Reinhard Malsch

Chromatographic and electrophoretic applications for the analysis of heparin and dermatan sulfate

Heparin and dermatan sulfate are highly sulfated polydisperse glycosaminoglycans. The methods to determine such compounds include chromatographic and electrophoretic techniques. Here we report on the performances of various analytical methods for the characterization and the determination of GAGs. Heparin, low-molecular-mass heparins, dermatan sulfate and low-molecular-mass dermatan sulfate were analyzed. High-performance size exclusion chromatography was used to determine the molecular mass, polydispersity, absorbance and the area under the absorbance-time curve. Polyacrylamide gel electrophoresis was used to determine the average molecular mass and the polydispersity. Heparin and dermatan sulfate preparations were analyzed by capillary electrophoresis using reversed polarity. The results obtained reflect different performances of various analytical methods used to characterize GAGs.

High-resolution capillary electrophoresis and polycrylamide gel electrophoresis of heparins

Abstract Heparin-oligosaccharides, low-molecular-mass heparins (LMMHs) and heparins were determined using high-resolution capillary electrophoresis (HPCE) and polycrylamide gel electrophoresis (PAGE). The conditions for HPCE were 20 mM phosphate buffer (pH3.5) and 20°C. The method was equivalent to that used previously with a borate-sodium dodecyl sulfate buffer (pH8.8). Six LMMHs were determined using PAGE. The method showed a standard deviation of the average molecular mass from 6.4 to 19.8% and of their polydispersity from 0.7 to 10.2%. HPCE and PAGE revealed different important structural and compositional differences of heparins.

Determination of heparin-induced IgG antibody by fluorescence-linked immunofiltration assay (FLIFA)

A fluorescence-linked immunofiltration assay (FLIFA) was developed for the determination of heparin-induced IgG in heparin-induced thrombocytopenia (HIT) type II patients. Protein A was immobilized on a nitrocellulose membrane to bind heparin-induced IgG of HIT type II patients. Fluorescein-5-isothiocynate (FITC)-heparin was added to platelet factor 4 present in normal serum to form the neo-antigen which was captured by heparin-induced IgG. The heparin-induced IgG was quantified by the relative fluorescence intensity (RFI) of bound FITC-heparin. Values were expressed as a RFI ratio (RFI patient / RFI normal) and were 1.965+/-0.413 in HIT type II patients (n = 36) and 1.064+/-0.162 in healthy controls (n = 50, p<0.0001). The intra- and inter-assay coefficients of variation were 4.9 and 10.4%, respectively. The heparin-induced IgG FLIFA will be useful in individual and epidemiological studies in patients during treatment with heparin. The FLIFA technique offers an alternative, rapid and sensitive methodological approach for studies on the interaction between antigen-antibody or ligand-receptor.

Magnetic bead protamine-linked microtiter assay for detection of heparin using iodinated low-molecular-mass heparin-tyramine

We have developed a competitive heparin binding assay employing protamine-coated magnetic beads for detection and measurement of heparin. The assay utilizes 125-iodine specifically bound to newly synthesized low-molecular-mass (LMM) heparin-tyramine. The tracer was stable over a period of 3 weeks, as demonstrated by gel filtration chromatography. The protamine-coated beads were found to be stable over at least two months. The heparin-tyramine bead assay had in buffer a lower detection limit of 0.04 microgram/ml and in plasma of 0.23 microgram heparin/ml. 50% binding was obtained at 0.7 microgram/ml and 20% binding at 4 micrograms/ml in plasma. The within assay coefficient of variation ranged from 9 to 28% for unfractionated, high molecular mass (HMM) heparin and from 12 to 15% for LMM-heparins in buffer system and in plasma. Various heparin fractions displaced the tracer from the protamine-coated magnetic beads to different extents. The validity of the assay was proven after intravenous administration of unfractionated and LMM-heparin in man. The elimination rate was similar using the heparin-tyramine bead assay compared with the anti-factor Xa coagulation assay. After intravenous dosing of LMM-heparin the maximal concentration was lower using the heparin-tyramine bead assay compared with the anti-factor Xa coagulation assay. The bead assay was found to be reproducible, valid, and rapid for measurement of the concentration of heparin preparations in purified systems and for HMM-heparin in plasma. Measurement of the concentration of LMM-heparin in plasma has a high coefficient of variation using the binding assay.

