DataSheet_1_Complement inhibition can decrease the haemostatic response in a microvascular bleeding model at multiple levels.xlsx

BackgroundHaemostasis is a crucial process by which the body stops bleeding. It is achieved by the formation of a platelet plug, which is strengthened by formation of a fibrin mesh mediated by the coagulation cascade. In proinflammatory and prothrombotic conditions, multiple interactions of the complement system and the coagulation cascade are known to aggravate thromboinflammatory processes and increase the risk of arterial and venous thrombosis. Whether those interactions also play a relevant role during the physiological process of haemostasis is not yet completely understood. The aim of this study was to investigate the potential role of complement components and activation during the haemostatic response to mechanical vessel injury.
MethodsWe used a microvascular bleeding model that simulates a blood vessel, featuring human endothelial cells, perfusion with fresh human whole blood, and an inducible mechanical injury to the vessel. We studied the effects of complement inhibitors against components of the lectin (MASP-1, MASP-2), classical (C1s), alternative (FD) and common pathways (C3, C5), as well as a novel triple fusion inhibitor of all three complement pathways (TriFu). Effects on clot formation were analysed by recording of fibrin deposition and the platelet activation marker CD62P at the injury site in real time using a confocal microscope.
ResultsWith the inhibitors targeting MASP-2 or C1s, no significant reduction of fibrin formation was observed, while platelet activation was significantly reduced in the presence of the FD inhibitor. Both common pathway inhibitors targeting C3 or C5, respectively, were associated with a substantial reduction of fibrin formation, and platelet activation was also reduced in the presence of the C3 inhibitor. Triple inhibition of all three activation pathways at the C3-convertase level by TriFu reduced both fibrin formation and platelet activation. When several complement inhibitors were directly compared in two individual donors, TriFu and the inhibitors of MASP-1 and C3 had the strongest effects on clot formation.
ConclusionThe observed impact of complement inhibition on reducing fibrin clot formation and platelet activation suggests a role of the complement system in haemostasis, with modulators of complement initiation, amplification or effector functions showing distinct profiles. While the interactions between complement and coagulation might have evolved to support haemostasis and protect against bleeding in case of vessel injury, they can turn harmful in pathological conditions when aggravating thromboinflammation and promoting thrombosis.

背景：止血（haemostasis）是机体阻断出血的核心生理过程，其依赖血小板栓的形成，并通过凝血级联反应（coagulation cascade）介导的纤维蛋白网予以加固。在促炎与促血栓状态下，补体系统（complement system）与凝血级联的多重相互作用已被证实可加重血栓炎症（thromboinflammatory）进程，提升动静脉血栓的发病风险。目前学界尚未完全明确，此类相互作用是否在止血的生理过程中同样发挥相关作用。本研究旨在探究补体成分及其激活状态在机械性血管损伤后的止血反应中的潜在功能。
方法：本研究采用模拟血管的微血管出血模型，该模型搭载人内皮细胞、新鲜人全血灌注系统，并可实现可诱导的血管机械损伤。我们针对凝集素途径（lectin pathway）的MASP-1、MASP-2、经典途径（classical pathway）的C1s、旁路途径（alternative pathway）的FD以及共同途径（common pathway）的C3、C5的补体成分抑制剂，同时纳入一种可同时抑制三条补体途径的新型三重融合抑制剂（TriFu），逐一研究其作用效果。通过共聚焦显微镜（confocal microscope）实时记录损伤部位的纤维蛋白沉积与血小板活化标志物CD62P的表达情况，分析其对凝血块形成的影响。
结果：靶向MASP-2或C1s的抑制剂未观测到纤维蛋白形成的显著减少；而在FD抑制剂存在的情况下，血小板活化程度显著降低。分别靶向C3或C5的两款共同途径抑制剂，均与纤维蛋白形成的大幅减少相关，且C3抑制剂存在时，血小板活化程度也有所下降。通过TriFu在C3转化酶（C3-convertase）水平对三条激活途径实施三重抑制，可同时减少纤维蛋白形成与血小板活化。在两名独立血液供体中直接对比多款补体抑制剂时，TriFu以及MASP-1与C3的抑制剂对凝血块形成的调控作用最为显著。
结论：本研究观测到补体抑制可减少纤维蛋白凝块形成与血小板活化，这提示补体系统在止血过程中发挥了调控作用，补体启动、扩增或效应功能的调节剂呈现出各异的作用谱。尽管补体与凝血系统的相互作用或许已进化为支持止血、在血管损伤时抵御出血的机制，但在病理状态下，它们可能会加重血栓炎症并促进血栓形成，从而产生有害影响。