Plasminogen lipid cargo drives macrophage TLR2 activation independent of protease activity

Plasminogen (Plg) is a highly abundant, liver-secreted plasma protein and zymogen precursor of plasmin, a key serine-protease in fibrinolysis. Zymogen Plg (Glu-Plg) has been reported to transport extracellular RNA, cholesterol, and oxidized phospholipids (oxPL) cargo which likely defines the biological functions of non-enzymatic Plg particles, including immunogenicity towards innate immune cells. Here, we demonstrate that native Plg dose-dependently activates NF-kB transactivity, pro-inflammatory gene expression and cytokine secretion in primary macrophages through toll-like receptors, i.e., toll-like receptor 2 (TLR2), as identified through a TLR screen. Plg stimulation of macrophages and NF-kB transactivity reporter cells were sensitive to small molecule inhibitors of TLR2, and Tlr2 and Myd88 genetic-deficiency. Plg treatments were found to stimulate both mouse and human primary macrophages but failed to activate microglial cells and vascular smooth muscle cells. Both C29, and its derivative ortho-vanillin, were found to inhibit macrophage TLR2 signaling and cytokine secretion in vitro, as profiled by proximity extension assays (PEA). Plg activation of TLR2 was observed to be independent of Plg’s conversion to plasmin or down-stream plasmin protease activity, as Plg stimulation of TLR2 were not affected by active-site mutagenesis, lysine analogues ε-aminocaproic acid and tranexamic acid, VPLCK peptide inhibitor, α2-anti-plasmin, α2-macroglobin, or urokinase plasminogen activator (uPA, Plau-/-) and tissue plasminogen activator (tPA, Plat-/-) macrophage deficiency. Mass spectrometry-based untargeted lipidomics demonstrated the full profile of bioactive lipids covalently bound or loosely associated with human Plg particles. Plg’s immunogenicity towards macrophages was found to be dependent, in part, through lipid cargo, as lipid removal with lipases and organic lipid extractions were found to reduce Plg’s capacity to activate macrophages. Plg-deficiency in hypercholesterolemic mice were observed to be associated with reduced atherosclerosis and intravenous injections of ortho-vanillin were found to significantly reduce lesion area and alter macrophage sub-phenotypes in mouse lesions, as per single cell RNA sequencing. Results from these studies support that Glu-Plg’s lipid cargo and TLR2 may be viable drug targets to reduce chronic inflammation without compromising fibrinolysis.