Synthesis and characterization of positively charged tPA as a prodrug using a heparin/protamine-based drug-delivery system
Journal Title: The AAPS Journal - Year 2000, Vol 2, Issue 1
Abstract
Positively charged peptides [(Arg)-Cys] were sucessfully linked to tissue-specific plasminogen activator (tPA) using cross-linking agent N-succinimidyl 3-(2-pyridyldithio) propionate. Specific amidolytic activity of this tPA/(Arg)-Cys (termed modified tPA, mtPA) was 3900 IU/μg as compared to 5800 IU/μg of the parent tPA. Both activation of plasminogen with mtPA (Km=2.7 mM−1) and tPA (Km=1.1 mM−1) in a purified system followed Michaelis-Menten kinetics. In addition, (Arg)-Cys modification did not result in significant changes in the fibrin-binding ability of tPA, and mtPA still retained a response to fibrinogen similar to that of the parent tPA. Compared with tPA, mtPA showed much stronger heparin affinity, and the heparin/mtPA complex was stable in human plasma. The activity of mtPA in such a complex was inhibited by heparin, and, unlike tPA, the heparin/mtPA complex did not cause statistically meaningful depletion of plasminogen, fibrinogen, and α2-antiplasmin in plasma. Using the chromogenic and the in vitro clot lysis assay, it was demonstrated that the heparin-induced inhibition of the mtPA activity was easily reversed following the addition of an adequate amount of protamine. To enhance the clot-targeting efficiency of the heparin/mtPA complex further, anti-fibrin immunoglobulin (IgG) was conjugated to heparin via an end-point attachment of heparin to the sugar moieties in the Fc region of the IgG. Results show that the activity of mtPA could also be blocked by the heparin/anti-fibrin IgG conjugate. These findings suggest the applicability of the heparin/protamine delivery system to abort the potential bleeding risks associated with clinical use of tPA.
Authors and Affiliations
Jun F. Liang, Yong T. Li, Maureen E. Connell, Victor C. Yang
Keywords
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