Aptamer-mediated modulation of PAI-1 function reveals a novel mechanism for restoring fibrinolytic balance

Plasminogen activator inhibitor-1 (PAI-1) is the principal regulator of fibrinolysis, acting by inhibiting tissue-type and urokinase-type plasminogen activators (tPA/uPA). Dysregulated PAI-1 activity contributes to diverse pathologies, including cancer, cardiovascular disease, and fibrosis, underscoring its therapeutic potential. We previously identified R10-4, an RNA aptamer that attenuates PAI-1's antiproteolytic activity against tPA by destabilizing covalent complex formation. To elucidate its inhibitory mechanism, we combined plasminogen activation assays with in silico modeling to map residue-level interactions. Systematic truncation of R10-4 revealed two regions critical for inhibition: (i) the reactive center loop, encompassing the s5A strand, and (ii) the vitronectin-binding domain. Removal of 17 nucleotides from the 3' end disrupted aptamer engagement, significantly altering R10-4's inhibitory activity. These findings define the structural basis for R10-4 modulation of PAI-1 function, revealing a novel aptamer-mediated mechanism of inhibition. By targeting key functional domains, R10-4 emerges as a promising prototype therapeutic for correcting fibrinolytic dysregulation in thrombotic and fibrotic disease.