Heparin is a linear, heterogeneous, highly sulfated polysaccharide belonging to the family of glycosaminoglycans, endowed with anticoagulant and antithrombotic properties. Its linear chains are made up of 15-200 disaccharide units of N-sulfated or N-acetylated D-glucosamine linked to hexuronic (glucuronic or iduronic) acid. Low molecular weight heparins (LMWHs), developed to circumvent some unwanted side effect of unfractionated heparins (UFH), are obtained from UFH by diverse chemical and enzymatic depolymerisation processes. Fragments generated by cleavage of heparin may therefore structurally differ from each other more than the original chains because of the modification induced by the depolymerisation methods and the heterogeneity of the UFH. Only about one-third of heparin chains contains a unique pentasaccharide sequence (AGA*IA), which specifically binds antithrombin (AT) thus promoting the inhibition of certain proteases of the coagulation cascade. Such a sequence, characterized by a central trisulfated glucosamine (A*), is believed to survive the depolymerising procedures used for the preparation of LMWHs. Functional assays performed in vitro, evaluating plasma protein binding and antiprotease activity AT mediated, showed wide variations among the commercially available LMWHs, indicating that their compositional differences have an important impact on function. With the aim of contributing to the elucidation of structure-activity relationship of LMWHs, the present work is focusing on the in depth study of the oligosaccharide composition of different LMWH preparations. Three of the most popular commercial preparations Enoxaparin, Dalteparin and Tinzaparin were analysed. Their compositional differences were determined by analyzing their oligosaccharidic populations by gel permeation chromatography, a very characteristic fingerprint for each sample was revealed. Affinity chromatography on AT-Sepharose was performed to separate and quantify the high affinity fraction,. Structural characterization of all samples was obtained by 1D and 2D NMR spectroscopy and all molecular weight parameters were evaluated through HP-SEC/TDA. All the HA fractions exhibited a considerably higher molecular weight and a reduced polydispersity with respect to NA fractions. To deepen the characterization of HA components, HA heparin chains of each LMWH were further fractionated into three subfractions with graded affinity toward AT HA1>HA2>HA3. All the above fractions were analysed via NMR evaluating the average content of all the monosaccharide components and, in particular, the percentage of A* (N-glucosamine tri-sulfated) and G-A* (disaccharide composed by glucuronic acid and A*) both regarded as markers of heparin active site for AT (AGA*IA). Selected oligomeric fractions and the HA1, HA2, HA3 fractions were analysed via ESI-TOF (as detector after a SEC chromatography). The molecular weight of all HA subfractions were estimated by two different methods: HP-SEC/ESI-MS and NMR. The results suggest that neither the molecular weight nor the sulfation degree calculated via NMR exhibited any correlation with the degree of affinity for AT. By combining information obtained by NMR (G-A* content) and the chain length (calculated by Mw evaluation)the AGA*IA content per chain was approximately calculated pointing out the presence of some chains containing more than an active pentasaccharide (HA1 of enoxaparin and dalteparin). Preliminary data on biological activity in vitro indicated that the different anti-Xa activities were directly related to the degree of AT affinity and the overall structural considerations. The present work represents the first insight into the detailed and comparative structural characterization of three commercial LMWHs differing in manufacturing process. Important and characteristic structural parameters were defined, including the precise oligomeric composition, the relative content of AT interacting species, and their molecular weight, together with the relative content of variously substituted monosaccharide components. Further studies are required to unravel the correlation of structural features with LMWH functional properties.
|Titolo:||Low Molecular Weight Heparins : in depth structural characterization to understand their different biological properties|
|Data di pubblicazione:||2008|
|Parole Chiave:||Low ; Molecular ; Weight ; Heparins ; Heparin ; Antithrombin|
|Settore Scientifico Disciplinare:||Settore BIO/10 - Biochimica|
|Citazione:||Low Molecular Weight Heparins : in depth structural characterization to understand their different biological properties ; A. Bisio ; tutor S. Sonnino, S.G. Pagani. - Milano : Università degli studi di Milano. DIPARTIMENTO DI CHIMICA E BIOCHIMICA MEDICA, 2008. ((21. ciclo, Anno Accademico 2007/2008.|
|Digital Object Identifier (DOI):||10.13130/vecchietti-davide-giovanni_phd2008|
|Appare nelle tipologie:||13 - Tesi di dottorato discussa entro ottobre 2010|