Niedermolekulare Heparine Prophylaxe und Therapie thromboembolischer Erkrankungen

LerninhalteDer Wert der Thromboembolieprophylaxe in der perioperativen Medizin und in der inneren Medizin ist eindeutig belegt. Eine Unterlassung der Prophylaxe bei Patienten mit erhöhtem Thromboembolierisiko stellt einen Behandlungsfehler dar. Die Therapie der akuten tiefen Beinvenenthrombose muß stationär erfolgen. Die subkutane Verabreichung von konventionellem Heparin in hoher Dosierung und aPTT-adjustiert ist der kontinuierlichen intravenösen Dauerinfusion gleichwertig. Subkutanes hochdosiertes niedermolekulares Heparin ist möglicherweise zur Therapie der frischen tiefen Venenthrombose dem konventionellen Heparin überlegen. Die Heparin-induzierte Thrombozytopenie Typ II ist eine seltene aber schwerwiegende Nebenwirkung. Maßnahmen zur Früherkennung dieser Nebenwirkungen gibt es nicht. Die Therapie besteht in einem Wechsel der Antikoagulation auf nicht heparinhaltige Antikoagulantien.

Purification of the monoclonal heparin antibody H-1.18

An antibody of the (immunoglobulin) IgG1 subclass against heparin was purified. Here we report on the purification of the heparin antibody. Ammonium sulfate precipitation was performed and showed a high purity of the precipitate. In the heparin radioimmunoassay it showed a high heparin binding. Capillary electrophoresis showed that albumin and other proteins were separated from the heparin antibody. The purification method allowed a large scale production of the heparin antibody.

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.

Intact Biological Activity and Binding to Granulocytes of LMM-Heparin-Tyramine-Fitc

Unfractionated and low molecular mass heparins are widely used for prophylaxis and treatment of venous thromboembolism (1–3). The biologic activities and the pharmacodynamic properties of heparins are quite well understood. However, direct measurement of their concentration encounters different problems. The radioactive labeling of heparins using the substitution with 3-(4-Hydroxyphenyl)-propionic-acid-N-hydroxy-succimideester may alter polysaccharide backbone. The binding of fluorescent compounds to heparin leads to a lipophilic character and also alter the polysaccharide backbone if a specific binding has not been used. Recently, we have reported on the endpoint attachment of fluorescein-5-isothyo-cianate (Fitc) to a low molecular mass heparin-tyramine. Fitc has been bound to tyramine, which has been coupled by endpoint attachment to the terminal anhydromannose group of LMM-Heparin (4, 5). This technique should provide an intact binding site of the LMM-Heparin to the binding sequences on proteins. Beside the anticoagulant activity, many non-anticoagulant activities of heparin are of clinical relevance (6). These may be induced by the binding of heparin to the endothelium, erythrocytes or monocytes (7–9).

Binding of Glycosaminoglycans to Leukocytes Using Fluorescent Labeled Gag-Derivatives

The non-anticoagulant properties of glycosaminoglycans (GAGs) include antithrombotic, antiatherosclerotic, antiinflammatory and antimetastatic actions and are thought to be caused by binding of the GAGs to positively charged groups of matrices and peptides (1, 2, 3). Heparins are the best known group of GAGs and they are linear polysaccharide chains with defined negatively charged sulfate and carboxylic groups. Fractions and fragments of heparins and heparin-derived oligosaccharides were obtained to investigate the metabolism and the structure function relationship with plasma and cellular proteins. Binding of heparin and heparin fragments to human endothelial cells was demonstrated using radiolabeled compounds (4). Decreasing the molecular weight of the heparin chains, the binding to endothelial cells decreased (5). Heparin has been reported to bind to human monocytes and to modulate their procoagulant activity (5). Blood macrophages were found to internalize and degrade heparin (6). Unfractionated heparin seems to bind to erythrocytes as demonstrated using radioactively labeled compound (7).

Inequivalence of Glycosaminoglycans Using High-Performance Size Exclusion Chromatography, Polyacrylamide Gel Electrophoresis and High-Performance Capillary Electrophoresis

Biologically active sulfated polysaccharides like heparin and dermatan sulfate are commonly known GAGs (1)* Although its primary application as anticoagulant heparin can be considered as polyelectrolytic drug displaying a variety of biological activities. Heparins can be easily neutralized in vitro and in vivo by an equigravimetric amount of protamine or polybrene.