体内单核细胞-血小板聚集物水平和促凝血小板亚群的比例变化。通过尾静脉注射小鼠GP1b抗体（R300）构建小鼠血小板清除模型，持续监测小鼠体质量和血小板数量，测定小鼠血浆TT及纤维蛋白原、D-二聚体水平。结果·H1N1病毒感染后，小鼠体质量持续性下降，凝血相关指标异常，表现为TT延迟、纤维蛋白原水平降低、D-二聚体水平升高等，血浆IL-6和TNF-α水平也升高（均P<0.05）；HE染色结果显示，病毒感染后的小鼠肺和肝组织血管内可见明显的血栓形成（均P<0.05）。流式分析结果表明H1N1病毒感染后促凝血小板比例显著升高，促凝血小板的比例与病毒使用滴度呈正相关，且与D-二聚体、纤维蛋白原水平以及血栓面积具有相关性（均P<0.05）。TSA染色结果发现血管内血栓主要由大量纤维蛋白和促凝血小板组成。将小鼠体内血小板清除后，感染H1N1病毒引发的肺和肝组织血栓形成水平明显降低，并且凝血异常程度得到缓解（均P<0.05）。结论·H1N1病毒可能通过增加促凝血小板比例，导致凝血功能异常，从而诱发小鼠多组织脏器中的血栓形成。

Objective ·To investigate the role of procoagulant platelets in thrombosis induced by H1N1 influenza virus infection in mice. Methods ·A viral infection model was established in mice by intranasal instillation of the H1N1 influenza virus. Body weight was continuously monitored post-infection, and whole blood was collected from the retro-orbital venous plexus to measure platelet counts. Plasma samples were prepared from abdominal aortic blood. Thrombin time (TT), fibrinogen, D-dimer, interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) levels were measured by using enzyme-linked immunosorbent assay (ELISA). Lung and liver tissue sections from infected mice were prepared, and hematoxylin-eosin (HE) staining was employed to observe thrombus formation in lung and liver tissues of mice. Tyramide signal amplification (TSA)-based multiplex immunohistochemistry was performed on lung and liver tissues to analyze thrombus composition. Flow cytometry was used to monitor the monocyte-platelet aggregates and the proportion of procoagulant platelets in mice after viral infection. A platelet depletion model was established by tail vein injection of anti-mouse GP1b (R300). Body weight and platelet counts were continuously monitored in infected mice. Plasma TT, fibrinogen, and D-dimer levels were measured to evaluate coagulation function in mice. Results ·Following H1N1 infection, mice exhibited progressive weight loss and abnormal coagulation parameters, including prolonged TT, decreased fibrinogen levels, and elevated D-dimer levels (all P<0.05).Plasma levels of IL-6 and TNF-α were also elevated. HE staining revealed prominent thrombus formation in the pulmonary and hepatic vasculature of infected mice (all P<0.05). Flow cytometry demonstrated a significant increase in the proportion of procoagulant platelets after infection. The proportion of procoagulant platelets showed a positive correlation with viral dose, and was associated with D-dimer levels, fibrinogen levels, and thrombus area (all P<0.05). TSA staining further confirmed that the thrombi primarily consisted of fibrin and procoagulant platelets. Depletion of platelets in mice prior to H1N1 infection markedly reduced thrombosis in lung and liver tissues and alleviated coagulation abnormalities (all P<0.05). Conclusion ·H1N1 virus infection may significantly elevate the proportion of procoagulant platelets in mice, leading to coagulation dysfunction and subsequent thrombosis in multiple tissues.