Synthesis and characterization of positively charged tPA as a prodrug using a heparin/protamine-based drug-delivery system
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 ($ K_{m} $=2.7 $ mM^{−1} $) and tPA ($ K_{m} $=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 $ α_{2} $-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..
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2000 |
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| Erschienen: |
2000 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:2 |
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| Enthalten in: |
AAPS PharmSci - 2(2000), 1 vom: März, Seite 59-67 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Liang, Jun F. [VerfasserIn] |
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| Links: |
Volltext [lizenzpflichtig] |
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| Themen: |
Plasmin |
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| Anmerkungen: |
© American Association of Pharmaceutical Scientists 2002 |
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| doi: |
10.1208/ps020107 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| PPN (Katalog-ID): |
SPR02463543X |
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| 245 | 1 | 0 | |a Synthesis and characterization of positively charged tPA as a prodrug using a heparin/protamine-based drug-delivery system |
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| 520 | |a 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 ($ K_{m} $=2.7 $ mM^{−1} $) and tPA ($ K_{m} $=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 $ α_{2} $-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. | ||
| 650 | 4 | |a Plasminogen |7 (dpeaa)DE-He213 | |
| 650 | 4 | |a Streptokinase |7 (dpeaa)DE-He213 | |
| 650 | 4 | |a Thrombolytic Therapy |7 (dpeaa)DE-He213 | |
| 650 | 4 | |a Protamine |7 (dpeaa)DE-He213 | |
| 650 | 4 | |a Plasmin |7 (dpeaa)DE-He213 | |
| 700 | 1 | |a Li, Yong T. |4 aut | |
| 700 | 1 | |a Connell, Maureen E. |4 aut | |
| 700 | 1 | |a Yang, Victor C. |4 aut | |
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