补体C5a信号通路在滤器相关血栓形成中的作用及机制研究

Blood purification therapy has been becoming more and more extensive application in clinic. However, the local thrombus formation on dialyzer has become the obstacle of blood purification clinical application. The research on the mechanism of dialyzer related thrombosis has been gradually becoming research hotspots and difficulties of multi-crossed disciplines. This project focuses on the latest research progress among complement C5a, thrombin (FⅡa) and platelet, combined with the previous research findings that the antithrombotic effect is still not ideal by inhibiting FⅡa. Furthermore, C5a plays a key role in the formation of dialyzer related thrombosis, but the mechanism is not clear. Therefore, we put forward a hypothesis that complement C5a signaling pathway mediates dialyzer related thrombosis. In this project, sheep and human are taken as research objects. From the overall, cell and molecular levels, we use specific antagonists including SCH79797, avacopan and complement factor H (CFH) to interve. Flow Cytometric Method, Western Blot, Calibrated Automated Thrombogram and other experimental techniques are used in this project, for exploring the role of C5a signaling pathway in the formation of dialyzer related thrombosis and its regulatory mechanism from two aspects of C5a-TF-FⅡa-Plt and CFH-C5a-Plt. This project will reveal the value of C5a/C5aR as the antithrombotic target, provide theoretical and experimental basises for developing the blood purification dialyzer with stronger antithrombotic.

血液净化治疗在临床上的应用日益广泛，而滤器局部血栓形成已成为制约其临床应用的“拦路虎”，滤器相关血栓形成机制的研究逐渐成为多学科领域交叉研究的热点与难点。本项目聚焦补体C5a、凝血酶（FⅡa）与血小板“分子对话”的最新研究进展，结合前期研究发现单纯抑制FⅡa的抗栓效果仍不理想，且C5a在滤器相关血栓形成中起关键作用，但机制不清，由此我们提出：“补体C5a信号通路介导滤器相关血栓形成”的假说。本项目以绵羊和人体为研究对象，从整体、细胞、分子层面，采用特异性拮抗剂SCH79797、avacopan，补体H因子（CFH）等干预，运用FCM、Western Blot、CAT等方法，从C5a-TF-FⅡa-Plt和CFH-C5a-Plt两方面探明C5a信号通路在滤器相关血栓形成中的作用及调控机制，揭示C5a/C5aR作为抗栓治疗新靶点的价值，为研发具备更强抗栓性能的血液净化滤器提供理论与实验依据。

在血液净化过程中，凝血或血栓堵塞滤器导致血液净化治疗中断的现象时有发生，严重影响其应用与疗效。本项目在前期基础上优化实验步骤，采用高分子材料领域成熟且先进的膜改性技术，实现了从“两步法”到“一步法”的跨越，将小分子高效抗凝剂阿加曲班接枝到滤器膜上，并对阿加曲班抗凝改性滤器膜进行抗凝性能及相关性能检测，发现阿加曲班抗凝改性滤器膜保留了其优良的理化性能，且具有良好的抗凝性能。本项目组以C5a-FⅡa-Plt信号通路为主线对滤器膜导致凝血的机制和阿加曲班抗凝改性滤器膜的抗凝机制进行探讨。研究发现：血液透析患者血浆中C5a的浓度显著高于正常对照组，滤器膜与血液接触后可诱导C5a活化，激活凝血酶和血小板，导致血栓形成，说明膜材料相关血栓形成存在C5a-FⅡa-Plt信号通路。阿加曲班改性滤器膜能有效抑制C5a激活、血小板活化及粘附，延长凝血时间，具有肯定的抗凝效应，表明阿加曲班改性滤器膜除直接抑制凝血酶外，还能通过抑制C5a-FⅡa-Plt信号通路减少血栓形成发挥抗凝作用，因此C5a是滤器相关血栓形成的关键干预靶点。本项目取得了阶段性成果，给新型血液净化滤器以及其它生物人工材料（如便携式血液净化装置、心脏瓣膜支架）的研发提供有价值的借鉴，为其成果转化奠定了重要的理论基础与实验依据。