Abstract Number: PB0748
Meeting: ISTH 2020 Congress
Background: The SERPIN plasminogen activator inhibitor-1 (PAI-1) regulates the fibrinolytic process as the primary inhibitor of tissue- and urokinase-type plasminogen activators (tPA and uPA). In contrast to other SERPINs, PAI-1 exhibits a relatively short functional half-life (~2h) before it loses inhibitory activity.
Aims: The goal of this study is to apply deep mutational scanning by coupling phage display with high-throughput DNA sequencing (HTS) to quantitatively map the mutational landscape of PAI-1 with respect to both its functional half-life, as well as the rate of target protease inhibition.
Methods: Fully functional PAI-1 was expressed as a fusion protein on the surface of M13 bacteriophage and mutagenized using error prone PCR. Phage-displayed PAI-1 was reacted with uPA, the phage-displayed PAI-1::uPA complex isolated by immunoprecipitation, and the corresponding phage genomes subjected to high-throughput DNA sequencing.
Results: A mutational library of ~107 independent phage-displayed PAI-1 clones was generated, with Sanger sequence analysis identifying ~3-4 mutations per clone, and HTS demonstrating coverage of ~74% of all possible single amino acid substitutions. uPA reactive phage were collected at multiple time points and either the functional half-life or second order rate constants for uPA inhibition calculated simultaneously from the fractional representation of each variant as determined by HTS. Comparison of these massively parallel determined half-lives and rate constants to those measured for a set of recombinantly-expressed PAI-1 variants each carrying a single amino acid substitution, demonstrated a high degree of correlation confirming the validity of this approach.
Conclusions: The quantitative fitness landscapes developed through this analysis serve as a useful tool for understanding the evolution and diversity of the SERPIN superfamily, as well as, for cataloging the potential clinically significance for the large set of human mutations that will be identified by the application of whole genome sequencing to large and rapidly expanding human cohorts.
To cite this abstract in AMA style:Haynes LM, Huttinger ZM, Ginsburg D. Deep Mutational Scanning and High-Throughput Kinetics to Quantitatively Assess Plasminogen Activator Inhibitor-1 Variants [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/deep-mutational-scanning-and-high-throughput-kinetics-to-quantitatively-assess-plasminogen-activator-inhibitor-1-variants/. Accessed January 28, 2022.
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