Hannah Digiovanni
Pronouns: she/her
Research Mentor(s): Laura Haynes
Research Mentor School/College/Department: Life Sciences Institute / Other
Program: UROPF
Session: Session 3 (11:00am – 11:50am)
Authors: Hannah DiGiovanni, Laura Haynes
Abstract
Plasminogen activator inhibitor-1 (PAI-1) is a member of the serine protease inhibitor (SERPIN) superfamily of proteins, which function as inhibitors of their target proteases. PAI-1 specifically inhibits urokinase-type plasminogen activator (uPA), a protein responsible for promoting the breakdown of blood clots. My laboratory has identified PAI-1 mutations that enhance its ability to inhibit clotting Factor XIIa, a procoagulant protein. The purpose of my research is to test potential mutations of PAI-1 to assess their ability to inhibit Factor XIIa in order to ultimately engineer a protein that can be used as a potential therapeutic to treat blood clotting disorders. First, a high throughput screen was done to identify mutations in PAI-1 that would lead to Factor XIIa inhibition, leading us to choose variants S331C, T251I, F98Y, and V247D as the mutations we would be further testing. Then, we transformed bacteria with plasmids containing cDNA of these PAI-1 variants, grew cultures to express the PAI-1 mutant proteins, and performed protein purifications of the cell lysates. Finally, we ran kinetic activity assays on each protein to obtain data on their abilities to inhibit both uPA and Factor XIIa. We have not yet obtained the final results of the assays of each of the Pai-1 variants, so we currently do not have any final results. However, based on the results of the high throughput screen, we expect that the assays will show that the S331C, T251I, and F98Y variants have higher inhibition of Factor XIIa than uPA and that the V247D variant will only inhibit Factor XIIa. Assuming we obtain our anticipated results, these results will show that these variants of the PAI-1 protein have improved inhibition of Factor XIIa compared to the wild type PAI-1. Thus, these variants could potentially be engineered and used as therapeutics to treat blood clotting disorders.
