Directed evolution and in vivo function of plasminogen activator inhibitor-1.

Plasminogen Activator Inhibitor-1 (PAI-1) is a member of the Serine Protease Inhibitor (SERPIN) family and an important regulator of fibrinolysis. Although similar in structure and mechanism, SERPINs each have unique specificity for target proteases. PAI-1 is the physiologic inhibitor of tissue-type Plasminogen Activator (tPA) and urokinase Plasminogen Activator (uPA), which activate plasminogen to its active form, plasmin. However, PAI-1 spontaneously transitions to a stable, inactive conformation with a half-life of 2 hours under physiologic conditions. In addition, PAI-1 is sensitive to oxidative inactivation. However, the physiologic significance of the transition to latency and oxidative inactivation are unknown. Several PAI-1 variants with increased functional half-lives have been identified through random mutagenesis. This study investigated the in vivo effects of PAI-1 loss of latency using a mouse model. Furthermore, a random mutagenesis approach was used in order to find additional PAI-1 variants with altered functional properties. A transgene was engineered to direct widespread expression of a stable variant of murine PAI-1 from a hybrid CMV/chicken beta-actin promoter, and 10 independent transgenic lines were generated. Plasma PAI-1 levels in the founders ranged from 3.1+/-0.1 to 1268.8+/-717.0 ng/mL, and PAI-1 mRNA levels across tissues were elevated from 2-fold to 2245-fold over wild-type. No evidence of thrombosis was observed. The 2 founders with the highest plasma PAI-1 levels failed to produce viable transgenic offspring, suggesting that high expression of a stable PAI-1 variant may be lethal in mice. These mice may serve as a useful model for future investigations of PAI-1 function in vivo. In addition, a phage library of randomly mutagenized human PAI-1 molecules was screened for altered activity, such as resistance to oxidative inactivation and inhibition of neutrophil elastase. However, isolated clones failed to demonstrate the selected function in vitro, and false positives are a potential concern. Nevertheless, this approach highlighted the potential utility of a high-throughput screen of an exhaustive and unbiased pool of mutants. PAI-1 variants found through screening the phage library may reveal structural elements responsible for unique PAI-1 properties and may be useful for engineering mouse models that yield new insight into PAI-1 biology.Ph.D.Biological SciencesGeneticsMolecular biologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/126683/2/3276152.pd