Engineering thrombin for selective specificity toward protein C and PAR1

Abstract

Thrombin elicits functional responses critical to blood homeostasis by interacting with diverse physiological substrates. Ala-scanning mutagenesis of 97 residues covering 53% of the solvent accessible surface area of the enzyme identifies Trp215 as the single most important determinant of thrombin specificity. Saturation mutagenesis of Trp215 produces constructs featuring kcat/Km values for the hydrolysis of fibrinogen, protease-activated receptor PAR1, and proteinC that span five orders of magnitude. Importantly, the effect of Trp215 replacement is context dependent. Mutant W215E is 10-fold more specific for protein C than fibrinogen and PAR1, which represents a striking shift in specificity relative to wild-type that is 100-fold more specific for fibrinogen and PAR1 than protein C. However, when the W215E mutation is combined with deletion of nine residues in the autolysis loop, which by itself shifts the specificity of the enzyme from fibrinogen and PAR1 to protein C, the resulting construct features significant activity only toward PAR1. These findings demonstrate that thrombin can be re-engineered for selective specificity toward protein C and PAR1. Mutations of Trp215 provide important reagents for dissecting the multiple functional roles of thrombin in the blood and for clinical applications. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.