Analysis of heparin binding to human leukocytes using a fluorescein-5-isothiocyanate labeled heparin fragment.
The binding of LMWH-tyr-FITC to granulocytes, monocytes, and lymphocytes was analyzed by flow cytometry using a low-molecular-weight heparin (LMWH) labeled with fluorescein-5-isothiocyanate (FITC). FITC was covalently bound to tyramine, which was synthesized to LMWH by endpoint-attachment (Malsch et al.: Anal Biochem 217:255-264, 1994). The binding was rapid, specific, dose-dependent, saturable, and reversible. To investigate the molecular weight dependence of heparins, heparin-derived di- to dodecasaccharides were used. With decreasing molecular weight, the amount of oligosaccharides increased; these were bound to granulocytes, monocytes, and lymphocytes (r = -0.77). The degree of sulfation of non-heparin glycosaminoglycans influenced the binding to leukocytes. Decreasing the degree of sulfation decreased the binding. The pentasaccharide did not bind as strongly as the other heparin-derived oligosaccharides, indicating an AT III-independent mechanism. Two classes of heparin binding sites were identified on granulocytes and one class of binding sites on monocytes and lymphocytes. The lowest amount of LMWH-tyr-FITC detected was 1 ng on granulocytes, 0.18 ng on monocytes and 0.01 ng on lymphocytes. The data suggest that heparin and other sulfated polysaccharides may play a role in the physiology of thrombosis, arteriosclerosis, and inflammation by binding to granulocytes, monocytes, and lymphocytes.
Harenberg
J
J
First Department of Medicine, Faculty of Clinical Medicine Mannheim, University of Heidelberg, Mannheim, Germany.
Malsch
R
R
Piazolo
L
L
Huhle
G
G
Heene
D L
DL
D001665:Binding Sites / Q000187:drug effects / Q000502:physiology; D001667:Binding, Competitive / Q000187:drug effects / Q000502:physiology; D002462:Cell Membrane / Q000378:metabolism*; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D006098:Granulocytes / Q000187:drug effects / Q000378:metabolism; D006493:Heparin / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D007962:Leukocytes / Q000378:metabolism*; D008214:Lymphocytes / Q000187:drug effects / Q000378:metabolism; D008970:Molecular Weight; D009000:Monocytes / Q000187:drug effects / Q000378:metabolism; D009844:Oligosaccharides / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*
D005456:Fluorescent Dyes; D009844:Oligosaccharides; D010446:Peptide Fragments; D006493:Heparin; D016650:Fluorescein-5-isothiocyanate