title: Synthesis and characterization of positively charged tPA as a prodrug using heparin/protamine-based drug delivery system.
abstract: Positively charged peptides [(Arg)7 Cys] were successfully linked to tissue-specific plasminogen activator (tPA) using cross-linking agent N-succinimidyl 3-(2-pyridyldithio) propionate. Specific amidolytic activity of this tPA/(Arg)7 Cys (termed modified tPA, mtPA) was 3900 IU/microg as compared to 5800 IU/microg 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)7 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 alpha2-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.affiliation: College of Pharmacy, The University of Michigan, Ann Arbor, MI 48109-1065, USA.
authors.forename: J F
authors.initials: JF
authors.lastname: Liang
authors.forename: Y T
authors.initials: YT
authors.lastname: Li
authors.forename: M E
authors.initials: ME
authors.lastname: Connell
authors.forename: V C
authors.initials: VC
authors.lastname: Yang
mesh_terms: D000906:Antibodies / Q000737:chemistry; D001120:Arginine / Q000737:chemistry; D003432:Cross-Linking Reagents / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D004355:Drug Stability; D005340:Fibrinogen / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D005343:Fibrinolytic Agents / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D006493:Heparin / Q000737:chemistry*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry*; D010958:Plasminogen / Q000378:metabolism; D010960:Plasminogen Activators / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D011355:Prodrugs / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D011479:Protamines / Q000737:chemistry*; D013388:Succinimides / Q000737:chemistry; D010959:Tissue Plasminogen Activator / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000494:pharmacology; D000979:alpha-2-Antiplasmin / Q000378:metabolism
chemical_list: D000906:Antibodies; D003432:Cross-Linking Reagents; D005343:Fibrinolytic Agents; D009842:Oligopeptides; D011355:Prodrugs; D011479:Protamines; D013388:Succinimides; D000979:alpha-2-Antiplasmin; C018151:N-succinimidyl 3-(2-pyridyldithio)propionate; D005340:Fibrinogen; D010958:Plasminogen; D006493:Heparin; D001120:Arginine; D010960:Plasminogen Activators; D010959:Tissue Plasminogen Activator; D003545:Cysteine