Plasminogen Activator Inhibitor-1 is Internalized by Endothelial Cells via A Pinocytosis-Like Mechanism and Evades Degradation.

Plasminogen activator inhibitor 1 (PAI1) is a secreted protease inhibitor, but accumulating evidence supports a role for PAI1 in regulating intracellular processes. PAI1 is secreted in an active conformation that serves as a bait for plasminogen activators (PA). Interaction between PAs and PAI1 results in a covalent linkage. PAI1/PA bind LRP1 and uPAR on the cell surface, triggering internalization and degradation. However, it was recently shown that exogenous PAI1 directly interacts with endothelial nitric oxide synthase inside endothelial cells. Our objective was to characterize the non-degradative pathway for PAI1 internalization. We use a hemagglutinin (HA)-tagged construct of PAI1 to track endocytosis of exogenous PAI1 in cultured endothelial cells via Western blotting of cell lysates and immunofluorescence microscopy. We demonstrate that PAI1-HA is actively internalized through a non-receptor-mediated mechanism, independent of clathrin-mediated endocytosis or caveolae. PAI1-HA internalization was partially inhibited by a derivative of amiloride, an inhibitor of macropinocytosis, though it was independent of Rac1 and CDC42, GTPases used for macropinocytosis. After internalization, PAI1-HA evades degradation for at least 3 h. By contrast, PAI1/uPA is degraded within 30 min of internalization. PAI1-HA internalization is independent of LRP1 and uPAR. We propose a model whereby PAI1/PA is endocytosed by LRP1- and uPAR-dependent endocytosis, whereas latent PAI can be internalized through a pinocytic-like mechanism and evade degradation, facilitating paracrine signaling.