Interplay of procoagulatory and neutrophil-derived anticoagulatory proteins in C1q-NET-driven blood coagulation.

Neutrophils interact with the external milieu in both tissue and blood microenvironments and are emerging as important regulators of blood coagulation. In this study, we explored whether complement induces neutrophil extracellular trap (NET) formation and related blood coagulation using human donor-derived neutrophils. Complement C1q induces NETosis in lipopolysaccharide (LPS) O127-primed neutrophils, whereas LPS alone does not induce NETosis. Bulk RNA sequencing revealed a unique LPS-driven altered neutrophil state, and complement sensitivity for NETosis was found to be transcriptionally dependent. Using an arrayed CRISPR knockout screen in the neutrophil-like differentiated HL60 cells, we identified that SCARF1 and complement receptor 3 are required for C1q-NETosis. Given NETs contain procoagulatory components such as DNA and histones, we investigated whether C1q-related NETs influenced blood coagulation. LPS+C1q-NETs were associated with reduced coagulation activity compared with LPS treatment alone. We further found that LPS upregulated tissue factor expression and coagulation-related activity in neutrophils. Furthermore, neutrophils secrete anticoagulant proteins, including protein C and tissue factor pathway inhibitors, during C1q-mediated NET formation that functionally regulate NET-related coagulation. C1q-NETs also activate the coagulation factors XII and XI, facilitating both intrinsic coagulation and kallikrein-dependent bradykinin production. This study elucidates how NETs regulate both procoagulatory and anticoagulatory components that may influence the pathophysiology of disease.