RESUMO
Background: Haemostasis 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. Methods: We 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. Results: With 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. Conclusion: The 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.
RESUMO
Background: Complement lectin pathway components, in particular mannan-binding lectin (MBL) and MBL-associated serine proteases (MASPs) have been shown to interact with coagulation factors and contribute to clot formation. Here we investigated the role of MBL and MASP-1 in the haemostatic response following mechanical vessel injury in a human microfluidic bleeding model. Methods: We studied haemostasis in a microvascular bleeding model in the presence of human endothelial cells and human whole blood under flow conditions. We monitored incorporation of proteins into the clot with fluorescently labelled antibodies and studied their effects on clot formation, platelet activation, and bleeding time with specific inhibitors. Platelet activation was also studied by flow cytometry. Results: Upon vessel injury, MBL accumulated at the injury site in a well-defined wall-like structure. MBL showed partial colocalisation with fibrin, and strong colocalisation with von Willebrand factor and (activated) platelets. Flow cytometry ruled out direct binding of MBL to platelets, but confirmed a PAR4- and thrombin-dependent platelet-activating function of MASP-1. Inhibiting MBL during haemostasis reduced platelet activation, while inhibiting MASP-1 reduced platelet activation, fibrin deposition and prolonged bleeding time. Conclusion: We show in a microvascular human bleeding model that MBL and MASP-1 have important roles in the haemostatic response triggered by mechanical vessel injury: MBL recognises the injury site, while MASP-1 increases fibrin formation, platelet activation and shortens bleeding time. While the complement lectin pathway may be harmful in the context of pathological thrombosis, it appears to be beneficial during the physiological coagulation response by supporting the crucial haemostatic system.
