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 1-3. 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, while 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 (dHL60), we identified that SCARF1 and Complement Receptor 3 are required for C1q-NETosis. Since NETs contain pro-coagulatory 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 to 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 regulates NET-related coagulation. C1q- NETs also activate the coagulation factors FXII and FXI, facilitating both intrinsic coagulation and kallikrein-dependent bradykinin production. This study elucidates how NETs regulate both pro-coagulatory and anti-coagulatory components that may influence pathophysiology of disease.