背景

补体系统为固有免疫的重要组成部分，其活化与血栓性疾病的发生及发展密切相关。补体系统通过三种途径活化，分别是经典途径、凝集素途径以及旁路途径，最终形成攻膜复合体（Membrane Attack Complex，MAC）。已有研究表明补体成分参与血栓形成，然而补体MAC及其成分C8在血栓中的作用未知。本研究的目的是确定MAC和C8在深静脉血栓中的作用和机制。

方法

对补体攻膜复合体成分6（C6）、7（C7）、8（C8）、9（C9）基因缺失小鼠实施下腔静脉限流诱发深静脉血栓形成；统计静脉血栓的发生率；收集静脉血栓进行栓长测量和称重；对静脉血栓进行病理染色，观察白细胞浸润情况；收集外周血进行外周血细胞计数、凝血四项反应、血栓弹力图检测和血小板聚集反应；使用流式细胞术检测外周血中血小板和中性粒细胞的激活情况，并检测血小板-中性粒细胞复合体（Neutrophil Platelet Aggregate，NPA）数目；使用酶联免疫吸附法检测小鼠外周血中炎症因子和补体分子的水平；使用微量分光光度计检测外周血DNA水平；使用高分辨率成像活体观察小鼠下腔静脉限流时，下腔静脉的中性粒细胞及血小板的变化情况。

结果

与野生型小鼠相比，C8缺失抑制由下腔静脉限流诱导的深静脉血栓的形成；其中C8敲除不影响血小板的聚集和体外激活、外源与及内源性凝血时间；通过病理及在体成像观察发现C8敲除能减少静脉血栓中白细胞浸润，伴随中性粒细胞胞外陷阱（Neutrophil Extracellular Traps，NETs）释放和NPA数量降低。同时，C8缺失显著抑制限流后外周血炎症因子IL-1β、TNF-α、IL-6、趋化因子CXCL8、C3a和C5a水平，有效降低外周血循环中的中性粒细胞数，抑制中性粒细胞激活。另外，在下腔静脉限流诱导的深静脉血栓形成过程中，小鼠缺失C6、C7和C9，均能抑制下腔静脉血栓形成，同时有效减少静脉血栓中的白细胞浸润、中性粒细胞释放NETs和NPA数量降低。

结论

MAC成分（包括C8）通过干预中性粒细胞的黏附、浸润、NETs的释放以及NPA形成，调节深静脉血栓的形成。靶向干预MAC形成和C8具有防治深静脉血栓的潜在应用价值。

Part 1 The role of complement component C8 in deep vein thrombosis

Abstract

Background

The complement system is an important part of innate immunity that activated closely related to thrombotic diseases. Complement system can be activated through three pathways, including the classical pathway, the lectin pathway and alternative pathway, and finally form the membrane attack complex (MAC). Previous studies shown that components of complement involved in thrombotic diseases. However, the role of complement MAC and its component C8 in thrombosis is unclear. The aim of this study was to determine the role and mechanism of MAC and C8 in deep vein thrombosis.

Method

Deep vein thrombosis was induced by partial flow stenosis in the inferior vena cava in complement component 6, 7, 8, 9 deficient mice. The incidence of venous thrombosis was calculated. Venous thrombosis was collected for thrombus length and weigh measurement. The venous thromboses were pathologically stained to observe the infiltration of white blood cells. Peripheral blood was collected for peripheral cell count, four coagulation reactions, thromboelastogram test and platelet aggregation study. Flow cytometry was used to detect the activation of platelets and neutrophils and neutrophil-platelet aggregates (NPA) in peripheral blood. The levels of inflammatory factors and complement molecules in peripheral blood of mice were detected by enzyme-linked immunosorbent assay. DNA level of peripheral blood was detected by microspectrophotometer. The motion of neutrophils and platelets in the inferior vena cava of mice was observed by high resolution in vivo imaging.

Result

Compared with wild-type mice, C8 deficient inhibited the formation of deep venous thrombosis, but did not affect platelet aggregation and activation, exogenous and endogenous coagulation time. Pathological and in vivo imaging observations showed that C8 deficient reduced leukocyte infiltration in venous thrombosis, as well as neutrophil extracellular trap (NETs) release and reduced NPA number. Meanwhile, C8 deficiency significantly inhibited the levels of inflammatory cytokines IL-1β, TNF-α, IL-6, chemokines CXCL8, C3a and C5a in peripheral blood after flow restriction, and effectively reduced the number and activation of neutrophils in peripheral blood. Additionally, C6, C7 and C9 deficient in mice also inhibited in the process of deep venous thrombosis induced by inferior vena cava restriction, and effectively reduce leukocyte infiltration, NETs release and NPA number in venous thrombosis.

Conclusion

MAC components (including C8) regulated deep vein thrombosis by interfering with neutrophils adhesion and infiltration, NETosis and NPA formation. Targeted intervention of MAC formation and C8 has potential application value in prevention and treatment of deep vein thrombosis.