Prorenin in Hepatic Stellate Cell Extracellular Vesicles Induces Platelet‐Dependent Thrombin Formation and Release of Profibrotic <scp>TGF</scp> ‐β

Dysregulated coagulation and platelet activation contribute to liver dysfunction and fibrosis, but mechanisms initiating these events are undefined. The hepatic stellate cell (HSC) agonist Concanavalin A (ConA) rapidly induces hepatitis, which progresses to hepatic fibrosis after serial exposure for 8 weeks. Extravascular platelets were intercalated throughout normal liver parenchyma, with ConA treatment activating resident platelets to degranulate and display P-selectin. HSCs matured from activated human induced pluripotent stem cells or the human LX-2 cell line released extracellular vesicles (EVs) that stimulated platelet aggregation, yet these particles lacked known platelet agonists, and this response differed from established behaviors. Enzymatic assays, biologic, chemical and aptamer inhibitors, immunohistochemistry, qPCR, REN siRNA, and western blotting elucidate a novel HSC-EV mediated pathway to platelet activation. The aspartyl protease inhibitor pepstatin, or specific inhibitors Aliskiren and VTP23999 of the aspartyl protease renin suppressed HSC EV-induced platelet activation, as did siRNA knockdown of prorenin. HSC maturation from mesenchymal cells increased prorenin transcripts, and HSC-EV contained surface-associated prorenin. Platelets expressed the prorenin receptor that overcomes prorenin auto-inhibition, and HSC EV interacting with platelets generated renin peptidolytic activity. This interaction stimulated extrinsic and common coagulation cascades, formation of thrombin over time, activated platelet PAR-1 thrombin receptors, and induced robust TGF-β release. This TGF-β stimulated the LX-2 cell TGFbR1 receptor, Smad phosphorylation, and profibrotic protein expression. We identify hitherto undiscovered pathways by which platelet and HSC interaction stimulates thrombosis through prorenin activation and show intrahepatic platelets are positioned to stimulate fibrotic protein deposition in a model of hepatic fibrosis.