Abstract 133: Engineering Plasminogen Activator Inhibitor-1 Variants with Novel Protease Inhibitor Activity

Plasminogen activator inhibitor-1 (PAI-1) is a member of the serine protease inhibitor, or SERPIN, protein superfamily that canonically inhibits the plasminogen activators, tissue plasminogen activator (tPA) and urokinase (uPA). PAI-1, however, also exhibits less potent inhibitory activity against other proteases, including thrombin, coagulation factor XIIa (FXIIa), and transmembrane serine protease-2 (TMPRSS2). To identify amino acid substitutions in PAI-1 that lead to enhanced inhibitory activity toward non-canonical protease targets, we constructed a phage-display PAI-1 mutational library using error-prone PCR that contained 89% of all possible single amino acid substitutions. The library was screened for PAI-1 variants that efficiently inhibit FXIIa, thrombin, or TMPRSS2 by incubating the phage-displayed PAI-1 with the target protease, immunoprecipitating the covalently linked protease-PAI-1 complexes, and isolating the phage DNA that encodes for the mutant PAI-1 fusion protein. The input and selected libraries were subjected to high-throughput DNA sequencing at a depth of >400 million PAI-1 sequencing reads. Missense mutations that both enhance and diminish PAI-1’s inhibitory activity toward a given non-canonical serine protease are identified throughout the PAI-1 structure—highlighting the importance of exosite-driven interactions and allosteric networks in the determination of SERPIN specificity. Detailed examination of the unique set of amino acid substitutions associated with improved PAI-1 reactivity toward a specific protease compared to uPA demonstrated a clear correlation with the evolutionary relationship between the given target protease and uPA. As FXIIa is the most closely related of the coagulation/fibrinolytic serine proteases to uPA, it was selected as a target to further engineer a potent and specific inhibitor on the PAI-1 scaffold. For this purpose, we are utilizing unbiased directed evolution coupled with functional characterization of the PAI-1 variants identified with either the most robust FXIIa inhibitory response or those that exhibit high FXIIa selectivity. The development of a novel FXIIa inhibitor may be of potential value as a novel therapeutic for thrombotic disorders.