Low-Molecular-Weight Heparin and Dermatan Sulfate End Group-Labeled with Tyramine and Fluorescein. Biochemical and Biological Characterization of the Fluorescent-Labeled Heparin Derivative

ABSTRACT

To improve the understanding of the biological functions and pharmacology of heparin and dermatan sulfate, low-molecular-weight heparin (LMWH) and low-molecular-weight dermatan sulfate (LMWDS) were labeled with tyramine (T) by covalently linking T to the terminal residue of 2,5-anhydromannose (or 2,5-anhydrotalose for dermatan sulfate). The covalent labeling was demonstrated by nuclear magnetic resonance spectroscopy. The tyramine-labeled LMWH (LMWH-T) was also labeled with fluorescein (F) by further reacting it with fluorescein isothiocyanate. The fluoresceinated LMWH-T (LMWH-T,F ) was used to analyze biological functions on blood coagulation and binding to leukocytes. The biological activities on factor Xa and thrombin inhibition remained unchanged compared with the parent compound. Flow cytometric analysis of leukocytes demonstrated binding of the modified heparin to granulocytes, monocytes, and lymphocytes, the half-live being twice as long as the antifactor Xa activity. F-labeled heparin was displaced by unlabeled heparin from all three populations of leukocytes. Binding of heparin to leukocytes may play an important role in inflammation and atherosclerosis.

KEYWORDS

Glycosaminoglycans - heparin - dermatan sulfate - tyramine- and fluorescent-labeled low-molecular-weight heparin - leukocytes

