As a consequence of aging, protein quality control systems become less efficient at maintaining protein homeostasis (proteostasis) which can result in a variety of serious protein misfolding diseases. In these cases, extracellular proteins misfold and aggregate to generate toxic protein oligomers and/or insoluble deposits which can disrupt tissue and organ function^1,2.

The plasminogen activation system may play an important role in extracellular proteostasis. Tissue plasminogen activator (tPA) can become activated by binding to aggregated protein fibrils³. Extracellular chaperones (eg. clusterin, alpha-2 macroglobulin) keep misfolded proteins soluble by forming complexes with them⁴.

Amorphous aggregates similar to fibrin clots were produced by heat-stressing ovotransferrin (ovo). Like tPA bound to fibrin, tPA bound to ovo aggregates significantly enhanced plasminogen activation. The level of activation was dependent upon the quantity of aggregate. Addition of plasminogen activator inhibitor type 2 abrogated plasmin generation as did tranexamic acid, confirming the specificity of tPA in the activation of plasminogen and indicating that the activation is mediated by tPA binding to lysine residues on protein aggregates.

In vitro, plasmin progressively digested ovo aggregates to release plasmin generated protein fragments (PGPFs); these were of a broad range of sizes and were shown to trigger reactive oxygen species (ROS) formation and cell death in EOC-13.31 and SVEC4-10 cell lines. In contrast, PGPFs generated by plasmin digestion of native ovo, and undigested ovo, did not produce these effects. Clusterin and alpha-2 macroglobulin were both shown to bind to PGPFs derived from ovo aggregates and to inhibit PGPF-induced ROS formation and cytotoxicity.

The results demonstrate that proteases and chaperones in extracellular fluids may act together to maintain extracellular proteostasis and to inhibit the development of age-related protein misfolding diseases.