Deborah L. Higgins

One Chain Variants of Tissue Plasminogen Activator have Increased Susceptibility to Inactivation by Plasmin

Abstract Tissue plasminogen activator (t-PA) is synthesised as a one chain molecule which can be converted to a two chain form by hydrolysis of the peptide bond after the arginine at position 275 by plasmin. Variants with non-basic residues at position 275 are resistant to this hydrolysis. However, incubation of these variants with plasmin can result in the formation of a ‘two chain’ molecule containing a new amino terminus at position G278. In R275E t-PA, the peptide bond to the carboxyl side of the lysine at position 277 was found to be over 100-fold less sensitive to plasmin than is the peptide bond following the arginine at position 275 in wild-type t-PA. Concomitant with the appearance of the ‘two chain’ variant was a decrease in the ability of the variant to activate plasminogen in the presence of a fibrin(ogen) cofactor. The ability of the ‘two chain’ variant to activate plasminogen in the absence of a cofactor was not decreased as significantly. An R275E/K277I variant of t-PA remained in the single chain form upon plasmin treatment and retained the ability to activate plasminogen in the presence of fibrin to a significantly greater extent than the other position 275 variants.

A Zymogenic Tissue Plasminogen Activator Variant: The Phe305→His Mutation Suppresses Fibrin(ogen) Stimulated Plasminogen Activation by One Chain t-PA

Abstract Tissue plasminogen activator (t-PA) is a unique serine protease because it does not require proteolytic activation to exhibit full enzymatic activity. In the presence of physiological stimulators related to fibrinogen and fibrin, the one chain form of the enzyme has equivalent activity to the two chain form against the physiological substrate, plasminogen. This report describes a Phe 305→His (F305H) variant of t-PA which displays kinetics more typical of a serine protease zymogen. Plasminogen activation by F305H t-PA in the presence of a fibrin-like stimulator was shown to exhibit non-linear kinetics, presumably due to the ability of plasmin in the reaction mixture to convert the one chain F305H t-PA to the more active two chain form. Comparison of the apparent kinetic constants of two chain F305H t-PA and a single chain variant (R275E/F305H), indicated that the two chain form of the enzyme had approximately 3-fold higher activity than the one chain form. The increase in activity was due primarily to an increase in the k cat of the reaction. Formation of a hydrogen bond between His 305 and Asp 477 in F305H t-PA, analogous to the His 40-Asp 194 hydrogen bond in chymotrypsinogen, is postulated to account for the partially zymogenic characteristics of this variant.