FⅡa-polyP-FⅫ级联反应在血液净化凝血中的作用及AG抗凝滤器对其影响

The fundamental research on the mechanism of blood purification related coagulation/thrombosis is a weak link, restricting the development of anticoagulation techniques during blood purification. Based on the recent advances in the field of contact activation coagulation pathway, thrombin and platelets, we proposed a hypothesis—FⅡa-polyP-FⅫ cascade boosting thrombin overproduction leads to local thrombosis in dialyzer. In our previous research, we have successfully constructed the Argatroban-modified Anticoagulation Dialyzer (the AG Dialyzer) by adopting the emerging technique of membrane modification, which is a new exploration of anticoagulation mode during blood purification in high risk bleeding patients. In this project, we will choose sheep and human as study object, use interventions such as FⅡa (activating platelets), TFPI (inhibiting thrombin generation at the startup of coagulation), specific antagonists SCH79797 and UHRA9 (blocking the polyP-FⅫ pathway), and adopt experimental methods such as CAT, TEG, FCM, so then, we can investigate the effect on platelet activation and polyP release by normal dialyzer and the AG Dialyzer, thus analyzing the role of FⅡa-polyP-FⅫ cascade in the process of thrombosis during blood purification. This project aims to expound the mechanism of blood purification related coagulation/thrombosis and anticoagulation/ antithrombosis of the AG Dialyzer from a completely new perspective, and as well as providing scientific basis for the clinical translation of the AG Dialyzer.

血液净化相关性凝血/血栓形成的机制研究相对薄弱，制约了血液净化抗凝技术发展。基于接触激活凝血途径、凝血酶与血小板的最新研究进展，我们提出“FⅡa-polyP-FⅫ级联反应助推凝血酶过量生成导致滤器局部血栓形成”假说。本课题组前期采用新兴膜改性技术，成功构建了阿加曲班抗凝改性膜滤器（AG滤器），对高危出血患者血液净化抗凝进行一种全新探索。本项目以绵羊和人体为研究对象，采用FⅡa（激活血小板），TFPI（抑制启动阶段的凝血酶生成）及特异性拮抗剂SCH79797、UHRA9（阻断polyP-FⅫ途径）等干预，运用CAT、TEG、FCM等方法，观察普通血液净化滤器与AG滤器对血小板活化、多聚磷酸盐（polyP）释放的影响，解析FⅡa-polyP-FⅫ级联反应在血栓形成过程中的作用。本项目将从新的视角阐明血液净化相关性凝血/血栓形成及AG滤器抗凝/抗栓机制，并为促进AG滤器临床转化提供依据。

高危出血患者的血液净化抗凝问题已成为制约血液净化临床应用的“瓶颈”，而血液净化滤器等生物材料所致的凝血/血栓形成研究相对薄弱与滞后是其重要原因之一。本项目针对血液净化凝血发生的关键部位——滤器膜/血液接触界面，采用新兴膜改性技术，构建一种能在膜/血界面局部发挥高效抗凝效能的具有特殊抗凝性能的阿加曲班抗凝改性膜，开创一种新型血液净化抗凝模式，并深入探究其抗凝机制。本项目在前期工作基础上，进一步优化了膜改性方法，通过浸没沉淀相转化法、多巴胺氧化自聚合反应、席夫碱反应等方法，成功构建出抗凝效果更为优越的新型阿加曲班抗凝改性膜/滤器（新型AG膜/滤器），对其进行全面性能检测，结果显示：其改性过程未损及膜本体结构，膜的理化性能良好，亲水性明显提高；具有良好的生物相容性与血液相容性。优化的膜改性工艺更为先进合理，利于产业化制作。本项目组以FⅡa-polyP-FⅫ级联反应为主线对滤器膜导致凝血的机制和新型AG膜/滤器的抗凝机制进行探讨，结果表明：与未改性的聚醚砜膜相比，AG膜/滤器能有效抑制滤器膜材料导致的血小板活化、粘附，延长APTT、PT、TT，具有肯定的抗凝效应。聚醚砜膜与血液接触后可诱导血小板释放polyP，并激活FⅫ，后者被polyP阻断剂显著抑制，说明在膜材料相关凝血过程中存在polyP-FⅫ级联反应的促凝机制。新型AG膜/滤器可显著抑制这一过程。上述结果表明：新型AG膜/滤器除直接抑制凝血酶外，还通过抑制polyP-FⅫ级联反应减少凝血酶过量生成而发挥抗凝作用。该级联反应为一个有价值的抗凝干预靶点。本项目计划总体完成良好，取得阶段性成果，并为后续更完善的抗凝改性膜/滤器的研制及其成果转化奠定了十分重要的实验和理论基础。