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.
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References
Cooney DA, Rosenbluth RJ. Enzymes as therapeutic agents. Adv Pharmacol Chemother, 1975; 12: 185–289.
Veronese FM, Morpurgo M. Bioconjugation in pharmaceutical chemistry. Farmaco, 1999; 54: 497–516.
Mumtaz S, Bachhawat BK. Conjugation of proteins and enzymes with hydrophilic polymers and their applications. Indian J Biochem Biophys, 1991; 28: 346–51.
Chang TM. Artificial cells, encapsulation, and immobilization. Ann NY Acad Sci, 1999; 875: 71–83.
Stricker RB, Wong D, Shiu DT, Reyes PT, Shuman MA. Activation of plasminogen by tissue plasminogen activator on normal and thrombasthenic platelets: effects on surface proteins and platelet aggregation. Blood, 1986; 68; 275–280.
Marder VJ, Shulman NR. High molecular weight derivatives of human fibrinogen produced by plasmin II. Mechanism of their anticoagulant activity. J Biol Chem, 1969; 244: 2120–2124.
Collen D, Stump DC, Gold HK. Thrombolytic therapy Annu Rev Med, 1988: 39: 405–423.
Collen D, Lijnen HR. On the future of thrombolytic therapy for acute myocardial infarction. Am J Cardiol. 1993; 72: 46G-50G.
Collen D. Towards improved thrombolytic therapy. Lancet. 1993; 342: 34–36.
Lijmen HR, Collen D. Strategies for the improvement of thrombolytic agents. Thromb Haemost, 1991; 66: 88–110.
Majerus PW, Bronze Jr GJ, Miletich JP, Tollefsen DM Anticoagulant, thrombolytics, and antiplatelet drugs In: Gilman’s The Pharmacological Basis of Therapentics 9th ed. New York: McGraw-Hill, 1995; 1341–1359.
Anderson HV, Willerson JT. Thrombolysis in acute myocardial infarction [see comments]. N Engl J Med. 1993; 329: 703–709.
Huber K, Runge MS, Bode C, Gulba D. Thrombolytic therapy in acute myocardial infarction update 1996. Ann Hematol. 1996; 73: S29–38.
Byun Y, Yang VC. Delivery system for targeted thrombolysis without the risk of hemorrhage. ASAIO J. 1998; 44: M638-M641.
Turpie AGG. Thrombolytic therapy in venous thromboembolism. In: Sobel BE, Collen D, Grossbard EB. eds. Tissue Plasminogen Activator in Thrombolytic Therapy. New York: Marcel Dekker. 1987; 131–146.
Bernstein H, Yang VC, Langer R. An investigation of heparinase immobilization. Appl Biochem Biotechnol. 1987; 16: 129–143.
Lanevschi A, Kramer JW, Greene SA, Meyers KM. Evaluation of chromogenic substrate assays for fibrinolytic analytes in dogs. Am J Vet Res. 1996; 57: 1124–1130.
Yun JH, Ma SC, Fu B, Yang VC, Meyerhoff ME. Direct potentiometric membrane-electrode measurements of heparin-binding to macromolecule. Electroanalysis. 1993; 5: 719–724.
Byun Y, Singh VK, Yang VC. Low molecular weight protamine: a potential non-toxic heparin antagonist. Thromb Res. 1999; 94: 53–61.
Ramamurthy N, Baliga N, Wakefield TW, Andrews PC. Yang VC, Meyerhoff ME. Determination of low molecular weight heparins and their binding to protamine and a protamine analogue using polyion-sensitive membrane electrodes. Anal Biochem, 1999; 266: 116–124.
Collen D, Verstraete M. Systemic thrombolytic therapy of acute myocardial infarction? Circul. 1983; 68: 462–465.
Fox KA, Bergmann SR, Sobel BE. Coronary thrombolysis: pharmacological considerations with emphasis on tissue-type plasminogen activator (t-PA). Biochem Pharmacol. 1984; 33. 1831–1838.
Owen J, Friedman KD, Grossman BA, Wilkins C, Berke AD, Powers ER. Quantitation of fragment X formation during thrombolytic therapy with streptokinase or tissue plasminogen activator. J Clin Invest. 1987; 79: 1642–1647.
Rao AK, Pratt C, Berke A, Jaffe A, Ockene I, Schreiber TL, et al. Thrombolysis in myocardial infarction (TIMI) trial phase 1: hemorrhagic manifestation and changes in plasma fibrinogen and the fibrinolytic system in patients treated with recombinant tissue plasminogen activator and streptokinase. J Am Coll Cardiol. 1988; 11: 1–11.
Verstraete M, Miller GA, Bounameaux H, Charbonnier B, Colle JP, Lecorf G, et al. Intravenous and intrapulmonary recombinant tissue-type plasminogen activator in the treatment of acute massive pulmonary embolism. Circulation, 1988; 77: 353–360.
Levine MN, Hirsh J, Weitz J, Cruickshank M, Neemeh J, Turpie AGG, et al. A randomized trial of a single bolus dosage regimen of recombinant tissue plasminogen activator in patients with acute pulmonary embolism. Chest, 1990; 98: 1473–1479.
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Published March 21, 2000
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Liang, J.F., Li, Y.T., Connell, M.E. et al. Synthesis and characterization of positively charged tPA as a prodrug using a heparin/protamine-based drug-delivery system. AAPS PharmSci 2, 7 (2000). https://doi.org/10.1208/ps020107
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DOI: https://doi.org/10.1208/ps020107