Plasminogen small RNA cargo promotes immunogenicity and inhibits enzymatic activity

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.