REFERENCES

• 1 Lam L H, Silbert J E, Rosenberg R D. The separation of active and inactive forms of heparin.  Biochem Biophys Res Commun . 1976;  69 570-577
• 2 Casu B, Oreste P, Torri G. The structure of heparin oligosaccharide fragments with high antifactor Xa activity containing the minimal antithrombin III-binding sequence.  Biochem J . 1981;  197 599-609
• 3 Harenberg J, de Vries X J. Characterization of heparins by high performance size exclusion liquid chromatography.  J Chromatogr . 1983;  261 287-292
• 4 Rosenberg R D, Damus P S. The purification and mechanism of action of human antithrombin-heparin cofactor.  J Biol Chem . 1973;  248 6490-6505
• 5 Linhardt R J, Loganathan D. In: Gebelein G ed. Biomimetic Biopolymers New York: Plenum 1990: 135-175
• 6 Lane D A, Lindahl U. Heparin: Chemical and Biological Properties, Clinical Applications London: Arnold; 1989
• 7 Kakkar V V, Cohen A T, Edmonson R A. Low molecular weight versus standard heparin for prevention of venous thromboembolism after major abdominal surgery.  Lancet . 1993;  341 259-265
• 8 Nurmohamed M T, Rosendaal F R, Büller H R. Low-molecular-weight heparin versus standard heparin in general and orthopaedic surgery: a meta-analysis.  Lancet . 1992;  340 152-156
• 9 Harenberg J, Heene D L. Pharmacology and special clinical applications of low molecular weight heparins.  Am J Hematol . 1988;  29 233-240
• 10 Harenberg J, Roebruck P, Heene D L, on behalf of the Heparin Study in Internal Medicine Group. Subcutaneous low-molecular-weight heparin versus standard heparin and the prevention of thromboembolism in medical inpatients.  Haemostasis . 1996;  26 127-139
• 11 Merli G J. Low-molecular-weight heparins versus unfractionated heparin in the treatment of deep vein thrombosis and pulmonary embolism.  Am J Phys Med Rehabil . 2000;  79 9-16
• 12 Harenberg J, Schmidt J A, Koppenhagen K, Tolle A, Huisman M V, Büller H R, and the EASTERN-Investigators. Fixed-dose, body weight-independent subcutaneous LMW heparin versus adjusted dose unfractionated intravenous heparin in the initial treatment of proximal venous thrombosis.  Thromb Haemost . 2000;  83 652-656
• 13 Harenberg J, Malsch R. German Patent P4217916.5-43, 1992 . 
• 14 Harenberg J, Löhr G, Malsch R. Magnetic bead protamine-linked microtiter assay for detection of heparin using iodinated low-molecular-mass heparin-tyramine.  Thromb Res . 1995;  79 207-216
• 15 Stehle G, Friedrich E A, Sinn H. Hepatic uptake of a modified low molecular weight heparin in rats.  J Clin Invest . 1992;  90 2110-2116
• 16 Malsch R, Guerrini M, Berti C. Synthesis and biological effects of N-alkylamine-labeled low-molecular-mass dermatan sulfate.  Semin Thromb Hemost . 1997;  23 99-107
• 17 Harenberg J, Malsch R, Piazolo L, Huhle G, Heene D L. Preferential binding of heparin to granulocytes of various species.  Am J Vet Res . 1996;  57 1016-1020
• 18 Malsch R, Guerrini M, Torri G. Synthesis of N′-alkylamine anticoagulant active low molecular mass (LMM) heparin for radioactive and fluorescent labeling.  Anal Biochem . 1994;  217 255-264
• 19 Harenberg J. Modified anti-factor Xa chromogenic substrate assay for heparin and low molecular weight heparins.  Ärztl Lab . 1987;  33 39-41
• 20 Harenberg J, Giese C, Knödler A, Zimmermann R. Comparative study of a new one-stage clotting assay for heparin and its low molecular weight derivatives.  Haemostasis . 1989;  19 13-20
• 21 Alban S, Harenberg J. Neutralization of heparins and heparin-related oligosaccharides by protamine and polybrene.  Pharm Pharmacol Lett . 1991;  1 37-40
• 22 Colas-Linhart N, Berthelot J L, Ducret A, Petiet A, Bok B. Technetium 99m labeled heparin: pharmacokinetics and tissue distribution in rats after vascular surgery.  Biomed Pharmacother . 1987;  41 189-191
• 23 Malsch R, Harenberg J. Analysis of acid mucopolysaccharides by HPLC techniques.  Krankenhauspharmazie . 1992;  13 67-75
• 24 Smith C W, Marlin S W, Rothlein R, Toman C, Anderson D C. Cooperative interaction of LFA-1 and Mac-1 with intercellular adhesion molecule-1 in facilitating adherence and transendothelial migration of human neutrophils in vitro.  J Clin Invest . 1989;  83 2008-2017
• 25 Malsch R, Harenberg J. Considerations of the correct nomenclature of glycosaminoglycans.  Thromb Haemost . 1993;  70 718-719
• 26 Linhardt R J, Hakim A A, Liu J. Acidic polysaccharides: their modifications and potential uses. In: Gebelein CG, ed. Biotechnology and Polymers New York: Plenum Press 1991: 155-165
• 27 Hoffmann A S, Larm O, Scholander E. A new method for covalent coupling of heparin and other glycosaminoglycans to substances containing primary amino groups.  Carbohydr Res . 1983;  117 328-331
• 28 Sie P, Ofosu F, Fernandez F. Respective role of antithrombin III and heparin cofactor II in the in vitro anticoagulant effect of heparin and of various sulphated polysaccharides.  Br J Haematol . 1986;  64 707-714
• 29 Piazolo L, Harenberg J, Malsch R, Casu B, Heene D L. Heparin-protein conjugates specifically increase thrombin inhibition in animals.  Ann Hematol . 1993;  66(Suppl 1) Abstr26
• 30 Young E, Cosmi J, Weitz B, Hirsh J. Comparison of the non-specific binding of unfractionated heparin and low molecular weight heparin (enoxaparin) to plasma proteins.  Thromb Haemost . 1993;  70 625-630
• 31 Young E, Cosmi J, Weitz B, Hirsh J. Comparison of the non-specific binding of unfractionated heparin and low molecular weight heparin (enoxaparin) to plasma proteins.  Thromb Haemost . 1993;  70 625-630
• 32 Sugiyama T, Itoh M, Ohtawa M, Natsuga T. Study on neutralization of low molecular weight heparin (LHG) by protamine sulfate and its neutralization characteristics.  Thromb Res . 1992;  68 119-129
• 33 Dawes J. Comparison of the pharmacokinetics of enoxaparin (Clexane) and unfractionated heparin.  Acta Chir Scand . 1990;  556(Suppl) 68-74
• 34 Stricker H. In: Martin A Swarbrick J Cammarata A Physikalische Pharmazie Wissenschafliche Stuttgart: Verlagsgesellschaft 1987: 350-352
• 35 Vanucchi S, Pasquali F, Chiarugi V, Ruggiero M. Internalization and metabolism of endogenous heparin by cultured endothelial cells.  Biochem Biophys Res Commun . 1986;  140 294-301
• 36 Gervasi G B, Farina C, Bartoli C. Disposition of a new heparan sulfate with fibrinolytic activity in rat.  Drug Res . 1993;  43 445-449
• 37 Lane D A, Adams L. Non-anticoagulant uses of heparin.  N Engl J Med . 1993;  329 130-131
• 38 Ahmed T, Garrigo J, Danta I. Preventing bronchoconstriction in exercise-induced asthma with inhaled heparin.  N Engl J Med . 1993;  329 90-95
• 39 Folkman J, Langer R, Linhardt R J, Haudenschild C, Taylor S. Angiogenesis inhibition and tumor regression caused by heparin or a heparin fragment in the presence of cortisone.  Science . 1983;  221 719-725