Lakbakbi Souad, Debrumetz Alexandre, Terryn Christine, Szymezak Jean, Rieu Philippe, Nguyen Philippe
Equipe d'accueil 3801 Hémostase et Remodelage Vasculaire Post-Ischémie, Université de Reims, France.
Centre Hospitalier Universitaire, Service néphrologie, Reims, France.
Thromb Res. 2016 Aug;144:218-23. doi: 10.1016/j.thromres.2016.05.017. Epub 2016 May 27.
End-stage renal patients present a high risk of thrombosis and bleeding. Consequently, it is challenging to prevent clotting during hemodialysis. If a contact system induces thrombin generation in the extra corporeal circuit, recent data suggest a role of tissue factor (TF) in hemodialysis-associated thrombosis. Using a method of elution, we collected adhering cells to an acrylonitrile membrane layered by polythyleneimine (AN69-ST). Using optic microscopy and flow cytometry, we observed that adherent cells were mainly constituted by activated polymorphonuclear neutrophils (PMNs). Using a sensitive fluorogenic method of thrombin generation, we found that adhering cells triggered thrombin generation in a TF-dependent manner. We next identified the presence of TF mRNA (Q-PCR) in adhering cells. Using immunofluorescence, we observed the presence of TF in PMNs and of TF-decorated neutrophil extracellular traps (NETs). As TF triggers thrombin generation after binding to serine protease FVIIa, we evaluated the effect of an inactivated human recombinant factor VIIa (hrFVIIai) in a sheep model of hemodialysis (HD). One single bolus of hrFVIIai maintained the full patency of the hemodialysis circuit without any measurable systemic anticoagulant effect. TF is a promising target for preventing thrombosis during HD.
终末期肾病患者存在血栓形成和出血的高风险。因此，在血液透析过程中预防凝血具有挑战性。如果接触系统在体外循环中诱导凝血酶生成，最近的数据表明组织因子（TF）在血液透析相关血栓形成中起作用。我们采用洗脱方法，收集附着在聚乙烯亚胺（AN69-ST）分层的丙烯腈膜上的细胞。通过光学显微镜和流式细胞术，我们观察到附着细胞主要由活化的多形核中性粒细胞（PMN）组成。使用一种敏感的凝血酶生成荧光方法，我们发现附着细胞以TF依赖的方式触发凝血酶生成。接下来，我们在附着细胞中鉴定出TF mRNA（定量聚合酶链反应）的存在。通过免疫荧光，我们观察到PMN中存在TF以及TF修饰的中性粒细胞胞外陷阱（NET）。由于TF与丝氨酸蛋白酶FVIIa结合后触发凝血酶生成，我们在绵羊血液透析（HD）模型中评估了灭活的重组人因子VIIa（hrFVIIai）的作用。单次推注hrFVIIai可维持血液透析回路的完全通畅且无任何可测量的全身抗凝作用。TF是预防HD期间血栓形成的一个有前景的靶点。
