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Plasminogen small RNA cargo promotes immunogenicity and inhibits enzymatic activity

ABSTRACT: Plasminogen (Plg), the pro-enzyme of the serine-protease plasmin, is a lipoprotein-like particle in biofluids. Plg transports (oxidized) phospholipids and has the ability to accept free cholesterol from macrophages, two processes normally observed with lipoproteins. We have previously reported the biological functions of cell-free small RNA (cf-sRNA) circulating on lipoproteins. We hypothesized that Plg also transports functional cf-sRNAs. Indeed, both human and bovine Plg samples were observed to transport sRNAs (25-60nt in length) in plasma. Small RNA sequencing analyses found that Plg carries both host and non-host (microbial) sRNA. Plg samples were observed to accept sRNAs from donor macrophages in efflux studies and were observed to have moderate affinity (Kd=µM range) towards candidate host and microbial sRNAs, as quantified by microscale thermophoresis. Using PCR and bulk RNA sequencing, we discovered that Plg-sRNAs confer, in large part, Plg’s pro-inflammatory cytokine responses in macrophages. RNase treatments of both human and bovine Plg were found to significantly increase Plg’s enzymatic activity, as observed through both activity assays and clot lysis assays. Collectively, results from this study suggest that Plg is a novel circulating lipoprotein-like ribonucleoprotein and support that sRNA cargo negatively regulate Plg’s enzymatic function and enhance Plg’s immunogenicity. Therefore, Plg-sRNA cargo and its receptors present new therapeutic targets to control fibrinolysis, as well as wound and systemic inflammation.

ORGANISM(S): Mus musculus

PROVIDER: GSE288432 | GEO | 2026/01/30

REPOSITORIES: GEO

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Project description: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.

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Plasminogen small RNA cargo promotes immunogenicity and inhibits enzymatic activity

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