血小板TLR4途径活化致其脱唾液酸与慢加急性肝衰竭血小板减少的关系

Due to lack of efficient therapy options, acute on chronic liver failure (ACLF), which is predominantly caused by chronic HBV infection in China, has high mortality. Thrombocytopenia are frequently seen, however the underlying mechanism is fully understood yet in ACLF. Our preliminary results were against the traditional concepts about thrombocytopenia in liver failure, which is generally derived from liver cirrhosis conditions. The percentages of immature platelet fractions were greater in ACLF than mild CHB or cirrhosis patients. Spleen areas did not increase as thrombocytopenia occurred while liver damages progressed. Obvious thrombocytopenia was even seen in one case developed hepatic failure after splenectomy. Apoptotic platelets were comparable between mild CHB and ACLF cases. All these suggested that other underneath mechanism(s) contribute to thrombocytopenia in ACLF individuals. In ASGR-/- or sialyltransferase-/- animal, chilled platelets survived well. Platelets treated by neuraminidase can easily be adhered and ingested by HepG2 cells via ASGR and its counterpart, Asialo-GP1bα on platelet membrane. Macrophage or Kupffer cells deletions do not alter the Asialo-PLT clearances. These elegant experiments demonstrated that platelets, mainly the Asialo-PLT, are cleared by hepatocytes. Considering the common endotoxemia in ACLF patients, we tried LPS (3.0μg/ml, 30 minutes at room temperature) to treat platelets from CHB patients, and surprisingly we found that the neuraminidase activity, the sialic acid contents and the adherence to HepG2 cell all increased. Platelets have functional toll like receptor 4 (TLR4), and its endogenous ligands such as hyaluronan and HMGB1 increased as well in ACLF. TLR4 pathway is indispensable for LPS-induced thrombocytopenia. Therefore we speculate that elevated contents of TLR4 ligands can increase neuraminidase activity on platelet, which changes to Asialo-PLT then adhered to or ingested by hepatocytes. Overwhelming adherence or ingestions of Asialo-PLT by hepatocytes could lead to thrombocytopenia, strengthen platelet aggregations and reduce sinusoidal microcirculation, releasing 5-HT, TGFβ and CXCL4 as well, to aggravate hepatitis, block hepatocyte regeneration and make fibrosis exacerbated. In vitro and in vivo experiments will be performed to clarify the underlying mechanism for increased neuraminidase activity on platelet after LPS treatment, observe the proliferation or apoptosis status of hepatocytes after the adherence or ingestion of Asialo-PLT, explore the impacts of neuraminidase inhibitor on platelet clearance in mice models with acute liver failure, and to build associations between Asialo-PLT and platelet counts and ACLF outcome in clinical cohort. The present project would help to clarify the mechanism for thrombocytopenia in ACLF cases and find the potentials of neuraminidase inhibitors to manage ACLF patients.

ACLF进展期常见血小板减少，一般认为是骨髓抑制、脾脏破坏等因素所致。但我们的初步观察结果不支持这些观点。最近研究证实，肝细胞通过ASGR粘附、内吞脱唾液酸血小板致血小板减少。我们的进一步结果显示，ACLF血小板唾液酸酶活性增高，肝炎患者血小板LPS刺激后脱唾液酸、结合肝细胞的比例增加。ACLF患者LPS、透明质酸、HMGB1等TLR4配体含量增加。根据临床观察、文献和预实验结果提出课题假说：肝衰竭时TLR4配体增多致血小板脱唾液酸化加重，被肝细胞粘附、内吞，血小板减少。粘附的血小板释放活性物质影响肝细胞增殖与凋亡。计划用多种实验手段确定TLR4途径引起血小板脱唾液酸的机制、肝细胞粘附/内吞血小板后的增殖凋亡、唾液酸酶抑制剂对肝衰竭鼠模型血小板清除的调控、血小板脱唾液酸与ACLF血小板减少及临床转归的联系。本课题可进一步阐明ACLF血小板减少的机制、探索唾液酸酶抑制剂治疗ACLF的可能性。

慢加急性肝衰竭死亡率高，缺少有效的预后判断指标。慢性肝病谱中血小板减少极为常见，通常认为由脾脏破坏增加，TPO生成减少，骨髓抑制等原因导致。慢加急性肝衰竭患者入院后血小板快速下降，但其具体机制及与临床事件的相关性并不清楚。本研究发现，ACLF患者入院后血小板大幅下降是短期死亡的独立危险因素，同时血小板下降比例还可预测ACLF的进展及新发肝外器官衰竭。ACLF患者的网织血小板、TPO水平，活化、凋亡血小板比例及血浆糖萼素水平与慢乙肝急性加重患者无异，但纤溶指标D-dimer及纤维蛋白原明显异常，在移除肝组织中可以检测到血小板聚集，提示凝血消耗可能是肝衰竭血小板减少的原因之一。在104例入院检测了D-dimer的患者中，我们发现D-dimer联合INR、腹水可以较好的预测ACLF患者28天的结局及新发器官衰竭，进一步提示ACLF可能存在高凝和纤溶，且与预后相关。在急性肝衰竭小鼠模型中，我们证实抗血小板药物可有效提高LPS/D-GalN诱导急性肝衰竭小鼠的存活。阻断VWF-GP1b相互作用一方面可减少血小板在肝内的聚集，恢复循环血小板计数，另一方面在一定程度上降低了细胞因子风暴的水平，因而改善了小鼠生存。结合临床观察和动物实验，我们提出凝血消耗可能是肝衰竭血小板减少的重要原因，活化的血小板或造成肝衰竭的“二次打击”，危及生命。临床上，D-dimer联合INR、腹水可简单方便的帮助判断预后，围绕血小板活化、凝血级联反应及纤溶系统的其他指标也有望成为新的预测指标。本研究提出的血小板减少及D-二聚体预测ALCF预后需要更大样本量、多中心的数据进一步验证，血小板的凝血消耗假说也需要更多组织学证据支持。总之，深入认识血小板在肝衰竭发生发展中的作用机制，将有助于治疗药物的开发，疾病转归的判断和患者管理。